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African Journal of 

Microbiology Research 

Volume 5 Number 16 18 August, 2011 

ISSN 1996-0808

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International African Journal Journal of Medicine of Microbiology and Medical Research Sciences 

Table of Contents: Volume 5 Number 16 18 August, 2011 

nces 

Research Articles 

ARTICLES 

Isolation and characterization of a psychrophilic bacterium producing cold 

active lactose hydrolyzing enzyme from soil of Mt. Himalaya in Nepal 2198 

E. S. Nam, Y. H. Kim, K. H. Shon, and J. K. Ahn 

Nigerian phages: The first bacteriophages from Tropical Africa 2207 

Adeniran S. Koko, Hans W. Ackermann, Monisola A. Taiwo 

and Sunday A. Omilabu 

Evaluation of real-time PCR for Mycobacterium ulcerans in endemic region 

in Côte d’Ivoire 2211 

Elise Solange Ngazoa-Kakou, Euloge Ekaza, Nguetta Aka, David 

Coulibaly-N’Golo1, Bakary Coulibaly and Mireille Dosso 

Contamination of cattle carcasses by Escherichia coli shiga like toxin with 

high antimicrobials resistence 2217 

Everlon Cid Rigobelo, Renato Pariz Maluta, Clarissa Araújo Borges, 

Lívia Gerbasi Beraldo, Manoel Victor Franco, Lemos Sirlei 

Aparecida Maestá and Fernando Antonio de Ávila 

Determination of insecticidal toxicity of three species of entomopathogenic 

spore-forming bacterial isolates against Tenebrio molitor L. (Coleoptera: 

Tenebrionidae) 2222 

N. Du Rand and M. D. Laing 

What changed in necrotizing fasciitis in twenty-five years? 2229 

Baris Yildiz, Derya Karakoc, Erhan Hamaloglu, Arif Ozdemir 

and Ahmet Ozenc


Table of Content: Volume 6 Number 23 21 June, 2012 

nces 

ARTICLES 

ARTICLES 

Aspects of bacterial colonization in newborn babies 2234 

Fariba Heshmati, Seyeed Amir Yazdanparast , Seyeed Akbar Moosavi , 

Influence of ciprofloxacin on glioma cell line GL26: A new application for 

Hussein Dargahi, and Farnaz Tabibzadeh 

an old antibiotic 

Abdolreza Esmaeilzadeh, Massoumeh Ebtekar, Alireza Biglari and 

Zuhair Mohammad Hassan 4891 

Evaluation of anticarcinogenicity effect of Artemia urmiana by Salmonella 

typhimurium TA 100 strain 2241 

Masumeh Abbasi, Saman Mahdavi and Sedigheh Mehrabian 

Identification of microbial diversity in caecal content of broiler chicken 

S. Nathiya, G. Dhinakar Raj, A. Rajasekar, D. Vijayalakshmi and T. Devasena 4897 

mRNA expression of iron metabolism relation genes in macrophages by 

infection with Salmonella typhimurium 2245 

Microbial quality of some non-sterile pharmaceutical products sourced 

Pan Xin 

from some retail pharmacies in Lagos, Nigeria 

Adeola Anifowoshe R., Opara Morrison I. and Adeleye Isaac A. 4903 

Expanding drug resistance through integron acquisition in Salmonella spp. 

isolates obtained in Iran 2249 

Molecular detection of adhesins genes and biofilm formation in methicillin 

Bahareh Rajaei, Seyed Davar Siadat, Mohamad Reza Razavi, 

resistant Staphylococcus aureus 

Mohammad Reza Aghasadeghi, Nahid Sepehri Rad, Farzad Badmasti, 

Karima BEKIR, Omayma HADDAD, Mohammed GRISSA, Kamel CHAIEB, 

Somieh Khanjani Jafroodi, Taraneh Rajaei, Arfa Moshiri, Saifuddin Javadian 

Amina BAKHROUF and Salem IBRAHIM ELGARSSDI 4908 

The synthesis and role of hydroxyectoine in halophilic bacterium 

Amylase production by moderately halophilic Bacillus cereus in solid 

Halomonas ventosae DL7 2254 

state fermentation 

Daochen Zhu, Chenxiang Wang, Shoko Hosoi-Tanabe, 

P. Vijayabaskar, D. Jayalakshmi and T. Shankar 4918 

Weimin Zhang and Shinichi Nagata 

Networking clusters and sequence characteristics of clustered regularly 

Virulence and characteristics of a new nucleopolyhedrovirus strain of 

interspaced short palindromic repeats (CRISPR) direct repeats and their 

Dendrolimus kikuchii (Lepidoptera: Lasiocampidae) 2261 

evolutionary comparison with cas1 genes in lactic acid bacteria 

Miao Miao Yang, Meng Lou Li, Yu Zhu Wang, Liang Jian Qu, Ke Yue Huai, 

Kaibo Deng, Fei Liu, Chuntao Gu and Guicheng Huo 4927 

Xue Wen Nie, Lu Qin Qiao, Jin Ying Ding and Yong An Zhang 

Characterization of Staphylococcus spp strains isolated from hospital, 

Antibacterial screening of the root, stem and leaf extracts of Terminalia albida sc. 

community and environmental in Puebla city, Mexico 2266 

elliot on selected pathogenic bacteria 

Ana Marta de los Ángeles Lobo-Sánchez, Patricia Lozano-Zaraín, 

S. M. Ayodele, G. Alpheus and O. M. Iruaga 1457 

Ygnacio Martínez-Laguna, Zita Gutiérrez-Cázarez, Antonio Rivera-Tapia, 

Carmen Torres and Rosa del Carmen Rocha-Gracia


nces 

ARTICLES 

Interaction of severe acute respiratory syndrome (SARS) nucleocapsid 

protein with macrophage migration inhibitory factor protein (MIF) 2273 

Yang Lin, Baohua Wang, Yijun Liu, Zhigang Yu, Manhua Cui and Haichun Ma 

Isolation and characterization of high caffeine-tolerant bacterium strains 

from the soil of tea garden 2278 

Fang-Yuan Fan, Yan Xu, Yue-Rong Liang, Xin-Qiang Zheng, 

Devajit Borthakur and Jian-Liang Lu 

Biopreservative potential of marine Lactobacillus spp 2282 

K. Indira, S. Jayalakshmi, A. Gopalakrishnan and M. Srinivasan 

Ephedra alata as biologically-based strategy inhibit aflatoxigenic 

seedborne mold 2297 

Al-Qarawi, A. A., Abd_Allah, E. F. and Hashem Abeer 

Primary resistance rates of Mycobacterium tuberculosis complex strains 

isolated from new tuberculosis cases: A 6-year observation 2304 

Servet Kayhan, Alper Akgüneş, Hikmet Tereci and Ümit Tutar 

Cytokine genes expression in mice hepatocytes during malaria infection 2311 

Saad Alkahtani, AL-Farraj S. A., Saud A. Alarifi, AL-Eissa Mohammed saad 

and Al-Dahmash B


nces 

ARTICLES 

Phylogenetic analysis of the nematicidal actinobacteria from agricultural 

soil of China 2316 

Xu Chuan Kun, Lou Xiao Jun, Xi Jia Qin, Gu Lei, Duan Chang Qun, Mo Ming He, 

Zhang Ke Qin, Yang Fa Xiang and Fang Dun Huang 

A new recommended disinfectant for dental instruments 2325 

Jamileh bigom Taheri, Mahin Bakhshi, Sedigheh Bakhtiari, Bahare Nazemi, 

Fateme Fallah, Sahand Rezaie, Hamed Mortazavi and Somayyeh Azimi 

The functional roles of the residue tyrosine at position 26 in staphylococcal 

enterotoxin C2 2329 

Hongbo Wang, Junyi Zhou, Mingkai Xu, Huiwen Zhang and Chenggang Zhang 

Prevalence study of cytomegalovirus (CMV) infection among foreign 

manpower in Jeddah Saudi Arabia 2338 

N. A. Redwan, M. M. M. Ahmed and M. S. H. AL Awfi 

Antibacterial and cytotoxic activity of Eremurus persicus 

(Jaub and Spach) Boiss 2349 

Mojdeh Hakemi Vala, Jinous Asgarpanah, Mohammad Hossein Hedayati, 

Jeilan Shirali and Fatemeh Bagheri Bejestani 

Optimization of ultrasound-assisted extraction conditions using orthogonal 

matrix design to enhance the antimicrobial activity of extracts from 

Cichorium intybus root 2353 

Quanzhen Wang, Haitao Liu, Jinhong Du, Jian Cui, Guo Chen and Yuyan Liu


nces 

ARTICLES 

Optimization of the production of exopolysaccharides by Bacillus thuringiensis 

27 in sand biological soil crusts and its bioflocculant activity 2359 

Z. R. Wang, J. P. Sheng, X. L. Tian, T. T. Wu, W. Z. Liu and L. Shen 

A simple and efficient method that uses low concentration fetal bovine 

serum to culture and purify Schwann cells 2367 

Han Feng, Qu Wei, Jiang Huajun, Fu Chongyang, Lu Ming, Zhang Weiguo and 

Lv Decheng 

First report on Enterobacter sakazakii from Sudanese patients 2374 

Humodi A. Saeed and Rania M. Musallam 

In search of enteroviruses in water media in Marrakech 2380 

Amina Hssaine, Jawhar Gharbi, Rafik Harrath, Rajae Harrak, 

Abderrahman Chait, Mahjoub Aouni and Jamal Hafid 

Laboratory analysis of a fatal meningococcal case due to serogroup B 

Neisseria meningitidis belonging to ST- 4821 complex 2385 

Zengguo Wang, Tiejun Hou, Zhijun Chen, Jinsong Li, Shouzhi Wu, 

Xiaoguang Wei, Yahui Sun and Quanli Du 

Genotyping of Hepatitis C virus (HCV) in infected patients from Saudi Arabia 2388 

Mohammed Ali M. Marie

African Journal of Microbiology Research Vol. 5(16), pp. 2198-2206, 18 August, 2011 

Available online http://www.academicjournals.org/ajmr 

DOI: 10.5897/AJMR10.609 

ISSN 1996-0808 ©2011 Academic Journals 

Full Length Research Paper 

Isolation and characterization of a psychrophilic 

bacterium producing cold active lactose hydrolyzing 

enzyme from soil of Mt. Himalaya in Nepal 

E. S. Nam 1 , Y. H. Kim 2 , K. H. Shon 2 , and J. K. Ahn 2 * 

1 Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, Republic of Korea. 

2 Department of Agricultural Sciences, Korea National Open University, Seoul 110-791, Republic of Korea. 

Accepted 18 February, 2011 

Psychrophilic bacteria, which grew on lactose as a carbon source, were isolated from soil of Island 

Peak area at Mt. Himalaya in Nepal, and identified. 148 bacterial isolates were isolated via X-gal 

hydrolysis on plate. Among these, three isolates, referred to as KNOUC 401, 402 and 403, displayed 

good cold-adapted growth and activity for hydrolysis of o-nitrophenyl- -D-galactopyranoside (ONPG) 

and lactose in cell free extracts. Strain KNOUC403 showing negative activity in -hemolysis was 

selected and identified. The strain was gram-positive bacilli, non-motile, catalase-positive and aerobic. 

Optimum growth was done at 20°C and pH 6.5~6.8. Anteiso-C15:0 (68.14%) was predominant in the fatty 

acid composition of cell. The physiological and biochemical properties, cellular fatty acid composition 

and sequence of 16S rDNA, suggest that strain KNOUC403 indicate a taxonomic affiliation to 

Arthrobacter psychrolactophilus. The optimal conditions for ONPG hydrolysis of cell free extracts from 

the strain were 15°C and pH 6.0, and the enzyme was stable at 4°C for 42 h. Zymogram assay of cell free 

extract from the strain in nondenaturing polyacrylamide gel showed a distinct band of X-gal hydrolysis. 

Key words: Psychrophilic bacteria, Arthrobacter psychrolactophilus, cold-active lactose hydrolyzing enzyme, 

Mt. Himalaya. 

INTRODUCTION 

Recently a wide diversity of cold adapted microorganisms 

have been found in various cold environments 

(Groudieva et al., 2004), and their enzymes attracted 

interests because of their biotechnological potential 

offering economical and ecological advantages such as 

energy saving, function in cold environment, minimizing 

undesirable chemical reactions that occur at high 

temperature, and inactivating easily by heating when 

required (Russell, 1988; Margesin and Schinner, 1999; 

Margesin et al., 2002; Cavicchioli et al., 2002). Owing to 

the fact that cold completely penetrates microorganisms, 

all components including enzymes of psychrophiles living 

in cold environment must be suitably adapted. Therefore 

psychrophiles can produce cold adapted enzymes which 

*Corresponding author. E-mail: ajk@knou.ac.kr. Tel: +82-2 

3668-4630. Fax: +82-2-3668-4187. 

are highly flexible in structure enabling increased 

complementarity between the active site and substrate at 

a low energy cost resulting in high catalytic activity at low 

temperature and labile at elevated temperature 

(Cavicchioli et al., 2002). A variety of useful cold active 

enzymes have been found in psychrophiles (Feller et al., 

1997; Marshall, 1997; Gerday et al., 2000; Groudieva et 

al., 2004). 

Cold active enzymes are attractive in food industry, e.g. 

processing of fruit juices and milk, as there is an 

increasing industrial trend to treat foodstuffs under low 

temperature conditions in order to avoid changes in taste 

and nutritional value, and to save energy (Margesin and 

Schinner, 1994; Russel, 1998). Cold active enzyme 

hydrolyzing lactose to glucose and galactose at 

refrigerating temperature is an important food-industrial 

enzyme, because it can be used to produce lactose 

hydrolyzed milk for the lactose intolerant people, and can 

also be used to convert lactose in whey, a by-product of

cheese industry, to more readily fermentable glucose and 

galactose. Until now, some psychrophilic bacteria 

producing -galactosidase have been reported, but none 

of them have been hitherto used practically in the food 

industry, meaning that more study is required to find 

better microbial source. The aim of this study was to 

isolate a psychrophilic bacterium, from Mt. Himalaya of 

Nepal, producing the cold-active lactose hydrolyzing 

enzyme practically useful in food industry. 

MATERIALS AND METHODS 

Isolation and cultivation condition of microorganisms 

A soil sample was collected from each of the 39 regions of Island 

Peak area (27°55'N, 86°56'E) of 4,500 to 6,000 m height in Mt. 

Himalaya of Nepal by using sterilized awls, sterilized spoons and 

sterilized Falcon tubes. To cultivate the psychrophilic bacteria, 5 g 

of soil samples were added to 45 ml of Brain heart infusion (BHI; 

Difco Laboratories, Detroit, Mich) broth containing 1% (w/v) lactose, 

and incubated at 4°C aerobically by shaking (200 rpm) for 30 days. 

1 ml of cells grown in this enrichment were spread onto BHI agar 

containing 1% (w/v) lactose and 0.01% (w/v) 5-bromo-4-chloro-3indolyl- 

-D-galactopyranoside (X-gal; Duchefa Biochemei, Holland). 

After incubation at 4°C for 15 days, blue colonies were selected, 

then cultivated in brain heart infusion broth or agar for identification 

and determining properties of lactose hydrolyzing enzyme. 

Morphological, biochemical and physiological properties 

Isolates showing good growth and good X-gal hydrolysis on BHI 

agar, and high activity for hydrolysis of ONPG and lactose were 

investigated for Gram staining, morphological, biochemical, and 

physiological properties. Cell was grown in BHI broth to determine 

growth conditions of various temperatures (5 to 40°C) and pH (4.0 

to 8.0). pH of media was adjusted with HCl or NaOH. Acid 

production from carbohydrate and utilization of sole carbon sources 

were determined using API 20E and API 50CH test strips (Bio- 

Merieux Inc., France). For the test of hemolysis, a colony was 

streaked on sheep blood agar (KOMED. Co. Ltd). After incubation 

at 15°C for 3 days, -hemolytic activity was detected by lysis and 

complete digestion of red blood cell contents surrounding colony. All 

biochemical and physiological tests were done at 15°C for 3 days 

Composition of cellular fatty acid 

The cell biomass for cellular fatty acid composition analysis was 

collected from BHI agar plates after incubation at 15°C for 3 days. 

Cells were harvested, and the cellular fatty acids were saponified, 

methylated and extracted, following the instructions in the manual 

for Sherlock Microbial Identification System (MIDI, USA). The fatty 

acids were analyzed by gas chromatography (Hewlett Packard 

6890, USA) and identified using the microbial identification software 

package (Sasser, 1990). 

16S rDNA sequence determination and phylogenetic analysis 

Isolation of genomic DNA, PCR amplification of the 16S rDNA gene 

and sequencing of the purified PCR product were carried out as 

described by Rainey et al. (1996). Universal primers of fD1 (5’gagtttgatcctggctcag-3’) 

and rD1 (5’-agaaaggaggtgatccagcc-3’) were 

used for PCR. PCR product was purified by ethanol precipitation, 

Nam et al. 2199 

and sequenced with a model 377 Genetic Analyzer (Perkin-Elmer 

Co.). The 16S rDNA sequence obtained in this study was aligned 

against the previously determined sequences of the genus of 

Arthrobacter available from the Ribosomal Database Project 

(Maidak et al., 1996). The phylogenetic tree for the data set was 

inferred using the neighbor-joining method (Saitou and Nei, 1987). 

The PHYLIP package (Felsentein, 1993) was used for constructing 

the tree. 

Assay of lactose hydrolyzing enzyme activity 

Microorganism was cultivated at 4°C aerobically by shaking (200 

rpm) for 7 days to the late of log phase, and harvested by 

centrifugation at 8,000 X g for 10 min at 4°C, suspended in sodium 

phosphate buffer (0.01 M, pH 6.8), washed 2 times by the same 

buffer, suspended in the same buffer, and sonificated at 4°C. Cell 

debris was eliminated by centrifugation at 12,000 g and 4°C for 20 

min. The cell free extracts were used for assay of lactose 

hydrolyzing enzyme. Lactose hydrolyzing enzyme activity was 

determined by measuring the rate of hydrolysis of o-nitrophenyl β- 

D-galactopyranoside (ONPG) or lactose as substrate. The 

procedure of Miller (1972) for hydrolysis of ONPG was used. An 

aliquot of cell free extracts (0.5 ml) was added to 2.5 ml of ONPG 

(0.04 M) dissolved in sodium phosphate buffer (0.01 M, pH 6.8) and 

incubated at 4°C for 2 h. The reaction was stopped by addition of 3 

ml of 0.5 M Na2CO3 dissolved in H2O and the absorbance at 420 

nm was measured. 

One unit of enzyme activity for hydrolysis of ONPG was defined 

as the activity hydrolyzing 1 mol of ONPG per min by cell free 

extract from 1 ml of culture that was concentrated to 8 of A600. The 

hydrolysis of lactose was assayed by measuring the release of 

glucose from lactose. The reaction mixture consisted of 1.6 ml of 

skim milk (lactose conc., 4.7%) and 0.4 ml of cell free extracts. The 

mixture was incubated at 4°C for 5 days followed by heating in 

boiling water for 2 min. After centrifugation, glucose concentration in 

the supernatant was determined by the colorimetric method with a 

commercial Glucose C-II test kit (Wako Chemical Co., Japan) at 

505 nm. One unit of enzyme activity for hydrolysis of lactose is the 

activity of hydrolyzing 1 mol of lactose per day by cell free extracts 

from 1 ml of culture that was concentrated to 8 of A600 . Zymogram 

assay for the cell free extracts of a selected strain was performed. 

After native-PAGE of cell free extracts on 10% (w/v) polyacrylamide 

gel (Laemmli, 1970), the gel was stained with 0.25 mM X-gal 

(Trimbur et al., 1994) at 4°C for 2 h. Hydrolysis of X-Gal was 

confirmed by blue band within the polyacrylamide gel. 

Effect of temperature and pH on lactose hydrolyzing enzyme 

The effect of temperature on the activity of cell free extracts for 

ONPG hydrolysis was analyzed by measuring the enzyme activity 

at various temperatures (5 to 40°C) in sodium phosphate buffer 

(0.01 M, pH 6.8). Effect of pH on enzyme activity was evaluated at 

pH ranging from 4.3 to 7.8 at 4°C was used for the pH from 4.3 to 

6.0, and Na-phosphates buffer (0.01 M) was used for the pH from 

6.0 to 7.8. Stability of enzyme was investigated by residual activity 

during incubation of cell free extracts in sodium phosphate buffer 

(0.01 M, pH 6.8) for 42 h at 4 and 37°C. 

RESULTS 

Isolation of strain producing cold active lactose 

hydrolyzing enzyme 

Psychrophilic bacteria, which grow and hydrolyze lactose

2200 Afr. J. Microbiol. Res. 

Table 1. Physiological and biochemical properties of isolated strains. 

Characteristics 

Strains 

KNOUC401 KNOUC402 KNOUC403 

Gram staining - + + 

Shape Rod Rod Rod 

Motility - + - 

Spore formation - + - 

Optimum temp for growth 20°C 20°C 20°C 

Optimum pH for growth 6.8 6.5-6.8 6.5-6.8 

Growth at 4°C + + + 

Growth at 37°C + + - 

Catalase + + + 

Oxidase + + - 

ONPG hydrolysis + + + 

PNPG hydrolysis + + + 

Nitrate reduction - - - 

Gas production + - - 

H2S formation - - - 

Citrate utilization + - - 

Growth on MacConkey agar + - - 

Utilization of 

D-glucose + + + 

D-mannitol - - - 

D-maltose + + - 

D-rhamnose + + - 

D-mannose - - - 

D-galactose - - - 

D-lactose + + + 

D-fructose - - - 

D-adonitol - + - 

D-raffinose + + - 

D-sorbitol + + - 

Sucrose + - - 

Xylitol + + - 

Ducitol + + + 

Gelatin - - - 

Sorbose - - + 

Glycerol + + - 

Erythrol - - - 

Genus Aeromonas Bacillus Arthrobacter 

+, Positive reaction; -, Negative reaction. 

at low temperature, were isolated from the soil of Island 

Peak area at Mt. Himalaya in Nepal. 148 bacterial 

isolates were isolated via X-gal hydrolysis after soil 

samples were fortified in BHI broth, spread and incubated 

on BHI agar at 4°C. Among these, three isolates, referred 

to as KNOUC 401, KNOUC402 and KNOUC403, showed 

good growth at low temperature (4°C), formed distinct 

blue colony on BHI plate containing X-Gal, and their cell 

free extracts showed practically useful activity for 

hydrolysis of ONPG and lactose. 

Identification of strain KNOUC403 

The strains of KNOUC401, KNOUC402 and KNOUC403 

were characterized for their physiological and 

biochemical properties to get information on their 

suitability for use in the food industry. KNOUC401, 

KNOUC402 and KNOUC403 were presumably identified 

as Aeromonas, Bacillus and Athrobacter respectively by 

phenotypic features as in Table 1. Isolate KNOUC 403 

was negative for β-hemolysis on sheep blood agar, but

Table 2. Properties of preliminarily selected strains. 

Area (M) Strain No. 

Hydrolysis of 

ONPG (Unit 1) ) Lactose (Unit 2) ) 


Identification Hemolysis 

4700 KNOUC401 3.3394 11.54 Aeromonas + 

4950 KNOUC402 0.1467 1.04 Bacillus + 

5550 KNOUC403 0.23319 2.76 Arthrobacter - 

1) 

One unit of enzyme activity is defined as the activity hydrolyzing 1 mol of ONPG per min by cell free extract from 1 ml of culture whose 

A600 is 8. 

2) 

One unit of enzyme activity is the one hydrolyzing 1 mol of lactose per day at 4°C by cell free extracts from 1 ml of culture whose A600 

is 8. 

Table 3. Composition of major cellular fatty acids of 

strain KNOUC403. 

Fatty acid Contents (%) 

14:0 iso 5.73 

15:0 iso 3.14 

15:0 anteiso 68.14 

16:0 iso 8.08 

17:0 iso 4.21 

KNOUC401 and 402 were positive (Table 2). Therefore, 

we selected strain KNOUC403 for further study. For 

further identification of strain KNOUC403, the strain was 

tested for its cellular fatty acid composition and the 

sequence of 16S rDNA. The cellular fatty acids of 

KNOUC403 were composed of anteiso-C15:0 (68.14%), 

iso-C16:0 (8.08%), iso-C14:0 (5.73%), anteiso-C17:0 (4.21%), 

iso-C15:0 (3.14%), with additional fatty acids comprising 

less than 1% (Table 3). 

The sequence of 16S rDNA (GeneBank accession No. 

HQ610444) of strain KNOUC403 determined as 1,492 bp 

is shown in Figure 1, and the phylogenetic tree 

constructed by the neighbor-joining method (Felsenstein, 

1993) is shown in Figure 2. Comparing 16S rDNA 

sequence of strain KNOUC403 with the sequences in 

Ribosomal Data base Project (Maidak et al, 1996) and 

NCBI, demonstrated that strain KNOUC401 should be 

classified as an Arthrobacter sp., and its closest relative 

was Arthrobacter psychrolactophilus (98 to 100%). 

Therefore, strain KNOUC403 was identified as A. 

psychrolactophilus, and the strain was named as A. 

psychrolactophilus KNOUC403. 

Properties of lactose hydrolyzing enzyme 

Cell free extracts of strain KNOUC403 was tested for the 

properties of lactose hydrolyzing enzyme related to pH 

and temperature to confirm the possibility as a source of 

cold active lactose hydrolyzing enzyme. The optimal 

temperature for hydrolysis of ONPG was 15°C (Figure 

3a). The enzyme exhibited ca. 87% of maximal activity at 

4°C, and the enzyme activity decreased to less than 80% 

of maximal activity with a further increase in temperature 

to 40°C. The optimal pH was pH 6.0, it exhibited activities 

higher than 80% of optimum activity at broad pH from 4.3 

to 7.8 (Figure 3b), meaning that this strain produces cold 

active lactose hydrolyzing enzyme useful at wide range of 

pH. To determine the stability at 4 and 37°C, the cell free 

extracts in Na-phosphate buffer (0.01 M, pH 6.8) was 

incubated at those temperatures for 42 h and residual 

activity were tested during incubation. It was stable at 

4°C but lost its all activity at 37°C in 42 h (Figure 4). To 

confirm the existence of lactose hydrolyzing enzyme in 

crude cell free extracts of strain KNOUC403, zymogram 

assay for X-gal hydrolysis was performed. There was a 

distinct band of X-gal hydrolysis (Figure 5). In native 

electrophoresis, the enzyme moved to the spot of around 

130 kDa. 

DISCUSSION 

Cold-adapted lactose hydrolyzing enzymes of high 

activity at low temperature would be useful for hydrolysis 

of lactose in refrigerated milk; enabling it to be consumed 

by lactose-intolerant individuals, and for converting whey 

to a sweetener by hydrolyzing lactose to sweeter sugars 

of glucose and galactose. Psychrophilic bacteria living in 

cold environments, such as glacier silts, place of high 

altitude in Himalaya, Arctic soils and Antarctic environments, 

are potentially useful sources of cold-adapted 

enzymes. Among strains isolated from soil of perpetual 

snow area at Island peak region of Mt. Himalaya in 

Nepal, we selected the strain KNOUC403 showing the 

good growth and lactose hydrolyzing activity at 4°C, and 

no activity of β-hemolysis. The growth physiology of this 

strain, growing at 4~30°C with optimum growth 

temperature of 20~30°C and not growing over 37°C, is 

well fitted to define this strain as a psychrophile (Kocur et 

al., 1991). The strain KNOUC403 has phenotypic 

characters of Gram positive, rod, nonsporing, nonmotile , 

and catalase negative, that are fitting to the genus 

Arthrobacter (Holt et al., 1994), as in Table 1. The


GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCCCGCTTG 

CGGGGGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTAACTC 

TGGGATAAGCCTTGGAAACGAGGTCTAATACTGGATATTGACTTTGCCTCGCATGG 

GGTTTGGTTGAAAGATTTATTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTG 

GTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACC 

GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG 

AATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACG 

GCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAACAAGGCCAGCATTTTTGTTGGTT 

GAGGGTACTTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAAT 

ACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGT 

TTGTCGCGTCTGCCGTGAAAGTCCGGGGCTCAACCCCGGATCTGCGGTGGGTAC 

GGGCAGACTAGAGTGATGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATG 

CGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCATTAACTGA 

CGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAT 

GCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAG 

CTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAG 

GAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAAC 

GCGAAGAACCTTACCAAGGCTTGACATGAACTGGAAATACCTGGAAACAGGTGCC 

CCGCTTGCGGTCGGTTTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAG 

ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTA 

ATGGTGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGAGGACG 

ACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGG 

TACAATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTT 

CGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGA 

TCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGT 

CACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAACCCCCTTGTGGGGAGGGA 

GCTGTCGAAGGTGGGACTGGCGATTGGGACTAAGTCGTAACAAGGTAGCCGTACC 

GGAAGGTGC 

Figure 1. 16S rDNA sequence of strain KNOUC403 (1,492 bp). 

cellular fatty acid composition profile of KNOUC403 with 

anteiso-C15:0 as a predominant fatty acid is typical for a 

member of genus Arthrobacter (Lee et al., 2003; Storms 

et al., 2003; Kotouckova et al., 2004; Margesin et al., 

2004). Heyrman et al. (2005) reported that predominant 

fatty acid of the genus Arthrobacter was anteiso-C15:0 and 

iso-C15:0. The predominant fatty acids of A. 

psychrolactophilus (sp. nov.) was also anteiso-C15:0 that 

comprised 73% of total fatty acids (Loveland-Curtze et 

al., 1999). Therefore “Loveland-Curtze et al. (1999) it has 

been reported that several Arthrobacter strains produced 

cold-active -galactosidase (Coker et al., 2003; 

Nakagawa et al., 2003). Arthrobacter sp. 20B - 

galactosidase was optimally active at pH 6.0 to 8.0 and


Figure 2. Phylogenetic tree based on 16S rDNA sequence showing the position of strain KNOUC403 as the type strain of Arthrobacter 

species and the representative of some other related taxa. Scale bar represents 0.01 substitutions per nucleotide. 

Relative Relative activity(%) activity (%) 

100 

90 

80 

70 

60 

0 10 20 30 40 

Temperature Temp. 

A


Relative actitvity(%) 

100 

90 

80 

70 

60 

Na-acetate buffer (0.01M) 

4 5 6 7 8 

pH 

B 

Na-phosphate buffer (0.01 M) 

Figure 3. Effects of temperature (A) and pH (B) on the activity of lactose hydrolyzing enzyme in cell free 

extracts of Arthrobacter psychrolactophilus KNOUC403 for the hydrolysis of ONPG. *Effect of temperature on 

enzyme activity was analyzed in Na-phosphate buffer (0.01 M, pH 6.8). *Values are means of triplicates ± S.D. 

*Effect of pH on enzyme activity was evaluated at 4°C in Na-acetate buffer (0.01 M, pH 4.3 to 6.3) and Naphosphate 

buffer (0.01 M, pH 5.8 to 7.8), *values are means of triplicates ± S.D. 

Residual activity(%) 

120 

100 

80 

60 

40 

20 

0 

0 6 12 18 24 30 36 42 48 

Hours 

Figure 4. Stability of lactose hydrolyzing enzyme in cell free extracts of Arthrobacter 

psychrolactophilus KNOUC403 at 4°C ( ) and 37°C ( ) in Na-phosphate buffer (0.01 M, pH 

6.8). *Values are means of triplicates ±S.D. 

25°C (Bialkowska et al., 2009). 

A -galactosidase of Arthrobacter sp. obtained from 

Antarctic dry valley soil was cold optimal at 18°C, showed 

50% activity of optimum activity at 0°C, and was an 

unstable one, inactivated within 10 min at 37°C (Coker et 

al., 2003). Arthrobacter reported by Nakagawa et al. 

(2006) produced cold active -galactosidase with 

optimum temperature of 10°C and optimum pH of 8.0. 

However, two -galactosidases of Pseudoalteromonas 

sp., a psychrophile, were not cold active enzymes whose 

optimum temperature were 26°C (Fernandes et al., 2002) 

and 45°C (Hoyoux et al., 2001) showing that all

KDa Ma 1 2 

X-gal hydrolyzing enzyme 

Figure 5. Native polyacylamide electrophoresis and zymogram of cell free extracts of 

strain Arthrobacter psychrolactophilus KNOUC 403. The protein was analyzed on a 

10% polyacrylamide gel. Ma: Kaleidoscope polypeptide standards marker. 1: The gel 

was stained with Coomassie Brilliant Blue R-250. 2: The gel was soaked in 0.1 M Xgal 

solution (in Z-buffer) at 4°C for 2 h. 

psychrophiles do not always produce cold active enzyme. 

The other -galactosidases of Athrobacter sp. B7 

(Trimbur et al., 1994) and Arthrobacter sp. C2-2 

(Karasova-Lipovova et al., 2003) were characterized also 

by high optimum temperature of 40°C. The lactose 

hydrolyzing enzyme in cell free extracts of strain 

KNOUC403 shows high activity at 4°C and in wide range 

of pH 4.3 to 7.8, is stable at 4°C, and has a moderate but 

valuable stability at 37°C, meaning that this strain has 

high possibility of producing cold active lactose 

hydrolyzing enzyme that is useful for hydrolysis of lactose 

in foods of wide range of pH at refrigerating temperature. 

Judging from these findings of physiological and 

enzymatic properties, A. psychrolactophilus KNOUC403 

is a psychrophile of useful source, for a cold active 

lactose hydrolyzing enzyme that would have advantages 

for application in the food industry. 

ACKNOWLEDGEMENTS 

This research was performed by the support of the Korea 

Institute of Planning and Evaluation for Technology of 

Food, Agriculture and Fishery. We thank the College of 

Agriculture and Life Science Alpine Association, Seoul 

National University for sampling soils at the area of Island 

Peak in Himalaya. 

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DOI: 10.5897/AJMR10.647 



Nigerian phages: The first bacteriophages from 

Tropical Africa 

Adeniran S. Koko 1,3 , Hans W. Ackermann 2 *, Monisola A. Taiwo 1 and Sunday A. Omilabu 1 

1 Department of Botany and Microbiology, University of Lagos, Lagos, Nigeria. 

2 Félix d'Hérelle Reference Center for Bacterial Viruses, Department of Microbiology, Faculty of Medicine, 

Laval University, Québec, QC, G1K 7P4, Canada. 

3 Quest Diagnostics West Hills, CA 91304, CA, USA. 

Accepted 28 March, 2011 

Fifteen morphological bacteriophage types were observed in enrichment cultures of sewage, surface 

water and human feces collected in Lagos, Nigeria. All phages were tailed and belonged to the 

Myoviridae, Siphoviridae or Podoviridae families. Most phages could be assigned to well-known 

morphotypes. Ten of them corresponded to phages of enterobacteria or pseudomonads. Four 

morphotypes (φKZ, T4 and χ, Jersey) prevailed in most samples. Most others occurred in traces only. 

Key words: Bacteriophages, ecology, identification, morphotypes. 

INTRODUCTION 

Phage ecology has essentially been investigated in North 

America, Europe and the oceans. Research into phage 

ecology in other parts of the world is almost nonexistent. 

As a result the phage flora of vast geographic areas, 

namely most of South America, Africa, Australia, China, 

India, all of Middle America, the Middle East and Siberia 

remains essentially unknown. The only reports on phages 

from African environments are limited to tailed phages in 

the subsoil of the Moroccan and Tunisian Sahara (Prigent 

et al., 2005) and the Namibian desert (Prestel et al., 

2008), RNA phages in sewage in South Africa (Grabow 

et al., 1993; Schaper et al., 2002), and unknown phages 

of nonidentified bacteria in Senegal (Bettarel et al., 2006). 

The Saharan phages were observed by electron 

microscopy, but not propagated or identified. The 

Namibian phages were observed by electron microscopy 

in bulk cultures, but they were not isolated and only one 

of the morphotypes present was identified. In addition, 

there are isolated reports of specific phages in various 

parts of the world, for example Vibrio phages from shrimp 

fisheries in Thailand (Pasharawipas et al., 2005), Bacillus 

*Corresponding author. E-mail: ackermann@mcb.ulaval.ca. Tel: 

+418 656-2131; 2558. Fax +418 656-7555. 

phages from deep sea wells in China (Liu et al., 2006), 

phages of particular actinomycetes in Australia (Kurtböke 

et al., 1993a, 1993b), and Thermus phages in Kamchatka 

hot springs (Yu et al., 2006). 

This situation is deeply insatisfactory from an ecological 

viewpoint. We intended to identify and diagnose our 

phages as precisely as possible. This is the first 

investigation of the bacteriophage flora in tropical Africa. 


We collected samples of sewage and surface water at the 

University of Lagos and in the Iddo area of the Lagos Lagoon, a 

natural lagoon of brackish water connected to the Atlantic Ocean 

through creeks. Nineteen water samples and one of human feces 

were enriched in Lagos, often three to four times in parallel, with 

common bacteria isolated from human wound and skin infections in 

Lagos hospitals (Bacillus spp., Escherichia coli, Klebsiella 

pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus 

and Xanthomonas sp.). Enrichment cultures were filtered, but single 

plaque purification was not attempted. Samples were examined in 

Canada by one of us (H.-W.A.). Approximately 75 enrichment 

cultures of 1 ml each were received. Although all vials were intact 

and none had leaked during transportation, all were heavily 

contaminated by viable enterobacteria and pseudomonads. This 

contamination had clearly occurred in Nigeria before shipment. 

Samples were too small for filtration and were clarified by simple 

decantation during storage for 1 month. Supernatants were 

centrifuged at 25,000 g using a Beckman J2-21 centrifuge and a


Table 1. Bacteriophage observations by sample. 

Sample Bacteriophage morphotype 

A41 φKZ Lu11 T4 χ χ (chi) Jersey N4 T7 Others 

1 * + + + + φ29, 44AJHD 

2 ++ + (+) * + 

3 +++ (+) * 

4 ++ * * * 

5 * ++ * * * 

6 + ++ * 

7 * ** * M1, S1 

8 ++ * ** * + 

9 * ++ + + ++ * 

10 ** + + ++ 

11 * + * ++ 

12 ** * ++ * * M1, S1 

13 * + * * 

14 + + * * ++ 121 

15 + * + +++ 

Iddo (+) * + * φ29 

OPP1 * * * * 

Berger (+) + 

Unilag (+) * (+) S2 

Feces * + * * (+) * T5, φ29 

+++, ++, +, (+), *: Variable quantities of phages; +++, many; *, traces. M1, S1, S2: Novel morphotypes of Myoviridae (M) and Siphoviridae (S) families. 

Table 2. Main dimensions of bacteriophage phage morphtypes. 

Family Type Head, nm Tail, nm 

YerA41 130 235 x 20 

φKZ 121 190 x 21 

Myoviridae 

Lu11 

121 

121 

116 

190 x 21 

115 x 17 

M1 133 156 x 17 

T4 111 x 78 113 x 16 

Siphoviridae 

Podoviridae 

χ (chi) 63 227 x 10 

Jersey 68 160 x 8 

T5 78 183 x 8 

S1 99 664 x 8 

S2 90 237 x 8 

N4 70 10 

T7 60 17 x 8 

φ29 54 x 42 36 x 12 

44JHD 45 35 x 12 

JA-18.1 fixed-angle rotor and were washed twice with 0.1 M 

ammonium acetate (pH 7.0). Sediments were deposited on copper 

grids with carbon-coated Formvar films, stained with 2% potassium 

phosphotungstate (pH 7.0) and examined in a Philips EM 300 

electron microscope. Magnification was calibrated by means of T4 

phage tails (length 114 nm). 

RESULTS AND DISCUSSION 

Fifteen morphological bacteriophage types, listed roughly 

by family and size in Tables 1 and 2, were observed. All 

were tailed and had contractile, long and noncontractile 

or short tails. They were thus identified as members of 

the Myoviridae, Siphoviridae or Podoviridae families, 

respectively. Twelve morphotypes (shortly "types") 

corresponded to well-known phages described in earlier 

reviews or books to which the reader is referred 

(Ackerrmann and DuBow, 1987; Ackermann, 1987). Part 

of the corresponding phage groups (φKZ, T4, T7, N4, φ29 

and AJHD) relate to phage genera or subfamilies defined 

by the International Committee on Taxonomy of Viruses 

or ICTV (Hendrix and Casjens, 2005; Lavigne et al., 

2008; 2009). Ten types corresponded to known 

enterobacterial and Pseudomonas bacteriophage species 

and only two corresponded to species of Bacillus or 

Staphylococcus phages (podovirus types φ29 and 

44AHJD, respectively). Most preparations were mixtures 

of 4 to 5, even 7 phage morphotypes (Table 1), 

suggesting that none of the bacteria used for enrichment 

had been pure. Most phage morphotypes could be 

related to well-known viruses with specific hosts. Four 

morphotypes were almost ubiquitous, namely : φKZ, T4, 

Jersey and χ (chi). Others occurred in traces only and 

were detected by intense scrutinizing only. This is not an

indication of their frequency or rarity in nature. No exact 

quantitation is possible here because the apparent 

frequency of phages in enrichment cultures depends on 

many variables (phage inoculum, host sensitivity, vitality 

of phages and nutrients), all of which may lead to 

overcrowding of some phages by others. 

Part of this may have occurred during transportation 

since, as noted earlier, no sample was sterile on arrival in 

Canada. Finally, the very procedure of negative staining 

often leads to uneven distribution of particles and 

gathering of different particles in different places of 

electron microscopical grids. Three particle types (M1, 

S1, S2; myovirus 1, siphovirus 1 and 2) could not be 

attributed to known bacteriophages and may represent 

novel species. Type S1 is particularly interesting. Types 

M1 and S2 are morphologically unremarkable and not 

illustrated for lack of space. 

Myoviridae 

Morphotype A41 corresponds to a giant phage of this 

name, observed in Yersinia enterocolitica in Canada 

(Stevenson and Airdrie, 1984). It has so far not been 

found elsewhere. Types φKZ and Lu11 correspond to 

giant Pseudomonas phages distinguished by the 

presence of long, curly tail fibers in the latter (Figure 2). 

Phages of type φKZ were previously isolated in Denmark, 

Germany, Georgia, Russia and the U.S.A. Type Lu11 is 

known from the Philippines only (Krylov et al., 2007). The 

relative frequency of the φKZ and Lu11 types is not 

surprising as representatives of these types multiply 

extremely well and may conceivably overcrowd other 

phages in mixed cultures. Enterobacterial phages of type 

121 were first observed in Romania (Nacesco et al., 

1969) and later in Canada by one of us (Ackermann and 

Nguyen, 1983). Type 121 is characterized by a large 

head and a relatively short tail. Type T4, easily 

recognized by its elongated head and a tail with six long 

tail fibers, seems to be truly ubiquitous and has been 

reported in many countries (Ackermann and Krisch, 

1997) (Figures 1 to 4). 

Type M1 does not, to our knowledge, correspond to 

any myovirus known. As types A41, φKZ, Lu11 and 121, 

It has an unusually large head. 

Siphoviridae 

Phages of the χ (chi) type are characterized by a 

relatively small head and a thick tail equipped with a 

single long, wavy tail fiber. The original phage χ is 

specific to mobile bacteria of E. coli, Salmonella and 

Serratia (Meynell, 1961). The tail fiber is difficult to see 

and apparently easily removed by centrifugation. Despite 

their relatively thick tails, χ and its relatives are 

Koko et al. 2209 

Figure 1-4. Myoviridae, morphotypes A41, 

T4, φKZ, and LU11, respectively. 

Phosphotungstate, final magnification x 

297,000; bars represent 100 nm. 

siphoviruses. Type Jersey has a conspicuous base plate 

with six spikes. It was first found in Salmonella paratyphi 

B and seems to be restricted to Salmonella bacteria. 

However, the orginal phage Jersey has a tail of 116 nm in 

length only (Ackermann and Berthiaume, 1969) while the 

Jersey-like particles here have tails of much longer length 

(Figures 5 to 6). It may be speculated that they are tail 

length variants of the normal phage Jersey, phage Jersey 

in nature. Type T5 is indistinguishable from coliphage T5. 

Type S1 is morphologically unremarkable, but apparently 

has not been encountered elsewhere and may be novel. 

Type S2 is very interesting as it displays a single, rigid, 

thick, spiral tail fiber of approximately 200 nm in length. 

The spirals have a periodicity of 47 nm. Prestel et al. 

(2008) observed similar particles in the sand of the 

Namibian desert. 

Podoviridae 

The N4 morphotype shows bundles of short tail fibers 

laying at the sides of the tail. Phages of this type seem to 

be very frequent in enterobacteria. Some of them 

arespecific to capsulated bacteria, for example, Klebsiella 

and Vi antigen-containing Salmonella bacteria. However, 

the rather uncharacteristic morphology of these phages 

suggests that "type N4" is heterogeneous and comprises 

several genomically unrelated phages. The T7 type is 

very frequent in Gram-negative bacteria, especially 

enterics, pseudomonads and vibrios. It seems to be 

ubiquitous and to have a world-wide distribution. So far,


Figures 5-10: 5-6, Siphoviridae; 7-10, Podoviridae. 5, 

morphotypes χ (chi, left) and Jersey (right). 6, type Sx with 

long spiral tail fiber and an adventitious particle of type φKZ. 

7-10, Podoviridae. Types T7, N4, 44AJHD, and φ29, 

respectively. Phosphotungstate. Final magnification x 

148,500 in Figure. 6 and x 297,000 in all others; bars 

represent 100 nm. 

all sequenced T7-like phages have shown genomic 

relationships (Lavigne et al., 2008). The types 

represented by Bacillus phage φ29 and Staphylococcus 

phage 44AJHD occur in traces only. They differ in head 

shape (prolate in φ29 and isometric in 44AJHD), but 

share genomic relationships (Lavigne et al., 2008) and a 

particular tail structure with a collar and collar 

appendages. The phages represent two different genera 

of the same phage subfamily, the Nanovirinae (Lavigne 

et al., 2008). Type φ29 is ubiquitous and occurs not only 

in bacilli, but also in Kurthia and Streptococcus bacteria. 

Phages of types A41, φKZ, Lu11, 121, T4, χ, Jersey, T7, 

φ29 and 44AJHD are known to be virulent. This indicates 

that most phages here reported derive from lytic phage 

infections in the environment. At least eight types (φKZ, 

121, T4, χ, Jersey, N4, T7 and φ29) seem to have a 

global distribution (Figures 7 to 10). This is consistent 

with metagenomic studies reporting the occurrence of T4 

and T7 genes in the Arctic Ocean, the Gulf of Mexico and 

the Sargasso Sea (Angly et al., 2006) but remains to be 

ascertained by further ecological studies, especially in 

Third-World countries. It should be interesting to study 

the geographical distribution of phages and to investigate 

whether all phages of similar habitats occur world-wide or 

are limited to certain regions. 

Electron microscopy for its rapidity and wealth of 

information it generates appears as the technique of 

choice for these investigations. We believe that we are 

just at the threshold of phage ecology and we have 

experienced that each ecological investigation of phage 

habitats in any country is likely to yield new and 

interesting phages. 

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DOI: 10.5897/AJMR10.767 



Evaluation of real-time PCR for Mycobacterium 

ulcerans in endemic region in Côte d’Ivoire 

Elise Solange Ngazoa-Kakou 1,3 *, Euloge Ekaza 1 , Nguetta Aka 2 , David Coulibaly-N’Golo 1 , 

Bakary Coulibaly 2 and Mireille Dosso 1,2 

1 Plateforme de Biologie Moléculaire, Institut Pasteur Cote d’Ivoire, BP 490 Abidjan, Cote d’Ivoire. 

2 Unité des Mycobactéries Tuberculeuses et Atypiques, Institut Pasteur Cote d’Ivoire, BP 490 Abidjan, Cote d’Ivoire. 

3 Institut des Nutraceutiques et des Aliments Fonctionnels, Université Laval, Qc, G1K 7P4, Canada. 

Accepted 19 May, 2011 

Buruli ulcer (BU) is caused by a mycobacterium called Mycobacterium ulcerans. The events of BU are 

the skin lesions. The lack of early diagnosis and treatment cause severe disability. Today the 

emergence to BU in Africa and particularly in Côte d’Ivoire needs faster diagnosis to control and to 

prevent the infection by M. ulcerans. The surveillance of BU is difficult, because the transmission of M. 

ulcerans occurs in rural regions where the transport of fresh collected sample is long, and the detection 

with culture technique needs several months. This study has allowed the application of polymerase 

chain reaction (PCR) technique in real time with two targets for molecular diagnosis of BU in Côte 

d'Ivoire. 63 samples (clinical, environmental, local strains and reference strains) were analyzed in realtime 

PCR by comparing the target of the Insertion Sequence (IS) 2404 and the sequence Ketoreductase- 

B (KR-B), located respectively on the chromosome and on the virulence plasmid. 49 samples (76%) 

were positive in real-time for both targets. The sensitivity of the PCR shows a detection limit of 0.25 

genome copy for both targets. The capacity, speed and sensitivity of real-time PCR assays improve the 

diagnosis and contribute to strengthening the eradication of BU in Côte d’Ivoire. 

Key words: Buruli ulcer, Mycobacterium ulcerans, real-time, insertion sequence, ketoreductase, Côte d’Ivoire. 

INTRODUCTION 

Mycobacterium ulcerans is the third mycobacteria 

infection after tuberculosis and Leprosy and it causes 

Buruli ulcer (BU), a severe skin disease that has 

irreversible consequences in rural populations 

(MacCallum et al., 1993). The WHO was esteemed that 

33 countries was endemic or semi-endemic for the Buruli 

ulcer (Asiedu et al., 2000; Janssens et al., 2005). The 

eradication of Buruli ulcer is difficult because, the lack of 

early diagnostic in rural endemic regions, and the 

unknown of the disease in medical national structures 

system. In the rural westlands and marshes of West 

Africa, children are most affected (Asiedu et al., 2000; 

WHO, 2008). The polymerase chain reaction (PCR) was 

become the gold method to confirm clinical and 

environmental samples (Ross et al., 1997a, b; Portaels et 

*Corresponding author. E-mail: ngazoa_solange@yahoo.fr. 

al., 2001; Marsollier et al., 2004; Portaels et al., 2008). 

The most using targets were the insertion sequence 

IS2404 or IS26406 and the Ketoreductase B (KR-B), 

these are respectively located on the chromosome and 

on the virulence plasmid pMUM001 (van Werf et al., 

2003). The cytotoxic mycolactone of M. ulcerans is produced 

from of three large multienzymes complexes called 

polypeptide synthases that are encoded by the genes 

mlsA1, mlsA2, and mlsB located on the plasmid (Stinear 

et al. 2004; 2005). The detection of M. ulcerans in clinical 

and in environmental samples was improved for the both 

most targets IS2404 and KR such aquatic insects, snails 

or in plants (Ross et al. 1994; Stinear et al. 2000; 

Eddyani et al. 2004; Philipps et al., 2005). In West Africa, 

the endemic region of BU is located in Côte d’Ivoire that 

has more 2000 confirmatory cases yearly (WHO, 2010). 

Molecular detection was the best method to confirm new 

human cases because of the low-growth of M. ulcerans in 

vitro (Portaels et al., 2001). Recently, de Vandelannoote


et al. (2010) have detected M. ulcerans using real-time 

PCR in small animals and in water sample in a BUendemic 

region. However, the sensitivity, the specificity 

and the capacity of real-time PCR are the major step 

forward to detect M. ulcerans. The application of real-time 

PCR in the Institute Pasteur contributes to increase the 

capacities of the national surveillance to eradicate the 

BU, by elucidating of the ecology and the transmission in 

the environment. The main aim of this study is to 

evaluate the performance of real time PCR as molecular 

diagnosis method, by using two targets IS2404 and KR-B 

to identify M. ulcerans in several types’ samples in Buruli 

ulcer-endemic region in Côte d’Ivoire. 


Mycobacterial strains 

Local strains were gathered from patients with Buruli ulcerative form 

in Cote d’Ivoire endemic regions. Those strains were isolated and 

maintained within Abidjan Pasteur Institute Mycobacterial 

Laboratory. Fresh swab clinical specimens were spilt for DNA 

extraction and culture technique. Environmental samples were 

collected in 2009 from different endemic regions of BU and were 

inclusive with strains isolated from water insects Reference strains 

were provided from the Institute of Tropical Medicine, Antwerp, 

Belgium (ITM) and from Centre Pasteur of Yaoundé, Cameroon 

(Table 1). 

DNA extraction 

Clinical specimen 

DNA from clinical samples was extracted using Phenol extraction 

method as previously described (Ekaza et al., 2004; Coulibaly et 

al., 2010). 

Bacterial strains 

DNA from bacterial strains was extracted by a phenol/chloroform 

method. Briefly five to ten colonies were harvested from 

Löwenstein-Jensen slants and suspended in 250 µl of lysis buffer. 

After overnight incubation at 37°C, phenol chloroform extraction 

was applied. The DNA was washed in ethanol and the pellet was 

dried and eluted in 50 µl of sterile water. 

Environmental samples 

Aquatic insects were placed in sterile box and add to liquid 

nitrogen, the matrix was crushed and 2 ml of 50 mM NaOH were 

added. 1 ml of sample was added to 150 µl of 0.1 M Tris and 

heated 20 min, 95°C. The extraction protocol of Mo Bio kit was 

applied following the instructions of the manufacturer. The pellet 

was eluted in sterile water and stored at -20°C. 

Real-time TaqMan PCR assays 

Real-time PCR was performed using PCR Reagents Kit (Promega, 

Madison, WI, USA) and the 7300 real-time PCR (Applied 

Biosystems, Forster City, USA). The reaction took place in a final 

volume of 25 µl and contained 5 µl of DNA, 0.3 µM of each primer, 

namely IS2404R and IS2404F (Rondoni et al., 2003) or KR-B-F and 

KR-B-F (Fyfe et al., 2007), and 0.25 µM of labelled IS2404 probe or 

KR-B probe, 20 µl PCR-Mix containing 25 mM MgCl2, Rox Dye 

Pure (Invitrogen, Cergy Pontoise, France), 10 mM dNTPs, 5X 

Buffer, 0.2 µl Go-Taq Flexi DNA Polymerase (Promega, Madison, 

WI, USA). 

PCR consisted of 35 cycles of melting at 95°C for 5 s, annealing 

and extension at 60°C for 1 min. A DNA segment of 58 and 65 bp 

for IS2404 and KR respectively in length was thus amplified. 

Fluorescence of FAM liberated from the probe by TaqMan was 

measured to determine the amplification threshold cycle (Ct), which 

was the first cycle at which fluorescent emission was 10-fold higher 

than the standard deviation of the mean baseline emission. 

Negative controls were performed with 5 µl of sterile water. A serial 

10-fold dilution of 2x10 7 CFU/µl extracted DNA was used to 

establish the standard curve. Each sample was duplicated and 

tested in real time assays. 

Sensitivity test of real-time PCR assays 

To determine the detection limit of both assays, serial dilutions of 

extracted DNA from 2.5 x10 4 CFU/µl from 2MU II strain 

(unpublished) and 5 µl were tested duplicate in real-time assays. 

RESULTS 

Sensitivity of the real-time PCR of M. ulcerans 

The serial dilution of DNA solution containing 2.5x10 4 

CFU/µl of M. ulcerans (2MUII) shows the detection by 

dilution (10 -6 ) with the target IS2404 by Ct 39. 27 and with 

the Ketoreductase-B by Ct 35.29 (Table 2) The detection 

limit of both assays was corresponding of 0.25 genome 

copies from M. ulcerans. The IS2404 is more sensitive 

with similar detection limit than the assay using 

Ketoreductase-B gen. 

Evaluation of real-time PCR for Insertion sequence 

IS2404 and Ketoreductase-B by different samples of 

M. ulcerans 

From 63 samples tested in real-time assays, 47 samples 

(74%) were positive by IS2404 and 40 samples (77%) by 

KR. Both assays show identical performance by the 

detection of local strains and clinical samples (Table 3). 

References strains show similar results by both real-time 

assays. No environmental sample was detected in both 

assays. 31 clinical samples (96%) were detected in both 

assays and confirm the sensitivity of real-time assays. 14 

samples (26%) were detected negative for both detection 

targets and 3 samples (5%) were negative by IS 2404 

real-time or by KR-B real-time assay. 

DISCUSSION 

Buruli ulcer was endemic in Côte d’Ivoire. The infection

Table 1. Types samples of M. ulcerans tested in this study. 

Samples Source or strain References 

ITM 9540 References strain Stieger et al., 2006 





ITM 990006 References strain Stieger et al. unpublished 

ITM 94821 References strain Stieger et al. unpublished 

ITM 5150 References strain Stieger et al. 2006 



CIV-B043 Local laboratory strain This study 

CIV-B058 Local laboratory strain This study 

CIV-01-0104 Local laboratory strain This study 






CIV-01-054a Local laboratory strain This study 

CIV-009 Local laboratory strain This study 







01-021b Local laboratory strain This study 

U064 Local laboratory strain This study 


02-43 Local laboratory strain This study 

B12 Local laboratory strain This study 




B043 Local laboratory strain This study 





08U-045 Local laboratory strain This study 



UB09-960 Clinical sample This study 









Ngazoa-Kakou et al. 2213


Table 1 Contd. 




UBE1 Environmental sample This study 

UBE2 Environmental sample This study 

UBES1 Environmental sample This study 



UBSE4 Environmental sample This study 




Table 2. Sensitivity of the both real-time TaqMan assays. 

Dilution of DNA CFU/µl IS2404 Ct KR-B Ct 

2.5.10 4 20.88 ±1.8 19.6 ±1.2 

2.5.10 3 24.85 ±1.7 22.03 ±0.7 

2.5.10 2 28.35 ±1.4 25.3 ±1.1 

2.5.10 1 31.82 ±1.3 28.46 ±1.4 

2.5.10 0 35.45 ±1.7 30.18 ±1.3 

0.25 39.27 ±1.8 35.29±1.2 

Table 3. Detection of different samples for M. ulcerans in real-time assays. 

Sample type 

IS2404 

KR-B 

Positive Negative Positive Negative 

Clinical 8 4 8 4 

Environmental 0 9 0 9 

Local strains 30 2 31 1 

References strains 9 1 10 0 

Total 47 16 49 14 

% 74 26 77 23 

can be eradicated if the national surveillance becomes 

effective in all regions. Early diagnostic is the only step to 

prevent ulceration that has irreversible consequences for 

the patients. The number of cases of M. ulcerans in Côte 

d’Ivoire continues to rise, with more 2000 new human 

cases reported in 2009 compared with 1440 cases in 

2008 and 1 case in 1978 (WHO, 2010). With increasing 

numbers of clinical specimens for diagnosis as well as 

the need to determine the mode of transmission and the 

natural reservoir of the mycobacteria, rapid, sensitive, 

and specific molecular tests are needed. Fyfe et al. 

(2007) have developed two multiplex, real-time TaqMan 

PCR assays for the detection of M. ulcerans DNA in 

clinical and environmental samples. Recently, de 

Vandelannoote et al. (2010) have detected M. ulcerans in 

small animals in Ghana. To ameliorate the capacity of 

molecular diagnostic, we apply the real-time PCR with 

two targets in monoplex reaction to detect M. ulcerans in 

clinical and environmental samples. The IS2404 is the 

most target for M. ulcerans PCR used in several labours 

(Stinear et al., 1999). The Ketoreductase B domain is 

present in 15 copies within the mycolactone synthase 

gen and the real-time assay has shown gut sensitivity in 

previous assay (Rondoni et al., 2003). Real-time PCR is 

more 10-10.000 sensitive than classic PCR, however the 

PCR is recommended by WHO as the confirmatory test 

for clinical diagnosis of M. ulcerans infection. 

The application in this study of real-time assays to

detect clinical samples of Buruli ulcer was chosen in 

rapidity and the capacity for the most endemic area of M. 

ulcerans in West Africa. Our results confirm the sensitivity 

of both assays and the detection limit was 0. 25 genome 

copies. However, 96% of clinical samples have been 

confirmed the infection of M. ulcerans in this study. This 

detection limit was consistent of the results of Rondoni et 

al. (2003) with the detection of 0.2 genome copy in realtime 

of IS2404. Fyfe et al. (2007) have demonstrated that 

IS2404 or KR real-time PCR assays are able to detect all 

geographic strains of the world including African strains. 

The combination of both assays has facilitated to detect 

simultaneous mycolactone producing strains by the target 

KR-B and non-mycolactone producing strains by the 

target IS2404. 

The application of several types of samples confirms 

the specificity of both real-time assays. Because of the 

long storage of references ITM strains DNA, several 

strains missed by real-time assays. No environmental 

sample shows the presence of DNA of M. ulcerans in 

both assays. The presence of inhibitors in environmental 

samples was investigated because of the correlation of 

the high incidence of BU in Côte d’Ivoire. Our results 

show gut performance of real-time assay for both targets 

to detect mycolactone producing strain and non 

producing strains. We will investigate in future studies to 

detect M. ulcerans in environmental samples. 

In view of the high number of the samples received in 

the case of the confirmation of the cases of Buruli ulcer in 

Côte d’Ivoire and the search for the environmental 

reservoir of M. ulcerans. This technology is going to allow 

the avoidance of not only the long manipulations, but also 

the contaminations after PCR, and also to increase the 

sensibility and the specificity of the detection. However, 

the application of real-time PCR for M. ulcerans in 

Institute Pasteur will permit us to elucidate the ecology 

and the transmission of BU in Côte d’Ivoire. 


The authors thank the Centre Pasteur of Yaoundé, 

Cameroon for the grateful gift of DNA of the strain MU2II. 

We thank the Institute for Tropical Medicine of Antwerp, 

Belgium for the references DNA using in this study. We 

thank the national surveillance program of Buruli ulcer 

and the World Health Organization. We thank Dr S. 

Bouzid for critical support of all parts in this work. This 

work was supported by a grant of Institut Pasteur de Côte 

d’Ivoire and Réseau International des Instituts Pasteur 

(RIIP). 

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24 march Geneva, Switzerland



DOI: 10.5897/AJMR10.782 



Contamination of cattle carcasses by Escherichia coli 

shiga like toxin with high antimicrobials resistence 

Everlon Cid Rigobelo 1 *, Renato Pariz Maluta 2 , Clarissa Araújo Borges 2 , 

Lívia Gerbasi Beraldo 2 , Manoel Victor Franco 3 , Lemos Sirlei Aparecida Maestá 1 and 

Fernando Antonio de Ávila 4 

1 Campus Experimental de Dracena- Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Brazil. 

2 Programa de Pós-Graduação em Microbiologia Agropecuária Universidade Estadual Paulista Júlio de Mesquita Filho 

(UNESP) Jaboticabal Brazil. 

3 Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal. Departamento de Biologia Aplicada a Agropecuária, 

Brazil. 

4 Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Patologia Veterinária, Brazil. 


During processing of cattle carcasses, contamination may occurs with the transfer of microbiota of 

animals feaces to carcasses. This contamination many times may be by Escherichia coli carriers of 

virulence factor as stx and eae genes being classified as Shiga like toxin. Shiga toxin-producing 

Escherichia coli (STEC) is recognized wordwide as human pathogen. A survey was performed to 

determine the sensibility profile to several antimicrobial drugs of STEC in carcasses obtained from an 

abattoir in Brazil between March 2008 and August at 2009. A total of 120 STEC were isolated. All 

isolates were confirmed as being E. coli by their biochemical analysis and submitted to polymerase 

chain reaction (PCR) for detection of stx, eae and ehly genes. No strains was isolated being carriers of 

ehly gene. The number of isolates carriers of eae gene were 48/120. The most frequent resistance was 

seen against cephalothin (84.0%), streptomycin (45.0%), nalidixic acid (42.0%) and tetracycline (20.0%). 

Multidrug resistance (MDR) to three or more antimicrobial agents was observed in 46 (38.3%) E. coli 

isolates. The findings of STEC and MRD show that cattle carcasses may be a reservoir of pathogenic 

bacterial for the consumer public. 

Key words: Multi-drug resistance, Escherichia coli, shiga toxin-producing Escherichia coli (STEC). 

INTRODUCTION 

Pathogenic Escherichia coli are classified at different 

groups of strains that cause a common disease using 

common and remarkable assortments of virulence factors 

(Kaper et al., 2004). One such pathotype, the STEC is 

the causative agent of severe clinical syndromes in 

humans such as haemolytic uremic syndrome (HUS) and 

haemorrhagic colitis. However the transmission of STEC 

*Corresponding author. E-mail: everlon@dracena.unesp.br. Tel: 

+ 55 (18) 3821- 8200. 

Abbreviations: STEC, Shiga toxin-producing Escherichia coli; 

PCR, polymerase chain reaction; MDR, multidrug resistance; 

HUS, haemolytic uremic syndrome; PR, Paraná. 

occurs by waterborne, from person to person and also 

may be transmitted by food borne (Nataro and Kaper, 

1998). E. coli is regarded as an indicator of fecal 

contamination when isolated from carcass processing. 

Levels of E. coli associated with cattle carcasses can 

increase or decrease during processing according to 

factors such as the levels of fecal contamination of live 

cattle, efficiency of evisceration and hygienic practices in 

the abattoir (Bell, 1997). 

Cattle, considered primary reservoirs of both O157 and 

non-O157 STEC bacteria (Bettelheim, 2000), frequently 

carry STEC without showing any pathological symptoms 

(Blanco et al., 1997). The full list of bacterial virulence 

determinants necessary for STEC's pathological effects is 

not known. Two types of Shiga like toxin, stx1 and stx2 

(encoded by stx1 and stx2 genes), are associated with


human disease. These toxins vary in their amino-acid 

sequence (Kaper et al., 1998) antigenicity, and in their 

activation and receptor specificity (Schmitt et al., 1999). 

E. coli acquire stx genes, and the subsequent ability to 

produce toxins, following infection with temperate 

bacteriophages (James et al., 2001). The ability of E. coli 

to adhere to intestinal epithe-lium is crucial in the 

colonization of the intestine, and therefore the 

progression of disease in humans. 

The protein intimin, encoded by the eae gene, enables 

intimate attachment of E. coli to intestinal cells 

(Donnenberg et al., 1992), causing characteristic 

attaching/effacing lesions (Paton et al., 1998). This 

attachment also enables Shiga toxins to be injected into 

the epithelial cytoplasm through a type III secretion 

system (Kaper, 2004). Other virulence factors such as 

intimin (eae) and hemolysin (hly A) are thought to 

enhance pathogenicity, but are not required for strains to 

produce severe disease, including HUS (Bonnet et al., 

1998; Acheson, 2000). Antimicrobial therapy is an 

important tool for infection treatment, resistance to 

antimicrobials is a cause of great concern in veterinary 

medicine (Monro and Polk, 2000). Indeed, a close 

association between the use of antimicrobial agents for 

the treatment of infections in animals and the observed 

levels of resistance exists (Chaslus-Dancia, 2001). The 

use of antibiotics in animal agriculture has been a 

controversial issue due to the potential transfer of 

antibiotic resistance from animals to humans. This could 

have several public health implications that may cause 

treatment failure, including death and illness prolongation, 

as well as increase in the associated costs (Kelly et 

al., 2004). 

The direct impact of resistance evolved from the use of 

antimicrobials in treatment of animal infection, is not 

clear. Since the antimicrobials routinely used for the treatment 

of infections in humans are also used in animals for 

both therapy and prevention or as growth promotion 

factors, it is not easy to describe the relative contributions 

of animal derived resistant strains to human E. coli 

disease (Maynard et al., 2004). Outbreaks have been 

associated with consumption of STEC contaminated and 

undercooked hamburgers, subsequent to both animal 

and foods (Erickson and Doyle, 2007). This probably 

occurs because during the processing of the carcass, 

fecal contamination or transfer of bacteria from the 

animal's hide to the carcass can facilitate transmission of 

pathogenic E. coli to food supplies (Bell, 1997; Barkocy- 

Gallagher et al., 2001). Some studies found a high 

prevalence of STEC in feces of healthy cattle, in Brazil, 

(Irino et al., 2005), Rio de Janeiro (Cerqueira et al., 

1999), Rio Grande do Sul (Moreira et al., 2003; Timm et 

al., 2007) and also in Paraná (PR), (Farah et al., 2007; 

Pigatto et al., 2008) and a prevalence of 1 to 2% of STEC 

in cases of diarrhea in humans was reported by Vaz et 

al. (2004), De Toni et al. (2009). The objective of this 

study was to determine the virulence profiles and the 

antimicrobial drug resistance of E. coli isolates from beef 

carcasses at an abattoir in Brazil. 


Carcass samples 

Six hundred carcass samples were collected an abattoir in São 

Paulo State, in southwestern Brazil, between March 2008 and 

August 2009. Samples studied were from carcasses cattle raised at 

pastures. Sampling of 150 feedlot cattle was done on four different 

occasions, two in the rain season and two in the dry season. Each 

sample was obtained using a Specie- Sponge (3M- Brazil) 

moistened with 25 ml of Brilliant Green (BBL/Becton Dickins on) in 

a stomacher bag. Sponges were wrung out as much as possible 

within the bag and used to swab each area. Each carcass was 

followed along the processing and sampled at three different stages 

always at the same site of the rump, near the anus over an area of 

10 × 30 cm, delineated by a sterile metal template, from the same 

half of each carcass. All samples were taken to the laboratory in an 

ice-cooled bag and kept for 12 h at room temperature. 

Bacterial isolates 

One hundred microliters of each sample was streaked on 

MacConkey agar plates (Oxoid Limited) and incubated at 37°C for 

24 h. Colonies showing E. coli characteristics were submitted to 

Gram staining and identified by standard biochemical tests; oxidase 

negative, indole positive, Simon's citrate negative, urease negative 

and hydrogen sulfide negative (Koneman et al., 1997). The isolates 

were serotyped for O157 using Latex Agglutination test kit (Oxoid, 

Basingstoke, UK). Negative strains were considered non-O157 

strains. 

PCR screening of samples 

Bacterial strains, grown overnight in nutrient broth (Sigma Chemical 

Company) at 37°C, were pelleted by centrifugation at 12, 000 g for 

1 min , resuspended in 200 µl of sterile distilled water and lyzed by 

boiling for 10 min. Lysate was centrifuged as described above and 

150 µl of the supernatants were used as DNA for the PCR (Wani et 

al., 2003). A total of 120 E. coli isolates were subjected to PCR. stx 

1, stx 2 and eae genes were detected using the primers and PCR 

conditions described by China et al. (1998) . 

Expression of E-Hly 

Expression of enterohemolysin was determined based on the 

method described by Beutin et al. (1989). Plates were incubated at 

37°C for 24 h and observed for hemolysis after 3 h (for expression 

of a -hemolysin) and 24 h (for E- Hly), respectively. The reference 

strains used in this assay were E. coli U4- 41 (positive control for a - 

hemolysin), E. coli 32511 (STEC O157: H7) (positive control for E- 

Hly), and E. coli K12 (negative control). 

Susceptibility testing 

In vitro susceptibility testing was performed by a standardized disk 

diffusion method (CLSI 2008). Staphylococcus aureus ATCC 29213 

and E. coli ATCC 25922 served as quality control strains. Four 

antimicrobial agents were selected for the tests: cephalothin, 

streptomycin, nalidixic acid and tetracycline. The antimicrobials 

used in this study were the same used by farmers in animal 

produce.

Table 1. Distribution of the Escherichia coli isolates at two different 

seasons collected between March 2008 and August 2009. 

Carcass 

Collection Season Stx genes eae 

1º Rainy 35/150 23/150 

2º Rainy 47/150 12/150 

3º Dry 17/150 5/150 

4º 

Total 

Dry 21/150 

120 Stx+ 

8/150 

48 eae+ 

Figure 1. Photograph of a 1.5% agarose gel stained with ethidium 

bromide. Columns: 1 = 100 bp DNA ladder; 2 = positive control; 3 = 

negative control; 4 = strain positive for eae; 4, 6 and 8 = strain 

negative for all studied genes; 7 = strain positive for stx1 and stx2. 

RESULTS 

All isolates, confirmed as being E. coli by their 

biochemical analysis, were submitted to PCR for the 

detection of sequences of virulence genes. From each 

MacConkey agar plate a loopful from a confluent 

bacterial growth was collected and analyzed. A total six 

hundred E. coli strains isolates the cattle carcasses were 

separate 120 isolates that carrying stx1, stx2 and eae 

genes. These isolates just 45 were carriers of eae gene 

(Table1 and Figure 1). All isolates were collected of 

preevisceration stage. There were not isolating of strains 

of pre-evisceration stages and neither of post-processing 

stage (data not show). The isolates number containing 

both stx and eae gene during rainy season were high 

than dry season (Table1) and also the isolates number 

that carried the eae genes were high than the number of 

isolates that carried eae genes. In no isolates was 

verified the expression of enterohemolisyn. All isolates 

Rigobelo et al. 2219 

were tested for this hemolytic toxin and also no isolated 

were isolates was serotyped as O157. E. coli strains 

were tested against ten antimicrobial agents. The 

resistance pattern observed was: cephalothin (84.0%), 

streptomycin (45.0%) and nalidixic acid (42.0%) and 

tetracycline (20.0%) (Figure 1), 24% of the isolates were 

resistant to all the antibiotics tested. Multidrug resistance 

was seen in 38.4% of the isolates and resistance to 2 or 

3 antibiotics was common (Figure 2 and 3). 

DISCUSSION 

Among 600 strains analyzed only three were 

enterohemolysin positive. These results were similar to 

Rigobelo et al. (2008) that analyzed 216 samples from 

bovine carcasses and all of the isolates were negative for 

ehly gene. During raining season were found a high 

prevalence of STEC than dry season; probably the 

presence of water increased the spread of bacteria 

STEC. Some authors as Rogerie et al. (2001) reported 

lower post processing of nonO157 STEC prevalence 

(1.9%) on carcasses sampled during the summer in 

plants in France. Similarly, the non-O157 STEC 

prevalence on carcasses processed in Hong Kong was 

reported to be 1.7% (Leung et al., 2001), however, Arthur 

et al. (2002) reported higher level (54.0%) of contamination 

with nonO157 STEC in carcasses processed in 

the United States. Major sources of pathogens in 

processing of carcasses have been the hide and hair 

(Barkocy-Gallagher et al., 2001). It is not clear what 

proportion of non-O157 STEC bacteria detected in cattle 

feces or on beef carcasses is able to cause disease in 

humans. Gyles et al. (1998) defend the idea that all 

STEC bacteria could be pathogenic under adequate 

circumstances. In the present study, the detected level of 

STEC strains (20%) did not match those reported by 

others (Rogerie et al., 2001; Leung et al., 2001; Mora et 

al., 2005). To the best of our knowledge, we could not 

find data from Brazil for comparison. Only Rigobelo et al. 

(2006) report of STEC (1.25%) and Rigobelo et al. (2008) 

report (1, 4%) of STEC. These differences were probably 

because of low hygienic conditions of abattoir where we 

collected the samples. 

Some authors have reported the detection of STEC 

strains in fecal samples of dairy cattle (Irino et al., 2005), 

from diarrheic (Leomil et al., 2003) and from mastitic 

cattle (Lira et al., 2004) but none from abattoir samples. 

In all of them, the stx 2 gene has been predominantly 

found, and the non-O 157 STEC strains detected. Only a 

small number of O157 strains have been detected among 

bovine fecal samples 0.6% as reported by Irino et al. 

(2005), they did not express the stx gene. Interestingly, 

the O157: H 7 strains isolated in São Paulo State from 

human infections, were all stx -producers (Vaz et al., 

2004 ), predominantly presenting the stx 1 gene. For 

more than four decades it has been a common practice 

on farms to use antimicrobial agents for disease


Figure 2. Antimicrobial resistance pattern of Escherichia coli isolate. CFL-cephalothin; 

STR - streptomycin – NAL-nalidixic acid; TET-tetracycline. 

Figure 3. Distribution of multidrug resistance to four antimicrobial drugs among 

Escherichia coli strains (n=120). 

prevention and growth promotion of animals. Widespread 

use of antimicrobial agents, select for resistance 

enhancement and may have promoted the increasing 

frequency of STEC strain's multidrug resistance in 

bovines. This could result in STEC population increases 

and perhaps greater shedding which could lead to higher 

contamination of animal food products with STEC (Zhao 

et al., 2001). 

Khan et al. (2002) reported resistance to one or more 

antibiotics in 49.2% of STEC strains in India, with some 

strains exhibiting multidrug resistance. Antimicrobial 

resistant bacteria from animals may colonize human 

population via the food chain; it is possible that resistant 

bacteria may be readily transferred to humans from 

animals used as food sources (Van den Bogaard and 

Stobberingh, 2000). During processing at an abattoir in 

Brazil we report a high level (20%) of occurrence of 

STEC strains on beef carcasses and also high

antimicrobial resistance suggesting poor hygienic 

conditions of slaughter of animals. 

ACKNOWLEDGEMENT 

FAPESP Fundação de Amparo Pesquisa do Estado de 

São Paulo financial support. 

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905.



DOI: 10.5897/AJMR11.069 



Determination of insecticidal toxicity of three species of 

entomopathogenic spore-forming bacterial isolates 

against Tenebrio molitor L. (Coleoptera: Tenebrionidae) 

N. Du Rand and M. D. Laing 

Department of Plant Pathology, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, Pietermaritzburg, 

Republic of South Africa. 

Accepted 30 July, 2011 

Bioassays were conducted using larvae of mealworms, Tenebrio molitor, to determine lethal 

concentration for five entomopathogenic strains of spore-forming bacteria. Lethal concentration was 

determined by feeding T. molitor larvae cabbage discs dipped in whole cell cultures of these five strains 

of bacteria. The strains of bacteria were isolates of Bacillus cereus, Bacillus thuringiensis and 

Brevibacillus laterosporus. An isolate of B. cereus required the highest concentration of viable spores 

(8.531 x 10 7 spores ml -1 ) to achieve its LC50, whereas an isolate of B. laterosporus required the lowest 

concentration of viable spores (3.388 x 10 6 spores ml -1 ) to achieve LC50. 

Key words: Mealworms, spore-forming bacteria, bioassays. 

INTRODUCTION 

The first isolates of B. thuringiensis Berliner subspecies 

effective against coleopterans were isolated from a 

mealworm larva, Tenebrio molitor L. (Coleoptera: 

Tenebrionidae). This subspecies was subsequently 

named B. thuringiensis subsp. tenebrionis (Kriege et al., 

1983). Another coleopteran specific strain was later 

isolated, B. thuringiensis subsp. san diego (Herrnstadt et 

al., 1986). These subsp. of B. thuringiensis are effective 

against one of the USA’s most important potato pests, 

the Colorado potato beetle, Leptinotarsa decemlineata 

Say (Coleoptera: Chrysomelidae) (Nault and Kennedy, 

1999). Colorado potato beetle has since been reported to 

have developed resistance to the Cry3A toxin (Whaldon 

et al., 1993). The development of resistance to pesticides 

is not uncommon in insects repeatedly subjected to a 

single pesticide, especially when only one toxin is 

involved (Ferré and Van Rie, 1992; Tabashnik, 1994). 

Various other coleopteran specific B. thuringiensis 

strains have been isolated and are effective against 

*Corresponding author. E-mail: 962082021@ukzn.ac.za. 

scarabaeid beetles such as Anomala cuprea Hope 

(Coleoptera: Dynastidae), A. rufocuprea Motschulsky 

(Coleoptera: Dynastidae) and Popillia japonica Newman 

(Coleoptera: Scarabaeidae) (Ohba et al., 1992). 

Bioassays on coleopterans are usually conducted on 

the Colorado potato beetle because these beetles are of 

economic importance and are a major pest in Asia, 

Europe and North America (Hare, 1990). These beetles 

are also the standard beetles used in bioassays to 

determine the International Units (IU) of toxicity (Navon, 

2000). However, it is absent from South Africa, and 

therefore T. molitor was used as the test organism 

because it is readily available in South Africa and easy to 

rear (Hinze, 2000). 

The aim of this research was to determine and 

compare the level of toxicity of five endospore-forming, 

entomopathogenic bacterial isolates, namely NDR1, 

NDR2, NDR3, NDR5 and NDR11. 


Samples 

Bacterial isolates were obtained from diseased insects such as T. 

molitor, Coleoptera adults and larvae of Schizonycha spp.

collected in sugarcane areas in KwaZulu-Natal (KZN), and debris 

from insect-rich environments such as compost, grain dust from 

grain storage bins and T. molitor cultures. 

Sample collection 

Mushroom compost was sampled from a mushroom farm (Karkloof, 

KwaZulu-Natal), compost from an urban garden (Hillcrest, KwaZulu- 

Natal) and grain dust was sampled from a chicken grain storage 

facility (Pietermaritzburg, KwaZulu-Natal). Diseased T. molitor 

larvae were collected from the insect rearing facility (University of 

KwaZulu-Natal). Adult beetles were collected from light traps in 

sugarcane areas in the KwaZulu-Natal midlands by employees of 

the South African Sugar Research Institute (SASRI, Mt. Edgcombe, 

KZN). White grubs were collected from sugarcane areas by staff 

members of SASRI and were delivered in plastic containers filled 

with soil. Diseased grubs were removed from these containers and 

placed into sterile Petri dishes. All samples were stored in a 

refrigerator kept at 4°C. 

Isolation of endospore-forming bacteria 

Isolation of Bacillus spp. was conducted using a similar 

pasteurization method to that described by Ohba and Aizawa 

(1978). Suspensions were shaken vigorously for 30 s at full speed 

on a vortex shaker. Mixed suspensions were then left to stand for 

1 h at room temperature. Suspensions were then vortexed a 

second time at full speed for 30 s and then subjected to a 

pasteurization process in a preheated water bath for 10 min at 

80°C. After pasteurization 0.1 ml aliquots of the suspensions were 

plated out onto nutrient agar (Biolab, Biolab Diagnostics, 259 

Davidson Road, Wadeville, Johannesburg) (Thiery and Frachon, 

1997). 

Plates were incubated for 24 h at 30°C in an incubator and 

examined for colonies with typical B. cereus morphology (fast 

growing, rhizoid, irregular, raised, smooth, and opaque white) or 

‘colonies with an ‘ice crystal’ appearance wider than 2 mm 

(Damgaard et al., 1997; Prescott et al., 1999; Selvakumar et al., 

2007). Various other white colored bacterial colonies larger than 

2 mm in diameter were selected for microscopic evaluation. The 

number of colonies isolated from each isolate was estimated and 

counted using a colony counter. Selected colonies were then 

purified by sub-culturing onto nutrient agar plates and incubated for 

3 days at 30°C in an incubator. Colonies were stored on nutrient 

agar slants. Not all colonies from each sample that fitted the 

description above were selected due to the large number of 

colonies formed. 

Selected isolates were subjected to Gram staining as well as 

Coomassie Blue staining [Coomassie Blue stain 0.133% (w/v) and 

methanol 50% (v/v)] for 1 min. Slides were then rinsed gently for 

30 s with distilled water and blotted dry with tissue paper (Ammons 

et al., 2002). Isolates were screened for the presence of crystal 

proteins using light microscopy with a 100 x oil immersion objective 

lens (Bernhard et al., 1997; Young et al., 1998). Crystal proteins 

stained as dark blue structures (Ammons et al., 2002). Isolates 

were screened for size of cells, spore morphology and the presence 

of crystal proteins. Isolates without crystal proteins, but with cells 

broader than 0.9 µm, were also selected. This was done in order to 

include B. cereus, which does not produce crystal proteins. Rodshaped 

bacterial cells containing oval spores were selected 

because they fit the description of B. cereus, B. thuringiensis and B. 

laterosporus cells. Selected cultures were purified by sub-culturing 

and were assigned numbers NDR1-NDR14. 

Multiple dose bioassay 

A pure culture of T. molitor was obtained from a pet shop in 

Rand and Laing 2223 

Pietermaritzburg, KZN, SA. The culture was reared on a diet of 

commercial chicken meal (Meadow Feeds, P.O. Box 426, 

Pietermaritzburg, SA) (Hinze, 2000). Bran was not used because 

this contains phytic acid that affects the absorption of calcium which 

is and essential mineral for a healthy T. molitor culture (Hinze, 

2000). T. molitor larvae were reared in 350 mm x 250 mm square 

plastic containers containing chicken meal to a depth of 70 mm. For 

moisture, potatoes skins, cabbage leaves and carrot peels were 

added once a week (Hinze, 2000). Carrots are an essential additive 

because most of the micronutrients required by T. molitor larvae are 

present in carrots (Hinze, 2000). Adults and pupae were removed 

carefully with forceps from the cultures on a regular basis and 

placed into in 350 mm x 250 mm square plastic containers 

containing chicken meal to a depth of 70 mm. After 14 mo of 

rearing, a population of T. molitor larvae was available that was 

large enough for the planned bioassays. It was not possible to 

determine the instars’ stages because meal worms have between 

10 and 14 instars, and several instars occur with larvae in the same 

size range (25 mm in length) (Anonymous, 2008a). Therefore, 

larvae sized between 18-20 mm were used in the bioassays. 

Test organism preparation 

T. molitor larvae were carefully removed from the media by gently 

sifting the chicken meal through a sieve. This procedure separated 

the T. molitor larvae and the chicken meal effectively. The T. molitor 

larvae, sized between 18-20 mm, were placed in a division of a 

plastic ice tray, for counting purposes, prior to being inoculated with 

the bacterial isolates (NDR1, NDR2, NDR3, NDR5 and NDR11). 

Isolates NDR1, NDR3 and NDR11 were isolates of B. thuringiensis. 

NDR2 was an isolate of B. laterosporus, formerly Bacillus 

laterosporus (De Oliviera et al., 2004). NDR5 was an isolate of 

B. cereus. 

Inoculum preparation 

Bacterial isolates NDR1, NDR2, NDR3, NDR5 and NDR11 were 

used to inoculate 150 ml of sterile tryptone soy broth (TSB) (Biolab, 

Biolab Diagnostics, 259 Davidson Road, Wadeville, Johannesburg) 

and incubated in a shaker water bath at 250 rpm for 5 d at 30°C 

(Meadows et al., 1992). A viable spore count was conducted using 

a standard viable spore technique (Wheelis and Segel, 1979). The 

concentration of the viable spores was measured in colony forming 

units (CFUs). The final whole culture (FWC), which consisted of 

spores and crystal proteins, was used for inoculation. For NDR5 

only spores are present because this isolate does not produce 

crystal proteins. 

Inoculation 

Discs with an 18 mm diameter were cut out of the inner leaves of 

freshly purchased cabbage using a pre-sterilized test tube cap. The 

FWC of NDR1, NDR2, NDR3, NDR5 and NDR11 were diluted with 

sterile distilled water to make up concentrations ranges of 20, 40, 

60, 80 and 100%. Cabbage discs were dipped into the whole cell 

bacterial suspensions and fed to the T. molitor larvae in Petri 

dishes. The trials were conducted in sterile Petri dishes. Control 

discs were dipped into sterile distilled water. Twenty larvae per 

dose in replicates of five were used for each bioassay for NDR1, 

NDR2, NDR3 and NDR11. A total of 28 larvae per dose in 

replicates of five were used for NDR5. Dead larvae turned black 

and liquefied internally. Koch’s postulate was used to determine if 

the isolates were responsible for the insect deaths (Prescott et al., 

1999). The amount of cabbage disc consumption (mm) was a 

general observation and was not recorded because larval mortality


Table 1. (LSTATS) PROBAN analysis of three replicate bioassays of five spore-forming bacteria tested against Tenebrio 

molitor, with regression parameters displaying the efficacy of these assays. 

Isolate No of larvae affected LC50 a 95% Fl b Slope X 2 (df-13) 

NDR1 n 

Assay 1 68 7.495 7.414 - 7.548 5.45 1.42 

Assay 2 55 7.391 7.256 - 7.497 2.54 3.47 

Assay 3 61 7.332 7.176 - 7.436 2.59 3.13 

Mean 61 7.406 7.282 - 7.494 3.53 - 

NDR2 n 

Assay 1 63 6.572 6.488 - 6.630 4.65 2.47 

Assay 2 68 6.507 6.358 - 6.603 3.02 0.36 

Assay 3 65 6.512 6.388 - 6.600 3.26 2.36 

Mean 65 6.530 6.411 - 6.611 3.64 - 

NDR3 n 

Assay 1 49 7.219 7.092 - 7.344 2.35 2.65 

Assay 2 46 7.281 7.170 - 7.403 2.56 1.32 

Assay 3 53 7.207 7.084 - 7.312 2.63 5.27 

Mean 49 7.236 7.115 - 7.353 2.51 - 

NDR5 n 

Assay 1 51 7.904 7.786 - 8.070 1.95 4.56 

Assay 2 49 7.927 7.809 - 8.101 1.97 2.04 

Assay 3 56 7.963 7.832 - 8.142 1.71 3.66 

Mean 52 7.931 7.809 - 8.104 1.88 - 

NDR11 n 

Assay 1 57 7.095 6.953 - 7.199 2.57 2.62 

Assay 2 48 7.178 7.087 - 7.266 3.35 2.48 

Assay 3 50 7.146 7.037 - 7.248 2.79 0.63 

Mean 52 7.140 7.026 - 7.237 2.90 - 

a Lethal concentration (LC50); b 95% fiducial limits were log transformed concentrations of bacterial spores per ml; n 20 larvae per 

dose in replicates of five (total of 100 larvae per bioassay). 

was the objective of this study. Results were recorded after 5 days. 

Statistical analyses 

The statistical computer programs (LSTATS) P/PROBAN Version 

2.1 (1992), as programmed by Van Ark (1983), and SPSS Version 

11.5, were used to calculate the regression parameters that 

included the determination of lethal concentration (LC50) and their 

fiducial limits. Each sample reflected three estimates of LC50 and 

fiducial limits. These were compared using a standard one-way 

ANOVA. A similar format to that of Hatting (2002) was used to 

depict the results obtained (Table 1). The LC50 values were 

converted back to concentrations of viable spores to determine the 

50% mortality dose. 

RESULTS 

Mortality of T. molitor larvae in the control Petri dishes 

was zero, and the cabbage discs were completely 

consumed after 5 days. The complete consumption of the 

cabbage discs indicates that no toxic effects were 

present in the control. Cabbage discs that were 

inoculated with different doses of the various bacterial 

isolates were consumed according to dose. An overall 

trend observed was that the more concentrated the 

inocula of spore-forming bacteria applied to the cabbage 

disc, the less the cabbage disc was consumed. Low 

bacterial dilutions resulted in levels of cabbage disc 

consumption similar to that of the controls but with a low 

level of T. molitor mortality. No further larval deaths were 

found to occur after 5 days. 

The minimum LC50 was 6.530 for NDR2 at a 

concentration equal to 3.388 x 10 6 spores ml -1 . The 

maximum LC50 was 7.931 (NDR5) at a concentration of 

8.531 x 10 7 spores ml -1 (Table 1). NDR5 could thus be 

considered the most effective isolate. A comparative 

bioassay with B. thuringiensis subsp. tenebrionis was not 

conducted because this strain is not a registered biocontrol


Table 2. (LSTATS) PROBAN hypothesis test used to determine the homogeneity between the five independent bioassays 

using NDR1, NDR2, NDR3, NDR5 and NDR11. 

Hypothesis Slopes equal Slopes and intercepts equal 

Isolate X 2 Df P X 2 df P 

NDR1 0.341 2 0.842 5.404 2 0.065 

NDR2 1.445 2 0.49 2.007 2 0.368 

NDR3 0.73 2 0.699 0.121 2 0.932 

NDR5 0.256 2 0.876 0.317 2 0.851 

NDR11 0.846 2 0.661 0.793 2 0.678 

Table 3. Results of a parallelism test between the three assays of the five bacterial isolates to determine homogeneity. 

Isolate No. of larvae affected Slope ± SE LC50 a 95% Fl X 2 (df-13) G b 

NDR1 184 2.82 ± 0.37 7.386 7.314 - 7.443 15.781 0.068 

NDR2 196 3.39 ± 0.40 6.530 6.468 - 6.580 7.271 0.055 

NDR3 148 2.50 ± 0.34 7.235 7.172 - 7.297 10.132 0.071 

NDR5 156 1.85 ± 0.22 7.933 7.861 - 8.021 11.091 0.053 

NDR11 155 2.87 ± 0.35 7.140 7.082 - 7.194 8.031 0.058 

a The data of the three bioassays were pooled to obtain a more accurate estimation of the LC50 and fiducial limits. b The Fiducial limits 

calculated by Fieller’s theorem as a measurement of variation. 

agent in SA and is not readily available. B. thuringiensis 

subsp. azawai and B. thuringiensis subsp. kurstaki are 

the only two subsp. of B. thuringiensis registered in SA 

for use as a bio-pesticide (Anonymous, 2008b). 

Numbers of larvae used in the assays were either 20 

for NDR1, NDR2, NDR3, NDR5 and NDR11 (per single 

dose). These were the largest numbers possible at the 

time, in this project, due to limited insect rearing facilities, 

financial resources and time constraints. 

One–way ANOVA was used to determine whether the 

slopes and intercepts of the three bioassays of each of 

the isolates were comparable. Deviations of the slopes 

and intercepts from the Probit lines were homogenous. 

These lines were therefore comparable (Tables 2 and 3, 

Figure 1). 

DISCUSSION 

Statistical results 

The chi-squared values obtained for each of the 

bioassays suggested that the deviations of the observed 

mortalities are within the range of accepted parameters 

for the calculated Probit line (Table 1) (Van Ark, 1983). 

Therefore, it may be concluded that the calculated Probit 

line is an acceptable representation of insect response to 

B. thuringiensis isolates. Parallelism tests showed that no 

significant differences could be detected between the five 

bioassays for each of the isolates. This allowed for the 

comparison of slopes and intercepts (Table 3). The 

slopes of the independent bioassays showed no 

significant differences and were found to be homogenous 

hence the lines were comparable (Table 2) (Van Ark, 

1983). The deviations from the observed mortalities were 

within the expected limitations of deviation thus rendering 

the Probit line acceptable (Table 2 and Figure 1) (Van 

Ark, 1983). Values obtained indicated that the lines for 

each of the five bioassays for each isolate were parallel 

and hence a similar response was observed for each of 

the isolates (Table 2 and Figure 1). The LC50 spore 

concentrations for each of the isolates were: NDR1 = 

2.432 x 10 7 , NDR2 = 3.388 x 10 6 , NDR3 = 1.718 x 10 7 , 

NDR5 = 8.579 x 10 7 and NDR11 = 1.380 x 10 7 . 

G is used as a measure of variation in the calculation of 

fiducial limits and is derived from Fieller’s Theorem (Van 

Ark, 1983). According to Finney (1971), in a good 

bioassay, the value of G will lie between 0.2 and 0.05. 

Van Ark (1983) suggested using G values of 0.25 and 

0.025. G values above 0.025 indicate that the variation of 

mortality is high. Values above 0.25 indicate that the 

experimental design is not appropriate and requires 

amendment. The fiducial limits cannot be calculated for 

assays where the G value is equal to one (Van Ark, 

1983). The G values for all five isolates fell within the 

parameters set by Van Ark as well as by Finney. 

However, the experimental precision was not ideal 

because the G values were larger than 0.025, indicating 

that the variation in mortalities was large. However, the 

values were still acceptable because they did not exceed 

0.25 (Table 3). 

These results could be improved by conducting six or


Empirical probit mortality 

12 

10 

8 

6 

4 

2 

0 

Series1 

Series2 

Series3 

Series4 

Series5 

y = 2.8157x - 15.798 

y = 2.5895x - 13.764 

y = 1.8497x - 9.6744 

y = 3.3919x - 17.15 

y = 2.8998x - 15.682 

0 1 2 3 4 5 6 7 8 9 

Log dose 

Figure 1. Separate Probit lines of the pooled data for each of the five isolates. 1. Series 1 = NDR1; Series 2 = NDR2; 

Series 3 = NDR3; Series 4 = NDR5 and Series 5 = NDR11. 

more bioassays per isolate. This would result in a better it 

of line and hence a lower G value (Van Ark, 1983). The 

number of insects required in a bioassay for reliable 

results depends on the experimental procedures, as well 

as the species of insect (Van Ark, 1983). 

Toxicity comparison of isolates 

The isolate with the best LC50 was NDR2, with a log 

value of 6.530 (3.388 x 10 6 spores ml -1 ) (Table 3). This 

isolate was identified previously as an isolate of 

B. laterosporus. Assays conducted with isolates of 

B. laterosporus did not exhibit any toxicity towards 

T. molitor. However, toxicity was evident against 

mosquitoes (Culex quinquefasciatus and Aedes aegypti) 

and a species of snail Biomphalaria glabrata (Favret and 

Yousten, 1985). Assays conducted by Rivers et al. (1991) 

on T. molitor with strains of B. laterosporus showed these 

strains to have toxicity values similar to those obtained 

from strains of B. thuringiensis subsp. tenebrionis. 

The isolate with the weakest LC50 was NDR5, with a 

log value of 7.933 (8.579 x 10 7 spores ml -1 ). This isolate 

was identified previously as an isolate of B. cereus. This 

species is not often used in entomopathogenic bioassays 

because it is considered an organism associated with 

gastrointestinal diseases and is often found as a food 

contaminant. However, B. cereus strains with insecticidal 

properties against coleopteran larvae have been isolated 

from species of white grub (Selvakumar et al. 2007; 

Sushil et al., 2008). The insecticidal properties of this 

organism lie in its ability to produce vegetative 

insecticidal proteins (VIP) (Estruch et al., 1996; Moar et 

al., 1994; Yu et al., 1997) which have been found to be 

effective against Western Corn Rootworm, Diabrotica 

virgifera LeConte (Coleoptera, Chrysomelidae) (Warren, 

1997). 

All five bacterial isolates (NDR1, NDR2, NDR3, NDR5 

and NDR11) displayed toxicity against T. molitor. The 

three B. thuringiensis isolates (NDR1, NDR3 and NDR11) 

displayed high levels of toxicity to T. molitor but were less 

toxic than the B. laterosporus isolate (NDR2) (Table 3). 

The dose response of the B. cereus isolate (NDR5) was 

found to stand alone from the other two species tested 

(Figure 1). This is indicative of substantially greater 

toxicity than the other isolates. 

Standardization assays often involve the use of purified 

or extracted crystal proteins (Cry and Cyt toxins) and do 

not include any of the other toxins and synergists (e.g., 

chitinases) produced by these species of bacteria

(Thamthiankul et al., 2001; Lee et al., 2007). B. 

thuringiensis and B. cereus are able to synthesize 

chitinases (Lee et al., 2007). Other toxins may play an 

important role in preventing target insects from becoming 

resistant because they would have to evolve resistance 

against more than one compound concurrently. In the 

bioassays used in this research, the FWC were used in 

toxicity determination. Hence, other unknown toxins and 

synergistic factors were included. Whole cultures 

consisting of spore and crystal suspensions are often 

used in bioassays to determine toxicity. The use of FWCs 

is applied where whole culture products will be 

commercialized, as opposed to pure protein crystals (De 

Oliviera, 2004; Lambert et al., 1992). Whole culture 

products have to be screened for beta-exotoxins because 

current registration of B. thuringiensis products requires 

the absence of these toxins (Prieto-Samsonova et al., 

1997). Continuous sub-culturing of B. thuringiensis can 

be problematic because it may cause a decline in toxicity 

(Sachidanandham and Jayaraman, 2003). 


Authors are thankful to M. Ohba and K. Kagoshima for Hserotyping 

of some of the isolates and to the team from 

The South African Sugar Research Institute (SASRI) for 

the collection and delivery of the white grubs and adult 

beetles. 

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DOI: 10.5897/AJMR11.108 



What changed in necrotizing fasciitis in twenty-five 

years? 

Baris Yildiz 1 *, Derya Karakoc 2 , Erhan Hamaloglu 2 , Arif Ozdemir 2 and Ahmet Ozenc 2 

1 Ankara Numune Teaching and Research Hospital, 6th General Surgery, Ankara, Turkey. 

2 Department of General Surgery, Faculty of Medicine, Hacettepe University, Ankara, Turkey. 

Accepted 30 June, 2011 

Necrotizing fasciitis (NF) is a deadly soft tissue infection characterized by necrosis of subcutaneous 

tissues. In this study, our aim was to identify variables affecting patient outcome and mortality in 

necrotizing fasciitis and their temporal changes. We reviewed records of 45 patients treated at our 

institution between 1979 and 2004. Data about gender, age, etiology, site of involvement, bacteriology, 

type of surgery, supportive treatment, accompanying diseases, mortality were collected. Factors 

contributing to mortality were sepsis, renal failure, liver failure, multi organ failure, disseminated 

intravascular coagulopathy and long term intubation. Mortalities accumulated in first 23 patients. There 

was not difference in microbiology, demographics, etiology, site of involvement, debridement technics 

between first patients and recent patients of the institution. Mortality in necrotizing fasciitis is mostly 

because of sepsis and associated disorders. Adequate control of the microbiological agent and 

preventing further contamination of the wound is cardinal part of treatment in NF. 

Key words: Necrotizing fasciitis, bacteriology, mortality, sepsis, Fournier’s gangrene. 

INTRODUCTION 

Necrotizing fasciitis (NF) is deeply located infection of 

subcutaneous tissue resulting in progressive destruction. 

Its histological patterns are large necrosis of the fascia, 

subcutaneous fat and skin with thrombosis of the 

microvasculature and sparing of the underlying muscles 

and the skin (Wilson, 1952; Giuliano et al., 1977; Misago 

et al., 1996). 

NF can be divided into two groups depending on the 

causative organism. In type I, at least one anaerobic 

species is isolated in combination with facultative 

anaerobic species and members of Enterobacteriaceae. 

In type II, group A streptococci or Staphylococcus aureus 

are isolated (Levine and Manders, 2005). 

There are a number of predisposing factors to NF such 

as diabetes mellitus (DM) (Salcido, 2007) immunocompromised 

state, corticosteroid use, intravenous drug 

abuse, trauma, malnutrition, burns and atherosclerosis 

(Thompson et al., 1993). Early diagnosis and prompt 

*Corresponding author. E-mail: baris104@yahoo.com. Tel: +90- 

532- 445 46 55. Fax: +90-312- 424 15 20. 

Abbreviations: NF, Necrotizing fasciitis; DM, diabetes mellitus. 

treatment consisting of surgery and antibiotics are key to 

improved patient outcome in NF. 

Depending on the depth of invasion, necrotizing soft 

tissue infections can cause extensive local tissue 

destruction, tissue necrosis, systemic toxicity and even 

death. Mortality rates for necrotizing soft tissue infections 

range from 6% to as high as 76% despite advances in 

management (Brandt et al., 2000; Singh et al., 2002; 

Bilton et al., 1998). 

In this study, our aim was to identify the temporal 

changes in incidence, etiology, bacteriology, management 

and outcome in NF at our institution to help define 

better strategies in treatment for this challenging 

condition. 


After ethics commitee approval was obtained, inpatient records of 

patients treated for necrotizing fasciitis between 1979 and 2004 in 

our university hospital general surgery department were retrospectively 

reviewed. Patients were identified from hospital archives 

and data was collected from hospital records and electronic 

database. There were 29 men and 16 women whose ages ranged 

between 16 and 88 years. We reviewed the charts of the patients


and recorded sex, age, predisposing factors, duration between the 

onset of symptoms and the first surgical debridement, bacteria 

isolated, duration of hospital stay, reconstructive proce-dures, and 

clinical outcome. Plastic and Reconstructive Surgery at our hospital 

was consulted for patients who needed reconstructive procedures 

after initial debridement was performed in General Surgery 

Department. Broad spectrum antibiotics were given initially and 

specific antibiotics were advised after confirmation of the organism 

isolated from the wound tissue. Patients with minor skin and softtissue 

loss or wound healing with secondary intention were referred 

for wound evaluation and wound care. The scrotal advancement 

flap, pudendal thigh fasciocutaneous flap, gracilis 

muscle/myocutaneous flap, or split-thickness skin graft was used as 

the reconstructive procedure to correct the defects arising from 

failure of secondary intention or primary closure. The statistical 

analysis was carried out with SPSS software (SPSS, Chicago, 

Illinois) using Chi-Square test. A P-value < 0.05 was considered 

significant. 

RESULTS 

There were totally 45 patients. The median age of the 

patients was 50 (minimum 16, maximum 88). Sixteen 

patients (35.6%) were female and 29 (64.4%) were male. 

The diagnoses of NF were established based on clinical 

symptoms and findings including combinations of fever, 

pain, swelling, erythematous change of the involved skin 

and purulent discharge. 

The most common etiology was perianal abcess (n=17, 

37.8%) followed by surgical site infection (n=9, %20) and 

others (perineal shaving, decubitus ulcer, strangulating 

inguinal hernia, vulvar trauma, intramuscular injection, 

pilonidal abcess, colocutaneous fistula, diabetic foot, gun 

shot wound, enterocutaneous fistula). It was seen that 

majority of cases were actually Fournier’s gangrene. This 

was supported by the analysis of the site of involvement 

as perianal area, scrotum/vulva with highest frequency 

(n=16), followed by combinations of localizations 

including scrotum, vulva, gluteus and inguinal region. 

Twenty-two percent of patients in our study had comorbidities 

while thirteen patients (28.9%) did not have 

any accompanying disease. The most common associated 

disease was DM (17.8%) followed by malignancies 

(8.9%), coronary artery disease (6.7%) and combinations 

of DM, hypertension, ulcerative colitis, paraplegia, 

chronic obstructive airway disease. During management 

46.7% of patients had sepsis and 11.1% had multiorgan 

failure. 

Polymicrobial bacterial strains were isolated from tissue 

and pus cultures. In 9 patients there was not any growth 

in bacterial cultures. The rest of isolated bacteria are 

listed in Table 1. 

Antimicrobial agents, surgery and nutritional support 

were the mainstay of treatment for patients with NF. 

Antibiotic therapy was started empirically and continued 

based on the microbiology of the wounds. In 3 patients 

antifungal medication was added to antibiotics. Median 

duration of antimicrobial therapy was 17 days (minimum 

3 – maximum 55). 

Surgery was performed as soon as the diagnosis of NF 

was suspected. There was delay in 18 patients between 

diagnosis and surgical intervention. Median time of delay 

was 1.5 days. Debridement under local anesthesia was 

performed only to 4.4% of the patients and 95.6% of the 

patients had debridement under general anesthesia. Of 

those who had debridement under general anesthesia, 

27 (60%) had only one debridement while 9 (20%) had 

twice and 4 (8.9%) had three times. Overall, colostomy 

was performed to 22.2% of patients and to 90% of the 

patients with Fournier’s gangrene. 

Twenty-six patients (48.8%) required reconstruction. 

Primary closure was performed in 15 (57.6%) patients 

whereas 4 (15.3%) patients had split-thickness skin 

grafting and 3 (11.3%) had flap reconstruction. 

Nutritional support was given to 17 patients. Of these, 

six patients received total parenteral nutrition whereas 8 

had enteral nutrition. 

The longest hospital stay was 92 days while the 

shortest stay was 9 days (median: 35.5). The overall 

mortality was 22.2% (n=10). Twenty-five percent of 

women and 20% of men died. Comparison of survivors 

and non-survivors are given in Table 2. Statistically 

significant factors contributing to mortality were sepsis 

(p=0.001), multiorgan failure (p

Table 1. Combinations of bacteriologic strains isolated from tissues in chronological order starting from 1979 to 2004. 

Case Year Microorrganisms 

Yildiz et al. 2231 

1 1979 Enterobacteriaceae spp. 

2 1979 Escherichia coli, Clostridium tetani 

3 1980 Coagulase positive Staphylococcus aureus (SCP) 

4 1983 No growth 

5 1983 Escherichia coli, Proteus, SCP, Diphtheroids 

6 1985 Escherichia coli, Diphtheroids 

7 1986 Escherichia coli, SCP, Pseudomonas aeruginosa 

8 1988 SCP, Acinetobacter 

9 1991 Klebsiella pneumonia, Pseudomonas aeruginosa 

10 1993 Streptococcus pneumonia, SCP 



13 1994 Pseudomonas aeruginosa 


15 1995 Enterobacteriaceae species 


17 1995 Proteus vulgaris 

18 1996 Klebsiella oxytoca, Escherichia coli 

19 1996 Escherichia coli, Streptococcus viridens 

20 1997 Escherichia coli 

21 1997 Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella Oxytoca 

22 1997 Escherichia coli, Streptococcus viridens 

23 1997 Escherichia coli, Klebsiella pneumonia, Coagulase positive Staphylococcus aureus 

24 1998 Escherichia coli, Staphylococcus aureus 

25 1998 Morganella morgagni, Pseudomonas aeruginosa 

26 1998 Enterobacteriaceae spp. 


28 1998 Streptococcus pneumonia, SCP 

29 1998 Staphylococcus aureus 

30 1998 Escherichia coli, Klebsiella pneumonia 


32 1999 Staphylococcus aureus, Serratia marcescens 

33 2000 Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus 

34 2001 Escherichia coli, Streptococcus agalactiae 

35 2001 Escherichia coli, Pseudomonas aeruginosa 


37 2002 Acinetobacter, Staphylococcus epidermidis 

38 2003 Acinetobacter, Escherichia coli 

39 2003 Staphylococcus epidermidis 

40 2003 Morganella morgagni 

41 2003 Methicillin resistant Staphylococcus aureus, Escherichia coli, Acinetobacter 


43 2003 Escherichia coli, Staphylococcus aureus 

44 2003 Streptococcus agalactiae 

45 2004 Escherichia coli, Enterococcus faecalis, Proteus mirabilis 

debrided area (Villanueva-Sáenz et al., 2002). Colostomy 

rate of survivors and non-survivors was similar in our 

study while there was an increase in tendency to perform 

colostomy in the second half of the study group. 

The defects after surgical debridement in NF can be 

managed with primary closure, grafting or flap 

reconstruction (Baharestani, 2008). Our center had a 

propensity towards primary closure whenever possible


Table 2. Comparison of survivors and non-survivors. 

Variable Non survivors Survivors 

Age (years) 59.7±16.9 45.6±12.6 

Gender 6 Males, 4 Females 23 Males, 12 Females 

Etiology Predominantly SSI with peritonitis Predominantly perianal abcess 

Duration of symptoms (days) 8±4.24 7.71±4.36 

Delay in surgery (days) 2.66±2.05 1.66±1.54 

Co-morbidity present 60% 60.7% 

Site of involvement Predominantly ABW Predominantly perianal area 

Number of debridements 1.4±0.51 1.34±0.787 

Colostomy 20% 22.9% 

Intubation 60% 8.6% 

Duration of Intubation (days) 3.66±3.2 0.087±0.288 

Red packed cell transfusion (Units) 4.5±1 1.7±2.2 

Antibiotic therapy (days) 14.8±10.56 23.18±14.1 

TPN administration 20% 20% 

Enteral nutrition 10% 20% 

Leukocyte count at admission ( /uL ) 21650±12834 16537±6943 

Neutropenia 1 patient 1 patient 

Renal failure 70% 34.3% 

Liver failure 50% 11.4% 

Sepsis 100% 31.4% 

SSI:Surgical site infection; ABW:Anterior abdominal wall; TPN: Total parenteral nutrition. 

Table 3. Characteristics of first 23 and last 22 cases. 

Variable First 23 patients (1979-1996) Last 22 patients (1997-2004) 

Age (years) 49.56±16.5 47.90±12.95 

Gender 65.2% Male 63.6% Male 

Etiology Predominantly SSI with peritonitis and perianal abcess Predominantly perianal abcess 

Site of involvement Predominantly perianal area Predominantly perianal area 

Number of debridements 1.42±0.676 1.28±0.78 

Colostomy 15% 33.3% 

Mortality 28.6% None 

SSI: Surgical site infection. 

and all reconstructions were done between 1997 and 

2004. 

Empirical antibiotic prophylaxis should be started in NF 

until the responsible pathogen is identified in cultures. In 

our study, when compared with respect to years, recent 

regimens started to contain piperacillin-tazobactam, 

teikoplanin and imipenem. Of note, 3 patients with sepsis 

in the second half of study received fluconazole along 

with antibiotics. 

Published mortality rates due to NF vary widely. Brandt 

et al. (2000) and Lille et al. (1996) from USA reported 

24% (Brandt et al., 2000) and 25% (Lille et al., 1996) 

respectively while Rietveld et al (1995) from New Zealand 

reported 43% (Rietveld et al., 1995). We found 

approximately the same result (22.2%) as those reported 

from USA. 

In our study, factors predicting mortality were mainly 

the end results of sepsis and multi-organ failure. In contrary 

to what we found, a study from Canada cited that 

the contributing factors for mortality were advanced age, 

immunocompromised state and presence of 

streptococcal toxic shock syndrome (Golger et al., 2007). 

In this study, we tried to reflect our 25 years of 

experience in NF between 1979 and 2004. Due to 

change in institutional policy our department stopped 

admitting and following NF patients after 2004. 

The first thing in analysis of cases was better mortality 

rate in more recent cases when compared to the earlier 

days of the institution. In this regard, when we look at the 

variables it seems that microbiology, demographics,

etiology, site of involvement, debridement technics are 

similar but frequency of colostomy procedure, type of 

antibiotics, utilization of antifungals seem to accumulate 

in the second chronological half of the series. The main 

outcome of our study is the fact that adequate control of 

the microbiological agent and preventing further contamination 

of the wound is the cardinal part of the treatment in 

NF and particularly in Fournier’s gangrene. 

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African Journal of Microbiology Research Vol. 5(16) pp. 2234-2240, 18 August, 2011 




Aspects of bacterial colonization in newborn babies 

Fariba Heshmati*, Seyeed Amir Yazdanparast , Seyeed Akbar Moosavi , Hussein Dargahi, 

and Farnaz Tabibzadeh 

Department of Microbiology, Tehran University of Medical Sciences (Hemmat Campus), Tehran, Iran. 


The study of colonization of microbial flora in neonate is very important and finding the causative agent 

may lead to explore these infections in neonate. In this research the colonization in neonate was 

measured at the beginning and a few hours after birth. Sampling was performed in operating room, 

rooming in and nursery section. Some of the neonates deliver by rapture of membrane in normal way 

and the other deliver by caesarean section. All the samples which are collected were from these sites: 

1) Forehead 2) Oral cavity 3) External ear 4) Auxiliary region 5) Umbilical region 6) Perennial region. 

Also we had some sampling from health care personnel who are worked in operating room or nursery 

section. At the time of sampling we used sterile gloves, mask and always sterile sets. Sampling was 

done by use of sterile swabs which were weltering by sterile phosphate buffer or sterile normal saline 

.Then this sterile swab scrubbed on the special surface of the body of neonate and directly transferred 

to BA plate, Mac agar plate, EMB agar plate and Sc agar plate and then streaked on these plates. The 

regions which were sampled ,numbered by 1 to 6 then in other hours once more the sampling were took 

place from the same site, therefore we began from number 7 to 12 and according to this method for 

example sampling from neonate A was showed by A1 to A12. The prevalent microorganisms in oral cavity 

were as follow: 63.5% GPC, 18.5% GPB and 11% GNC. Staphylococcus epidermidis was the major 

microorganism found in neonate forehead. No bacteria growth was found in 50% of samples through 

caesarean section. Staphylococcus and Diphtheroied constitute the highest colonization of neonate 

skin's microorganisms and their growth rates were low at the onset of the birth, but increased after 12 

hours. Colonization of gram-negative bacteria in infant hours after birth considered to be an important 

issue that was studied in this research. Hence hand washing of health-care personnel’s can reduce 

such bacteria in infant. There was a significant decrease in (GNB) number due to hand washing, hence 

application of disinfectant solution in hand washing play an important role in controlling 

microorganisms in care unites. 

Key words: Colonization, GPC (Gram- positive cocci), GPB (Gram-positive bacilli), GNC (Gram- negative 

cocci), GNB (Gram- negative bacilli), Microorganism. 

INTRODUCTION 

Microbial colonization of newborn infant begins 

immediately after birth. Infants’ skin is colonized by flora 

derived from the body of the mother and other human 

contacts and from various inanimate objects. Initial 

colonization is fortunately, depending on the first suitable 

organism to arrive at a particular site as well as factors 

such as the type of delivery, the amount of vernix 

*Corresponding author. E-mail: 

heshmati_99_1999@yahoo.com 

caseosa persent at birth, the type of nourishment 

received (breast milk or formula) and the degree of 

exposure in the hospital environment (Guenthner et al., 

1987). Moreover exclusively formula-fed infants were 

more often colonized with E.coli, C.difficle, Bacteroides 

and Lactobacillus, when compared with breast-fed 

infants. Other factors like hospitalization and prematurity 

were also associated with higher prevalence of C. difficil 

(Guenthner et al., 1987; Penders et al., 2006). However, 

in most cases, after a few days the representation of 

microbial species with in the neonatal flora was 

remarkably similar to the adult pattern of colonization.

Interest in the bacterial colonization of the newborn was 

developed in mid 1940s when epidemic of 

Staphylococcal pyoderma began to appear in substantial 

number in the developed countries (Falle and Schleifer, 

1981; Pezzati et al., 2002; McConnell et al., 2004). 

Staphylococcal disease continues to be a serious 

problem in some nurseries, but gram-negative bacilli are 

currently the major encountered pathogens in hospital 

acquired infection (Pass et al., 1980). Infant born through 

caesarean section had lower number of Bifidobacteria 

and Bacteroides, but more often colonized with C.difficle, 

when compared with those borne vaginally (Penders et 

al., 2006). Occasionally infections are transmitted by a 

member of the nursery staff who harbors pathogenic 

bacteria on the hands or by contamination of vaginal, 

rectal bacteria. However, the hand transmission is more 

common in infant to infant (Goldmann et al., 1978). The 

number of nosocomial gram-negative infections has 

increased dramatically in the past decade but pathogenic 

E.coli, Klebsiella, Enterobacter and Pseudomonas 

organisms have been responsible for majority of the 

gram-negative infections 8 . Gram-negative bacilli (GNB) 

cause more than 50% of hospital acquired infections. 

These organisms are not thought to be part of the 

resident flora of the skin perhaps because of factors 

such as desiccation inhibition by skin lipids or 

interference by normal cutaneous bacterial flora 

(Guenthner et al., 1987). Most studies have assumed that 

GNB are part of the transient flora only and easily 

removed by hygienic hand washing with soap (McGarrity 

and Coriell, 1973). GNB remaining after removal of 

transient hand flora with soap and water have not been 

studied extensively (McGarrity and Coriell, 1973). The 

purpose of this study was to determine the prevalence of 

GNB as a part of the non transient flora on the hands of 

hospital personnel during the period of nosocomial 

infections after hand washes and also analyzing the 

influence of working a shift in an intensive care unit on 

hand carriage of GNB by nurses, the contribution of a 

broad range of external influences and mode of delivery 

on neonate's skin micro biotic composition in early 

infancy. 


Subjects (infants) 

Twenty- two newborn infants born at Amin hospital part of Esfahan 

University were included in this study. This research was conducted 

in 2 stages. The colonization was measured in the beginning and a 

few hours after birth. In the pilot study, 12 infants born by vaginal or 

cesarean delivery were studied. In final work, 10 infants (2 infants 

were premature) at operating room, rooming in and nursery section 

were studied. 

Sampling and culture of the skin micro flora 

In pilot study and final work, 96 and 80 samples were collected 

Figure 1. Sampling on blood agar plate. 

Heshmati et al. 2235 

respectively from forehead region, oral cavity, perineum, and 

external ear, auxiliary and umbilical regions. Samples from the skin 

flora were taken 3, 4, 5, 8, 14 and 20 h after vaginal or caesarean 

delivery by using cotton-tipped swab weltering in sodium phosphate 

buffer that were scrubbed on the surface of the skin and 

immediately streaked on to the blood agar, Mac conkey, EMB, and 

Sc (sabburo cholramphenicol) plates. Samples were brought to the 

laboratory and incubated at 37°C within 24 h after collection (Figure 

1). Media were incubated in candle jar at 37°C for 48 h. The 

bacterial colonies were analyzed in terms of number and 

morphological characteristics and then preserved in BHI and 15% 

glycerol for determining their species. Upon discharge from the 

newborn nursery at the Amin Hospital for maintaining the 

microorganisms, brain heart infusion broth (BHI) and 15% glycerol 

was used. 

Identification of isolates 

Bacteria were stained by Gram stain and also 3% H2O2 solution we 

used to detect the catalase enzyme then identified by standard 

bacteriological and biochemical methods such as acid production 

from carbohydrates, using of MSA (Manitol salt agar medium), 

DNAse medium, sensivity to Furazolidon disk(100 µg), Bacitracin 

disk(0.04U)for differentiation Micrococcus from Staphylococcus, of 

medium, SIM, 10% Nacl nutrient agar, MR-VP, TSI, Simmon citrate 

agar, urea agar, Nitrate reduction test. Any bacterial colonies which 

resembled -hemolytic streptococci were tested for sensivity to 

bacitracin with disks containing 0.04U of bacitracin, if the zone of 

inhibition was greater than 15 mm, the streptococci would 

presumed to be group A and then precise serological grouping was 

carried out. For Fungi (Candida sp) we used Sc medium and germ 

tube and by using of Corn meal agar medium for production of 

clamidospore (Figure 2). 

RESULTS 

In the first phase 12 neonates were used to obtain 

bacteria flora samples at various time of day. A variety of 

microorganisms were isolated from the samples. 

However, some samples demonstrated no bacterial 

growth. We observed that neonates during the birth 

would have colonization with a single bacterial species. 

Nevertheless.after a period of time several species of 

bacteria would be intermixed together. Several gram 

negative bacilli strains isolated at various hours after birth 

were characterized (Figures 3, 4, 5, 6 and 7). 

Oral cavity was found to be sterile in most of times 

especially during birth and consequently colonization of


Figure 2. Isolate sub cultured. 

Figure 3. Frequency of isolated organisms from forehead at the birth time. 

Figure 4. Frequency of isolated organisms from forehead after birth time. 

Staphylococcus epidermidis 

Staphylococcus aureus 

Corynebacterium xerosis 

Streptococcus spp. 

E. coli 

Enterobacter agglomerans 



Staphylococcus saprophyticus 

Micrococcus Luteus 

E. coli 

Corynebacterium xerosis 

Corynebacterium renale

Figure 5. Frequency of isolated organisms from perinea area at the birth time. 

Micrococcus luteus 



Bacillus spp. 

E. coli 


Plesiomonas shigelloides 

E. coli 




Micrococcus roseus 


Micrococcus varians 

Fungi 

Figure 6. Frequency of isolated organisms from perinea area after birth. 

Figure 7. Frequency of isolated organisms from oral cavity 6 h after birth. 


Rhodococcus equi 

Moraxella catarrhalis 

Aurococcus viridans 



Streptococuus beta hemolytic 

Bacillus lentus 

Heshmati et al. 2237


Figure 8. Percentage of infant with organisms in various anatomic sites. * KEC (Klebsiella, Enterobacter, or Citrobacter). 

bacteria was initiated by ingesting food and other 

environmental factors (Figure 7). 

Although 8 to 15% of cultures yield -hemolytic 

streptococci group B which was detected by CAMP and 

Hippurate hydrolysis test, group A streptococci are also 

encountered. During a two-day period 16 of 22 neonates 

in one nursery had organisms sensitive to bacitracin and 

penicillin cultured from their umbilical areas. None of the 

infant showed clinical signs or symptoms of disease. 

These organisms were presumptively identified as group 

A streptococci and the positive cultures were first 

interpreted as evidence of group A streptococcal 

infection. Several weeks later, organisms from 14 of 

these infants were identified as a single strain of - 

hemolytic Sterptococcus (Figure 8). 

DISCUSSION 

Various factors may influence the role of microorganisms 

in relationship to colonization such as antibacterial agent 

(bacteriocin), bacterial interference and competitive 

inhibition. Production of bacteriocin by a bacterial species 

can inhibit colonization of other bacterial species. It is 

also known that a floral bacteria growth can be 

inhibited by another bacterial species through 

competitive inhibition. For instance pathogenic 

microorganisms like Staphylococcus aurous can 

proliferate on the neonatal skin in the absence of skin 

normal flora such as coagulase-negative Staphylococci. 

External factors can also alter the skin normal flora 

population: 

1) The climate: augmentation in humidity and 

temperature can increase the bacterial growth and hence 

modifying their population ratio. 

2) Anatomical condition, body site: the distribution of skin 

normal flora on the body depends on different anatomical 

places. The transient bacteria grow extensively on 

exposed body parts such as face, neck and hands. 

3) Hospitalization, in this study has revealed that long 

term hospitalization can increases the bacterial 

colonization in neonates. 

4) The disease effect: The presence of systemic 

disease can also make the body susceptible to 

bacterial growth. In this study has revealed diabetic 

neonates born from diabetic mother have greater 

potential for bacterial growth than the normal neonates. 

5) Disinfectant effect: Base on this study organisms such 

as Pseudomonas was the most common, and the E.coli 

and Entrobacter were isolated repeatedly from washing 

liquid. Nonetheless, it was shown that none of these 

disinfectant washing liquid would change the coagulase- 

negative staphylococcus population, but using disinfecting

soap increased the Propionobacterium population 

extensively. 

6) Irradiation: this study show that bacterial growth was 

susceptible to U.V light radiation and staphylococci were 

the most susceptible one among all the studied 

organisms. 

One of the controversial points in this study was the 

level of colonization for group B Streptococci among the 

neonates. 

According to the study in the Cooper Green Hospital, the 

rate of colonization for sterp B was established during 28 

months. They emphasize that strep B was the most 

common cause of infection in neonate and twines in 

which being considered as high risk group (William et al., 

1999). Nevertheless we found variation in the amount of 

these bacteria in forehead and perinea areas (14.2 and 

3.5%).Therefore it can be concluded that the main cause 

of strep B colonization in neonate was due to exposure of 

birth canal. 

Base on reported studied (Falle and Schleifer, 1981). at 

this study ,bacterial growth were increased in still-birth 

,low-weighted birth neonates and also in some twins 

and single-born babies ,this increase however was more 

profound in twins due to complex delivery involved. We 

also have found that there is an increasing rate of 

colonization of bacteria in immature and low weight 

neonate. 

Some studies showed a significant difference exist in 

colonization of bacteria on skin of neonates during the 

first, fourth and tenth weeks. We have demonstrated that 

although the rate of colonization of bacteria after birth is 

undetectable but colonization of gram- negative 

organisms was more prevalent. 

Nobel and Jackman reported that colonization of 

staphyloccocus in axillaries area can decrease rate of the 

Coryneforms of bacteria (William et al., 1999). 

But we have showed different result and that the rate of 

colonization of these bacteria was similar both during and 

after birth. 

We also concluded that the highest rate of colonization 

for various species of staphyloccocus was in axillary's 

areas .and the highest rate of colonization for Coryneform 

bacteria was reported in forehead and auxiliary’s areas. 

The S. aureus and S. epidermidis were the most common 

organisms in forehead area. 

The most common organisms of external ear were 

reported to be S. epidermidis; we found the same result 

which confirmed pervious studies. 

Some of earlier researches and literatures have 

reported that anaerobic Sterptococcus species are the 

first cause of infection. It seems that specific species 

constitute the normal flora of vaginal and oral cavity. 

While being born the skin of neonates with cesarean 

surgery was found sterile during delivery, but neonates 

born through birth canal was found to be colonized with 

the normal flora. 

Heshmati et al. 2239 

Based on other results.the Satphylococcus and 

Coryneforms were reported as the most common 

organisms.found in neonate skin and E.coli and Proteus 

species (10%) were also reported. In this study the same 

result has obtained (Sarkany and Gaylarde, 1967). 

The S. epidermidis was colonized in upper parts of 

body and consisted more than 50% the of 

Staphylococcus species. We found the same result too. 

The S. 0hominis and S. hemolyticus were found in 

auxiliary’s and perinea areas. Other findings indicated 

that coagulase negative staphyloccocus was colonized in 

perinea area. According to other researches, intestine of 

neonates were colonized with high rates of klebsiella, E. 

ntrobacter or Citobacter species (William et al., 1999). 

We found that the most common organisms in neonate 

s, stool were gram- negative bacteria such as E.coli and 

Entrobacter species. 

We also observed that gram -negative bacilli were 

mostly colonized in neonates care units. Therefore, we 

disregarded studying bacteria colonization in the first 

hours of birth in care units. 

Larson reported an inverse relation between the bacteria 

colonization and continuous hand washing of personals 

and nurses of hospitals. 

Moreover, hands can be a transmission bacteria portal 

between the neonates. Therefore, it is highly 

recommended, to use disinfectants daily and hand 

washing should be done in neonates care units and ICU 

as well (Pass et al., 1980). 

Ethical aspects 

Informed consent was obtained from the parents of the 

babies. 

REFERENCES 

Guenthner S.H, Owen Hendley J, Wenzel RP (1987). Gram-Negative 

Bacilli as No transient Flora on the Hands of Hospital Personnel. J. 

Clin. Microbiol., 25: 488-490. 

Penders J, Thijis C, Vink C, Stelma FF, Snijders B, Kummeling I, Brandt 

PAvan den, Stobberingh EE (2006). Factors Influencing the 

Composition of the Micro biota in Early Infancy. Pediatrics, 118: 511- 

521. 

Falle A, Schleifer KH (1981). Modified Oxidize and Benzidine Tests for 

Separation of Staphylococci from Micrococcci. J. Clin. Microbiol., 13: 

1031-1035. 

Pezzati M, Biagioli EC, Martelli E, Gambi B, Biagiotti R, Rubaltelli FF 

(2002). Umbilical Cord Care The Effect of Eight Different cord-Care 

regimens on: Cord Separation Time and Other Outcomes. Biol. 

Neonate, 81: 38-44. 

McConnell TP, Lee CW, Couillard M, Westbrook Sherrill W (2004). 

Trends in Umbilical Cord Care: Scientific Evidence for Practice. 

Newborn Infant Nurs. Rev., 4: 211-222. 

Pass MA, Khare S, Dillon HC (1980).Twin pregnancies: Incidence of 

group B streptococcal colonization and disease. Brief clinical and 

laboratory observations. J. Pediatr., 4: 635-637. 

Goldmann DA, Leclair J, Macone A (1978). Bacterial colonization of 

neonates admitted to an intensive care environment. J. Pediatr., 

93(2): 288-293.


McGarrity GJ, Coriell LL (1973). Bacterial Contamination of Children’s 

Soap Bubbles. Am. J. Dis. Child., 125: 224-227. 

William EB, Jeffrey BG, Maurice LD (1999). Risk Factors for Early-onset 

Group B Streptococcal Sepsis: Estimation of Odds Ratios by Critical 

Literature Review. Pediatrics, 103: e77. 

Sarkany I, Gaylarde CC (1967). Skin flora of the newborn. Lancet, 1: 

589-590.



DOI: 10.5897/AJMR11.234 



Evaluation of anticarcinogenicity effect of Artemia 

urmiana by Salmonella typhimurium TA 100 strain 

Masumeh Abbasi 1 , Saman Mahdavi 2* and Sedigheh Mehrabian 3 

1 Islamic Azad university- Malekan Branch, Iran. 

2 Islamic Azad university- Maragheh Branch, Iran. 

3 Department of Biology, Faculty of science, Teacher Training University, Tehran, Iran. 


Free radicals are produced during oxidative metabolism in body and they can attack all components of 

cells and cause cellular changes and early aging. Antioxidant materials role for balance maintenance in 

cells has currently attracted more attention. The aim of this study is investing of antimutation effect of 

Artemia, cyst and decapsulated egg extract by Ames test and Salmonella typhimurium TA 100 strain. 

These extract prevent reverse mutation caused by carcinogenic materials. In comparison with positive 

controls (sodium azid, KMno4, cresol) and negative control (distilled water) antimutagenicity properties 

of Artemia was evaluated and confirmed. In addition, the value of astaxanthin in those extracts was 

assayed. For quality analysis and separation of astaxanthin, we used thin-layer chromatography (TLC) 

chromatography and column chromatography, respectively. Then, the presense of astaxanthin in these 

extracts was confirmed by spectrometer IR test. The results showed that antimutagenicity properties of 

Artemia urmiana are the same with other salty water Artemia. Antimutagenicity and anticarcinogenicity 

properties in decapsulated egg, dried Artemia and cyst are 85, 70 and 100%, respectively. 

Key words: Ames test, anticarcinogenicity, antioxidant, Artemia urmiana, Salmonella typhimurium TA 100. 

INTRODUCTION 

Investigation of anticancer features of Artemia urmiana 

by using the Salmonella typhimurium TA100 strain has 

been done. However, human has discovered the 

presence of Artemia in salty rivers long a time ago. The 

first report about Artemia was registered by Schlosser in 

1755 A.D. He observed Artemia in the samples of the 

water that he has provided from salty rivers in a place 

near Lymington. He called it Artemia linneaus by the 

scientific name of the crab of salt water and in 1819 a 

person whose name was Litch recalled it Artemia salina. 

At last Artemia in Urmia sea was called Artemia urmiana 

by Clark and Bawn (1976), and by the time being, many 

*Corresponding author. E-mail: S.mahdavi@iau-maragheh.ac.ir. 

Tel: 009809144150454. 

Abbreviations: ISA, International study of Artemia; TLC, thinlayer 

chromatography; ROS, reactive oxygen species; FT-IR, 

fourier transform infrared. 

researches and investigations have being done about 

different subjects of Artemia by ISA (International Study 

of Artemia) in different countries (Lorenz and Cysewski, 

2000). Now a day Artemia (that is the famous shrimp of 

salty water) is a good source of protein for human, a food 

for marines and is used in drugs (Magbool and Suresh, 

2003). Because of it contains Caretonoids such as 

Astaxantin, Kantanxantin and Autooxicuein, Artemia is 

able to do different biological performances such as 

protection against light, removing the injuries caused by 

oxygen, immune adjusting in body, and antioxidant activities. 

Also reactive oxygen species (ROS) may play a 

major role as endogenous initiators of degenerative 

processes such as deoxyribonucceic acid (DNA) damage 

and mutation that may be related to cancer (Nair and 

Risch, 2000) cancer (Nair and Risch, 2000). Test of 

carcinogens on rats, is expensive and time consuming. 

Recently, bacteria have been using for assessment of the 

carcinogenicity of suspected materials which are not only 

cost- effective but also give prompt and excellent results.


Table 1. Survey of genotype results of Salmonella typhimurium TA 100 strain. 

Strain rfa mutation UVRB mutation R-Factor 

Salmonella typhimurium TA100 + + + 

One of the methods of assaying carcinogenic effects via 

bacteria is Ames test by use of Salmonella strains which 

because of mutation have lost the histidine synthesis 

potential and in the medium without histidine will face a 

reverse mutation which leads to histidine synthesis. By 

measuring the developed colonies in the medium, the 

carcinogenicity effect will be identified (Motorelmansk 

zeger, 2000). This article discusses different steps of 

growth of Artemia that has antioxidant and more 

anticancer activities so that in proper step we can use it 

as foodstuff and drug for human, poultries and marines. 


Artemia was caught from Urmia Lake and transferred to 

the laboratory on summer 2005. The dried Artemia, cyst, and 

decapsulated egg gathered and froze at -10°C until utilization. 

Salmonella typhimurium TA 100 strain was received directly from 

Professor Ames (University of Beverly, CA). 

Confirming genotypes of tester strains 

The tester strains genotype should be confirmed, so fresh overnight 

nutrient broth cultures were used for this purpose. Salmonella 

typhimurium strain has deficient in dark repair of mutations (UVrB), 

and unable to synthesize a portion of the cell wall (rfa). 

rfa mutation 

TA 100 strain was tested for crystal violet sensitivity. For this test, 

nutrient agar plates were seeded with cultures of the motional strain 

and sterile filter paper disc soaked in crystal violet was placed on 

the surface of each cultured plate. After 12 h incubation the clear 

zone of inhibition (approximately 14 mm) was appeared around the 

disc indicating the presence of the rfa mutation which permits large 

molecules such as crystal violet to enter and kill the bacteria. 

UvrB (UVrB) mutation 

The UVrB mutation was confirmed by demonstrating UV sensitivity 

in TA100 strain which showed this mutation. 

R- factor 

The R- factor of TA 100 strain was tested for the presence of the 

ampicillin resistance factor. It is a convenient marker that makes it 

possible to test for the presence of the R- factor plasmid. The 

specific regions of the R-factor plasmid PKM 101 DNA that are 

essential for enhancement of UV, chemical mutagens, replication 

and ampicillin resistance have been identified. 

Astaxanthin exploitation 

10 g of each dried Artemia, cyst, and decapsulated egg separately 

were placed into glass flasks. Petroleum ether, acetone and water 

were added at a ratio of 1.5:7.5:1 to the samples and left under 

cover for 2 h. Then the solvents were evaporated in rotary 

evaporator until sample dryness. The resulting pigments were 

redissolved in an adequate volume of diethyl ether. These solutions 

were analyzed by thin-layer chromatography (TLC) analysis and 

Fourier transform infrared (FT-IR) spectroscopy. In TLC, mixture 

25% acetone in n- hexane was used as developers (Kobayashi 

and Katsuraji, 2001). In addition, the values of Astaxantin in 

those extracts were assayed. To quality analyze and 

separate Astaxantin, we used TLC chromatography and 

column chromatography respectively. Then we proved this test IR in 

these extracts. 

Ames test 

In antimutagenicity test, the inhibitions of mutagenic activity of 

sodium azide and permanganate potassium by the test samples 

were determined. 1 ml of solution of the tested compounds and 0.1 

ml of an overnight bacterial culture suspension cultivation for 16 h 

at 37°C, approximate cell density (2-5)×10 8 cell/ml and 0.1 ml of 

solution of the positive mutagens were carefully mixed with 3 ml 

of melted top agar containing 50 µmol/l of histidine- biotin, and 

poured onto minimal glucose agar plates. Positive and negative 

controls were also included in each assay. Sodium azide and 

potassium permanganate were used as diagnostic mutagens 

(0.1 ml) in the positive control and plates without mutagens and 

test samples were considered as negative control. Their revertants 

were counted after incubation of the plates at 37°C for 48 h. 

Tester strains were checked routinely to confirm genetic features by 

using the procedure described by Maron and Ames (Negi et al., 

2003). 

RESULTS 

Salmonella typhimurium TA 100 strain has special 

mutation in its operon histidine that makes it dependent 

to outer histidine source. In relation to mutagenic material 

this mutation is reversible, so independent bacteria to 

histidine will come out. According to Tables 2, 3 and 4, 

the antimutation properties of dry Artemia extract, 

decapsulated egg and cyst extract in the Presence of 

Positive mutations were confirmed. By using of 

Salmonella typhimurium TA 100 strain was shown that 

the positive control containing the mutating materials like 

azid sodium, Permanganat Potasium and cresol causes 

reverted mutation in operon histidine (His+). According to 

Tables 2, 3 and 4, in presence of anti-mutagenic material 

comparing with control sample, the number of reverted 

colonies reduced. Indeed, rate of mutation suppression 

was evaluated higher than 40% that was indicating 

strong antimutagenicity effect of comparison with three

Table 2. The results of investigation of preventing and moderating of mutagenesis effects of Azide sodium by dried 

Artemia, cyst and decapsulated egg by using Salmonella typhimurium TA 100. 

Examined material 

Number and percent of reverted colonies of bacterium 

Salmonella typhimurium TA 100 

1 2 3 

Positive control (Azid sodium) 1600 - 1500 - 1440 - 

Negative control 440 - 512 - 524 - 

Dried Artemia extract 600 86% 720 78% 800 69% 

Decapsulated egg extract 500 94% 724 78% 632 88% 

Cyst extract 480 96% 360 100% 400 100% 

Abbasi et al. 2243 

Table 3. The results of investigation of preventing and moderating of mutagenesis effects of permangeant potasium by dried 




Salmonella typhimurium TA 100 

1 2 3 

Positive control (Permangeant potasium) 1680 - 1760 - 2000 - 




Cyst extract 500 87% 480 100% 360 100% 

Table 4. The results of investigation of preventing and moderating of mutagenesis effects of cresol by dried 




Salmonella typhimurium TA100 

1 2 3 

Positive control (cresol) 1560 - 2400 - 2800 - 




Cyst extract 480 100% 800 85% 360 100% 

promotagen materials (Azid sodium, Permanganat 

Potasium and cresol). The result coming out of 

chromatography of thin layer TLC has showed that the 

progressing of the samples produced from the extract of 

dried Artemia, cyst and decapsulated egg on the silica 

gel paper leads to the formation of a dark orange band 

with Rf=40%. This band from color and RF 

measurement point of view looks like astaxantin 

reference, and the main created stigma was in result 

of the separation of the primary samples. Five weak 

spots from dried Artemia and two weak spots from cyst 

and one weak stigma from decapsulated egg have been 

observed (Figure 1). 

At first it was assumed that the light colored bands are 

related to the ester from astaxantin or even to the rest 

of the combinations in it, but after doing spectrographic 

with spectrophotometer and comparing them, it was 

observed that these spots are like to the spectrums in 

result of the primary bands and the light colored bands 

are related to astaxantin extract, because astaxantin 

can combine with oxygen in the air and change to other 

isomers. 

Comparison of spectrums related to the main band 

of dried Artemia extract, cyst and decapsulated egg 

showed that the depth and intensity of created pigments 

are almost the same. It showed that the thickness 

of pigment in Artemia egg was the most and in dried 

Artemia and cyst were fewer than the expected sample.


Figure 1. Chromatography of thin layer of dried Artemia 

extract (A), Decapsulated egg (B) and cyst (C) on 

Chromatographic paper of silica gel. 

DISCUSSION 

One of the main reasons of death in industrialized 

societies is cancer. In the last two decades, different 

kinds of mutating, cancer making and chemical materials 

in foods have been known (Sarmineto and Ochoa, 2002). 

Today scientists believe these genetic damages and 

changes caused by succession of mutating DNA or 

mutation in genes and other genetic changes in chromosomes 

structure have role in cancer. According to Ames 

theory 80% of mutating material cause cancer (Gerber et 

al., 2002) and most of these mutating and cancer 

makings show their damaging effects by producing the 

free radicals and active oxygen. Free radicals are related 

to various physiological and pathological events such as 

inflammation, immunization, aging and etc. It has been 

suggested that compounds which possess antioxidant 

activity, can inhibit mutation and cancer because they 

can scavenge free radicals or induce antioxidant 

enzymes (Mikula and Ikova, 2003). Thus, daily use of 

antioxidants has an important role in protecting body 

against the free radicals, even most of the antioxidants 

are known as anticancers (Rosenkraz, 2003). Antioxidant 

materials such as carotenoids, astaxantin, fixed vitamin 

C, autooxicuien, lipids, fish oil and etc has shown 

anticancer features of them. TLC 

analysis showed that astaxantin and its isomers seemed 

to be the dominant carotenoied in Artemia (Duarte and 

Lunec, 2005). Epidemiological studies indicate that there 

is a close relationship between diet, life style and human 

cancer (Nair and Risch, 2000; Mehrabian and 

Shirkhodaei, 2006). The result of this research is in 

accordant with other researches. According to Ong 

formula, antimutagenicity effect is higher than 40%, so 

recent examined materials are high antimutagenicity 

ability (Ong et al., 1980). Presence of carotenoid pigments 

such as β-caroten, kantaxanthin and astaxanthin 

have been found in Artemia structure that most of them 

are antioxidant properties (Duarte and Lunec, 2005). 

Compounds such as glutation in marine food materials 

play major role in elimination and limitation of free 

radicals (Trusheva, 2006). 

REFERENCES 

Clark LS, Bowen ST (1976). The genetics of Artemia saliva V 

reproductive isolation. J. Helved., 47(6): 385-390. 

Duarte T, Lunec J (2005). When an antioxidant is not an antioxidant? A 

review of novel actions and reactions of vitamin C and free radicals. 

Free Radic. Res., 39(7): 671-686. 

Gerber GB, Leonard A, Hantson PH (2002). Carcinogenicity, 

mutagenicity and teratogenicity of manganese compounds. Crit. Rev. 

Oncol. Hematol., 42: 25-34. 

Kobayashi M, Katsuraji T (2001). In larged and astaxathin 

accumulating cyst cells of the green algae hematoccocus 

pluvialis. J. Biosci. Bioeng., 92: 565-568. 

Lorenz RT, Cysewski GR (2000). Commercial potential for 

hematococcus microalgae as a natural source of astaxanthin. Trends 

Biotechnol., 18. 

Magbool TK, Suresh Kumar S (2003). Microbial quality of shrimp 

products of export trade produced from aquaculture shrimp. Int. J. 

Food Microbiol., 82: 213-221. 

Mehrabian S, Shirkhodaei E (2006). Modulation of mutagenicity of 

Various mutagens by Shrimp Flesh and Skin Extracts in Salmonella 

Test. Pak. J. Biol. Sci., 9(4): 598-600. 

Mikula M, Ikova B (2003). Modulation of mutagenicity of various 

mutagens by lignin derivatives. Mut. Res. Genet. Tox., 535: 171-180. 

Motorelmansk zeger E (2000). The Ames salmonella 

microsomemutagenicity assay. Mutares, 455(1-2): 29-60. 

Nair B, Risch H (2000). Cancer epidemical biomarker. J. Genet., 

1: 3-28. 

Negi PS, Jayaprakasha GK, Jena BS (2003). Antioxidant and 

antimutagenic activities of pomegranate peel extracts. Food Chem., 

80: 393-397. 

Ong TM, Wolf CR, Zeiger E (1980). Diferential effects of cytochrome 

p45 inducers on promutagen activation capabilities and enzymatic 

activities of S9 from rat liver. J. Environ. Pathol. Toxicol., 4: 55-65. 

Rosenkraz HS (2003). Synergy between toxicity and genotoxicity 

relevance to human cancer risk. Mut. Res., 529: 117-127. 

Sarmineto M, Ochoa M (2002). Mutagenicity and antimutagenicity 

studies of lipidic extracts from yellow tail fish, lise fish and Cason fish. 

Food Chem. Toxicol., 40: 1469-1474. 

Trusheva B (2006). Bioactive constituents of Brazilian red proplis. 

Evidence-based complementary and Alternative Medicine. Ecam. 

Oxford University press, 3(2): 249-254.



DOI: 10.5897/AJMR11.549 



mRNA expression of iron metabolism relation genes in 

macrophages by infection with Salmonella typhimurium 

Pan Xin 

Department of Microbiology, Second Military Medical University, Shanghai 200433, China. E-mail: xinpanpx@163.com. 

Tel: 86-21-81870998 or 13524211027. Fax: 86-21-81870998. 


A total number of 13 iron metabolism relation genes expression profiles of RAW264.7 murine 

macrophages infected or uninfected with Salmonella typhimurium were tested by real-time polymerase 

chain reaction (RT-PCR) to evaluate the metabolism of iron in host-pathogen interplay. The living wildtype 

S. typhimurium induces expression of the transferrin receptor (Tfr1) in host cell macrophages, 

which results in a sustained increase of the labile iron pool inside the host cell after 1 or 24 h infection. 

Gene expression analysis showed that wild-type S. typhimurium drives an active iron acquisition 

program with induction of ferrireductase (Steap3), iron membrane transporter Dmt1, and iron regulatory 

proteins (Irp1 and Irp2), while not too much iron efflux changing through ferriportin (Fpn1). The spiA - 

Salmonella mutant strain used in our studies also caused an increase in Tfr1 at 1 or 24 h, but leading to 

decrease in Fpn1 at 24 h as compared with 1 h. The assessment of the labile iron pool after infection 

with spiA - Salmonella after 24 h shows an increase. The same of these two phenotypes allowed iron 

overload in macrophage and became one of the reasons for Salmonella survival inside the macrophage. 

Key words: Macrophage, Salmonella, iron metabolism. 

INTRODUCTION 

Iron is a critical determinant in host-pathogen interaction. 

It is required for full virulence expression by a wide 

variety of intracellular bacteria. Deprivation of iron in-vivo 

and in-vitro severely reduces the pathogenicity of 

Mycobacterium tuberculosis, Coxiella burnettii, Legionella 

pneumophila, and S. typhimurium (Braun, 2001). In fact, 

attempts to withhold iron by sequestering free iron during 

infection is a major defense strategy used by many 

species (Bullen et al., 2006). On the other hand, hostcells 

require iron for first-line defense mechanisms. Iron 

is needed for Haber-Weiss redox chemistry to produce 

hydrogen peroxide or hydroxy radicals (Eaton and Qian, 

2002). Iron and proteins involved in iron metabolism have 

interesting links to the immune system (Hentze et al., 

2004). 

How the bacterium tricks the host cell into providing 

iron and how the host, at the same time, keeps a delicate 

balance between having enough iron for redox reactions 

and withholding iron from intracellular bacteria, however, 

is not well understood. Here we investigated the 

expression profile of macrophage iron homeostasis 

following the infection of RAW264.7 murine macrophages 

with S. typhimurium. 


Bacterial strains and cell 

Wild-type S. typhimurium strain ATCC 14028 (ST) and S. 

typhimurium mutant strains spiA::kan (EG5793) (STA) were used. 

RAW264.7 murine macrophage-like cell line was obtained from 

ATCC. 

Primers (synthesized by Bioasia Company) 

The primers in Table 1 were used in this study. 

Infection assay 

Several colonies of Salmonella were collected, washed twice with 

cell culture complete medium (DMEM was supplemented with 10% 

fetal bovine serum), and resuspended with the same medium. 

Multiplicity of infection was adjusted to 50 using a standardized 

calibration curve of OD600 / colony-forming units (cfu). Bacteria 

were added to RAW264.7 cells in 100×15 mm cell culture dishes. 

Infections were initiated by centrifuging the bacteria onto the cell 

monolayers at 170×g for 5 min and then incubated for 1h at 37°C 

5% CO2. In the second hour, gentamicin was added to a final 

concentration o f 100 g/mL, after which, it was lowered to 10


Table 1. Primers used for real-time quantitative PCR assay. 

Gene name Accession number Forward primer (5’ 3’) Reverse Primer (5’ 3’) 

Irp1 NM_007386 ACTTTGAAAGCTGCCTTGG CTCCACTTCCAGGAGACAGG 

Irp2 NM_022655 TGAAGAAACGGACCTGCTCT GCTCACATCCAACCACCTCT 

Tfr1 BC054522 TGCAGAAAAGGTTGCAAATG TGAGCATGTCCAAAGAGTGC 

Dmt1 NM_008732 GCCAGCCAGTAAGTTCAAGG GCTGTCCAGGAAGACCTGAG 

LcnR BC062878 GCAAGGCTACCCCATACAAA TCTTTGGGCATAGGATGGAG 

Lcn2 NM_008491 CTGAATGGGTGGTGAGTGTG TATTCAGCAGAAAGGGGACG 

Steap3 BC037435 CTCTCTGTGCAGTCTCGCTG TGCAGAGATGACGTTGAAGG 

Hmox1 NM_010442 CCTCACTGGCAGGAAATCAT CCAGAGTGTTCATTCGAGCA 

Fpn1 AF226613 TGCCTTAGTTGTCCTTTGGG GTGGAGAGAGAGTGGCCAAG 

Hamp1 NM_032541 GAGAGACACCAACTTCCCCA TCAGGATGTGGCTCTAGGCT 

Ftl1 NM_010240 AAGATGGGCAACCATCTGAC GCCTCCTAGTCGTGCTTGAG 

Ftl2 NM_008049 TGACTTCCTGGAAAGCCACT GCCTCCTAGTCGTGCTTGAG 

Fth1 NM_010239 CTCATGAGGAGAGGGAGCAT GTGCACACTCCATTGCATTC 

GAPDH NM_008084 CCCACTAACATCAAATGGGG CCTTCCACAATGCCAAAGTT 

g/mL. At different time intervals, the RAW264.7 cells were washed 

three times with DMEM, and harvested for RNA analysis. 

Uninfected cells were used as control. 

RNA analysis 

Total RNA was isolated 1 h later and DNased using the Microto- 

Midi Total RNA Purification System (Invitrogen) according to the 

product instructions. RNA concentrations were determined by a 

RiboGreen assay (Molecular Probes). The reverse transcription 

reactions were carried out with 20 units of MMuLV reverse 

transcriptase (Fisher Scientific); 20 units RNase inhibitor (Fisher 

Scientific); RT-PCR buffer containing 10 mM Tris-HCl and 50 mM 

KCl, 2.5 mM MgCl2, 10 mM dithiothreitol, and 1 mM of each dNTP. 

The concentration of each reverse primer was 5 M. 100 ng of total 

RNA from each sample was reverse transcribed using reverse 

primers. The reverse transcription reactions were incubated for 1 

min at 48°C, 5 min at 37°C, 60 min at 42°C, and then 5 min at 

95°C. 

Real-time RT-PCR was based on the high affinity, doublestranded 

DNA binding dye SYBR Green using a Bio-Rad IQ SYBR 

Green Supermix according to manufacturer's instructions. A total of 

2 L of cDNA was used in the qPCR reactions (1×SYBR green 

PCR master mix, 500 nM gene specific forward and reverse 

primers). All qPCR reactions started with 2 min at 95°C followed by 

40 cycles of 15 s at 94°C and 20 s at 55°C and 30 s at 72°C in an 

Applied Biosystems 7900HT Fast Real-Time PCR System, utilizing 

SDS 2.1 software. All reactions were run three times. Differences in 

mRNA concentrations were quantified by the cycles to fluorescence 

–[ Ct experimental gene- Ct housekeeping 

midpoint cycle threshold calculation (2 

gene] 

), using GAPDH as the housekeeping gene. 

Statistical analysis 

Data were expressed as mean ± SD. An analysis of variance was 

performed with Statview 4.0 statistical analysis software, and P < 

0.05 was considered statistically significant. 

RESULTS 

The realtime PCR results are listed in Table 2. 

DISCUSSION 

We sought to characterize the metabolic changes of 

RAW 264.7 macrophage iron homeostasis following the 

infection with S. typhimurium. The SPI-2 encoded SpiA 

protein is a component of the outer membrane ring of the 

type three secretion systems (TTSS) needle complex 

(Tobar et al., 2006). A spiA - mutant strain rendered 

bacteria unable to translocate effector molecules into 

macrophage cytoplasm (Ochman et al., 1996) and 

displayed levels of phagosome-lysosome fusion that 

were much higher than those displayed by wild-type 

Salmonella and similar to those exhibited by heat-killed 

Salmonella. So we used STA as non-virulent S. 

typhimurium strain to compare with wild-type strain in 

studying iron metabolism. Iron is delivered to 

macrophages primarily through the transferrin receptor 1 

(Tfr1). Tfr1 mRNA levels were significantly increased 

following 1 h both of ST and STA infection by 2.20-fold 

(F=2036.27, P=0.0001) and 1.72-fold (F=8.64, P=0.0424) 

as compared with non-infected RAW264.7 cells. 

However, after 24 h of infection, the mRNA level for Tfr1 

in ST group decreased 0.7-fold (F=11.94, P=0.0259) as 

compared with infected 1 h, but increased 1.88-fold 

(F=14.11, P=0.0198) during infection with STA. 

We also investigated the expression of other genes that 

regulate host cell iron levels by Real time PCR. There are 

two main eukaryotic iron-regulatory proteins, Irp1 and 

Irp2, which sense changes in the labile iron pool (Galy et 

al., 2008). They both act post transcriptionally by 

stabilizing their respective target mRNA and by affecting 

initiation of translation. When investigating Irp1 mRNA 

levels in RAW264.7 phagocytes, we found significant 

difference between controls and 1 h of two Salmonella 

strains infected cells (2.37-fold for ST, F=27.44, 

P=0.0063; 2.51-fold for STA, F=45.85, P=0.0025); when 

we compared 1 h with 24 h infected samples, there was

Table 2. mRNA expression of iron metabolism-related genes in RAW264.7 cells after infection with viable Salmonella. 

Gene name Uninfected 

ST infection sample 

STA infection sample 

Infection 1 h Infection 24 h Infection 1 h Infection 24 h 

Irp1 1 2.37±0.45 2.15±0.68 2.51±0.39 0.68±0.06 

Irp2 1 2.77±0.32 3.73±1.01 2.52±0.78 2.76±0.32 

Tfr1 1 2.20±0.04 1.83±0.10 1.72±0.43 4.15±0.35 

Dmt1 1 12.36±2.24 3.22±0.17 5.36±0.35 12.26±3.41 

LcnR 1 3.06±0.41 0.66±0.12 2.23±0.36 0.24±0.05 

Lcn2 1 2.07±0.38 17.44±1.41 1.18±0.15 11.63±0.54 

Steap3 1 2.86±0.18 1.87±0.43 5.20±0.68 3.78±1.19 

Hmox1 1 1.33±0.12 6.81±0.39 2.87±0.31 6.34±0.58 

Fpn1 1 1.04±0.12 0.62±0.09 1.42±0.13 0.57±0.12 

Hamp1 1 3.88±0.45 11.85±1.73 2.64±0.33 1.42±0.25 

Ftl1 1 0.81±0.05 1.17±0.09 1.16±0.04 1.46±0.07 

Ftl2 1 0.88±0.03 1.23±0.07 1.18±0.01 1.49±0.04 

Fth1 1 0.77±0.07 1.58±0.17 1.34±0.10 1.66±0.13 

Xin 2247 

Mean values shown were normalized to GAPDH mRNA levels and compared to correlated values found in uninfected macrophages (=1). 

no significant difference in ST-infected cells (1.11-fold, 

F=0.60, P=0.4831), but Irp1 expression decreased (0.27fold, 

F=3996.75, P=0.0001) in 24 h STA -infected cells. 

Irp2 mRNA levels was also induced (2.77-fold, F=92.96, 

P=0.0006) in 1 h ST-infected cells as compared with noninfected 

RAW264.7 cells, and no substantially alter in 24 

h ST-infected cells (1.11-fold, F=0.60, P=0.4831) as 

compared with 1 h infection. In STA-infected samples, 

Irp2 mRNA levels at 1 h increased 2.52-fold (F=11.37, 

P=0.0280) as compared with controls, while the mRNA 

levels also no substantially alter (0.90-fold, F=0.40, 

P=0.5599) after 24 h of infection as compared with 1 h 

infection. 

After uptake of iron and vesicle acidification, iron is 

reduced to its ferrous form by the ferrireductase sixtransmembrane 

epithelial antigen of the prostate 3 

(Steap3) prior to being transferred across the endosomal 

membrane (Ohgami et al., 2005). After 1 h of infection 

with wild-type Salmonella, mRNA levels for Steap3 were 

increased 2.86-fold (F=313.07, P=0.0001) as compared 

with controls. After 24 h infection with ST, Steap3 

moderately decreased 0.66-fold (F=10.41, P=0.0321) as 

compared with 1 h infection. In STA infection sample, 

mRNA levels for Steap3 at 1 h were increased 5.20-fold 

(F=115.98, P=0.0004) as compared with controls, but 

declined at 24 h infection period (0.67-fold, F=10.85, 

P=0.0301) as compared with 1 h infection. 

After reduction, ferrous iron is transported into the 

cytosol by Divalent metal transporter 1(Dmt1) (Andrews 

and Schmidt, 2007). After 1 h of infection with wild-type 

Salmonella, mRNA levels for Dmt1 is increased 12.36fold 

(F=64.49, P=0.0013) as compared with controls. 

After 24 h infection with ST, Dmt1 decreased 0.27-fold 

(F=793.44, P=0.0001) as compared with 1 h infection. In 

STA infection sample, mRNA levels for Dmt1 at 1 h 

(5.36-fold, F=465.08, P=0.0001) were significantly 

different from control. This increase continued over a 24 

h infection period (2.11-fold, F=14.66, P=0.0186) as 

compared with 1 h infection. 

The lipocalin system provides the host with another 

way of scavenging iron. At the same time, lipocalin2 

(Lcn2) can interact with bacterial siderophores to inhibit 

bacterial growth and has now been recognized as an 

important arm of the innate immune response (Flo et al., 

2004). The secreted gene product Lcn2 is induced 2.07fold 

(F=22.88, P=0.0088) when cells are infected with ST 

at 1 h as compared with controls. This increase continued 

over a 24 h infection period (8.6-fold, F=70.14, P=0.0011) 

as compared with 1 h infection. In STA infection sample, 

mRNA levels for Lcn2 at 1 h did not alter (1.18-fold, 

F=4.00, P=0.1161) as compared with control, but 

increased (11.63-fold, F=1183.82, P=0.0001) at 24 h 

infection as compared with 1 h infection. Wild type 

Salmonella also raised LcnR mRNA levels 3.06-fold 

(F=78.44, P=0.0009) at 1 h as compared with control, but 

decreased (0.21-fold, F=7956.57, P=0.0001) at 24 h as 

compared with 1 h infection. In STA infection sample, 

mRNA levels for LcnR at 1 h (2.23-fold, F=19.26, 

P=0.0118) were increased as compared with control, but 

also decreased (0.07-fold, F=76729.00, P=0.0001) at 24 

h infection period as compared with 1 h infection. 

Infected host cells can restrict the intracellular iron pool 

available for intracellular parasites by transporting iron 

out of the cells via ferroportin 1 (Fpn1), a transmembrane 

iron efflux protein (Knutson and Wessling-Resnick, 2003). 

Fpn1 is negatively regulated by hepcidin antimicrobial 

peptide (Hamp1), thus effectively reducing iron efflux 

(Nguyen et al., 2006). Hepcidin is significantly 

upregulated 3.88-fold (F=118.54, P=0.0004) in infections 

with ST at 1 h with accompanying unchanged in Fpn1


(1.04-fold, F=0.32, P=0.6030) as compared with control, 

and Hamp1 increase continued (3.1-fold, F=32.90, 

P=0.0046) at 24 h infection with decreased in Fpn1 (0.60fold, 

F=192.64, P=0.0002) as compared with 1 h 

infection. Hamp1 is increased (2.64-fold, F=73.42, 

P=0.0010) in infections with STA at 1 h with 

accompanying increased in Fpn1 (1.42-fold, F=30.77, 

P=0.0052) as compared with control, and Hamp1 

moderately decreased (0.51-fold, F=41.16, P=0.0030) at 

24 h infection with decreased in Fpn1 (0.33-fold, 

F=498.78, P=0.0001) as compared with 1h infection. 

This suggests a stronger effect for iron retention in the 

host cell during infection with ST. Heme oxygenase 

(Hmox1) catalyzes the conversion of heme to biliverdin, 

iron, and carbon monoxide. In macrophages it has an 

important antioxidative protective function, presumably by 

reducing pro-oxidant or proapoptotic hemoproteins 

(Orozco et al., 2007). The mRNA level for Hmox1 is 

moderately increased 1.3-fold (F=23.42, P=0.0084) in 

macrophages infected by ST at 1 h. This increase 

continued over a 24 h infection period (5.13-fold, 

F=886.43, P=0.0001) as compared with 1 h ST infection. 

In STA infection sample, mRNA levels for Hmox1 at 1h 

were increased (2.87-fold, F=109.54, P=0.0005) as 

compared with control, and the increase continued (2.21fold, 

F=1324.96, P=0.0001) at 24 h infection as 

compared with 1 h infection. 

The components of the ferritin iron storage system, 

ferritin heavy chain 1(Fth1), ferritin light chain 1 (Ftl1) and 

ferritin light chain 2 (Ftl2), are moderately decrease by 

infection with ST at 1 h (0.77-fold, F=32.39, P=0.0047; 

0.81-fold, F=51.57, P=0.0020; 0.88-fold, F=61.71, 

P=0.0014, respectively) as compared with control, and 

increased over a 24 h infection period (2.05-fold, 

F=2037.88, P=0.0001; 1.45-fold, F=867.86, P=0.0001; 

1.40-fold, F=369.23, P=0.0001, respectively) as 

compared with 1h ST infection. In STA infection sample, 

these three mRNA levels at 1 h were increased (1.34fold, 

F=31.65, P=0.0049; 1.16-fold, F=45.63, P=0.0025; 

1.18-fold, F=972.00, P=0.0001) as compared with 

control, and still increased (1.24-fold, F=576.00, 

P=0.0001; 1.24-fold, F=782.29, P=0.0001; 1.27-fold, 

F=182.25, P=0.0002) at 24 h infection as compared with 

1 h infection. 

Thus, wild-type S. typhimurium appears to drive an 

active transferrin-mediated iron uptake program after 

infection 1 or 24 h. This was supported by increased 

mRNA levels for Irp1 and Irp2, induction of genes 

required for transfer of iron to the cytosol via Dmt1 and 

Steap3, and by not too much iron efflux changing through 

Fpn1 and Hamp1. The spiA - Salmonella mutant strain 

used in our studies caused an increase in Tfr1 at 1 or 24 

h, and led to decrease in Fpn1 and Hamp1 at 24 h as 

compared with 1 h. The assessment was that the labile 

iron pool increased after infection with spiA - Salmonella 

for 24 h. Of interest in this context is that the spiA - 

Salmonella mutant strain used in our studies behaves 

quite different to the spiC - strain by lacking an increase in 

Tfr1, but leading to an increase in Fpn1 (Pan et al., 

2010). Macrophage iron overload inhibits the transcription 

of iNOS and the generation of NO (Weiss et al., 1994) 

and became one of the reasons for Salmonella survival 

inside the macrophage. 

ACKNOWLEDGMENT 

This work was supported by the National Natural Science 

Foundation of China (Contract No. 30972633). 

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DOI: 10.5897/AJMR11.570 



Expanding drug resistance through integron 

acquisition in Salmonella spp. isolates obtained in 

Iran 

Bahareh Rajaei 1 , Seyed Davar Siadat 1,2* , Mohamad Reza Razavi 3 , 

Mohammad Reza Aghasadeghi 2 , Nahid Sepehri Rad 1 , Farzad Badmasti 4 , 

Somieh Khanjani Jafroodi 3 , Taraneh Rajaei 5 , Arfa Moshiri 6 and Saifuddin Javadian 7 

1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 

2 Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. 

3 Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. 

4 Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran. 

5 Department of Immunology and Allergy, Mashhad University of Medical Sciences, Mashhad, Iran. 

6 Department of Biotechnology, Faculty of Allied Sciences, Tehran University of Medical Sciences, Tehran, Iran. 

7 Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran. 


A total eighty four epidemiologically unrelated clinical isolates of Salmonella enterica serovars were 

subjected to antimicrobial susceptibility testing and molecular detection of class 1 and 2 integrons. 

Eleven isolates (13.1%) which were resistant to at least 4 groups of antimicrobial agents considered 

as MDR (multidrug resistant) Salmonella serovars. PCR assays detected intI1 and intI2 genes in 50 

(59.5%) and 14 (16.7%) of Salmonella clinical isolates respectively. Emergence of MDR Salmonella 

serovars demonstrates that antimicrobial selection pressure is widespread and increased distribution 

of integron carrying gene cassettes which confer resistance to different antibiotics confirms that 

integron-mediated antibiotic resistance is considerable in our clinical settings. 

Key words: Salmonella spp., integron, multidrug resistance (MDR). 

INTRODUCTION 

Salmonella is involved in a wide variety of infections 

ranging from life-threatening typhoid to gastroenteritis 

and bacteremia (Boyd and Hartl, 1998). Antibiotic 

resistance in Salmonella is an emerging problem during 

the last decades. The intensive use of antibiotics in both 

human and veterinary medicine, as well as in agriculture, 

has caused bacteria to develop resistance mechanisms 

against therapeutic drugs (Stokes and Hall, 1989; 

Rodriguez et al., 2008). Dissemination of antibiotic 

resistance genes by horizontal gene transfer has led to 

the rapid emergence of antibiotic resistance among 

bacteria, thus complicating the treatment of infections 

(Gu et al., 2008). The worldwide emergence of multidrugresistant 

(MDR) phenotypes among Salmonella 

*Corresponding author. E-mail: d.siadat@gmail.com. Tel: 

00982144865100. Fax: 00982144865105. 

serotypes is of increasing concern for both the scientific 

community and the general public (Havlickova et al., 

2009; Yang et al., 2009). 

Acquired resistance evolves via horizontal transfer of 

antimicrobial resistance genes located on various types 

of mobile DNA elements (Yang et al., 2009). A key genetic 

system involved in spreading antibiotic multiresistance 

is the integron, a genetic element that, although normally 

immobile itself, can be transferred in companion with 

mobile genetic elements (Tamang et al., 2007) 

Integrons were first identified by virtue of their 

important role in the acquisition and expression of genes 

conferring antibiotic resistance. Integrons are composed 

of three key elements necessary for the capture of 

exogenous genes: a gene (intI) encoding an integrase 

belonging to the tyrosine-recombinase family; a primary 

recombination site (attI) located immediately adjacent to 

intI; and a strong promoter (Pc) that directs transcription 

of the captured genes (Carattoli, 2001; Mazel, 2006).


Table 1. Information about number of MDR and resistant isolates on the basis of the MIC determination according to guidelines of CLSI 

(2009). 

Integron positive isolates 

No. of resistant isolates to the following antibiotics a on the basis of MIC 

SMX SXT TMP CIP NAL AMP CAZ CHL STR 

intI1 positive isolates(50) 20 45 22 1 32 5 2 21 20 11 

intI2 positive isolates(14) 10 12 11 0 11 0 1 4 6 1 

a Abbreviation of mentioned antibiotics and breakpoint for resistance (µg/ml) : AMP, ampicillin ( 32); CAZ, ceftazidime ( 16); CHL, chloramphenicol 

( 32); CIP, ciprofloxacin ( 4); NAL, nalidexic acid ( 32); STR, streptomycin ( 64); TMP, trimethoprim ( 4); SMX, sulfamethoxazole ( 512); SXT, 

sulfamethoxazole-trimethoprim ( 4/76). Breakpoints were adopted from CLSI (Clinical and Laboratory Standards Institute), except for streptomycin, 

which has no CLSI breakpoint. 

b In this study isolates which were resistant to at least 4 groups of antimicrobial agents considered as MDR Salmonella isolates. 

On the basis of nucleotide sequence of the integrase 

gene (intI), nine classes of integrons have been identified 

(Moura et al., 2007; Macedo-Vinas et al., 2009) but, to 

date, only those of class 1 and 2 have been reported in 

S. enterica (Rodriguez et al., 2008). 

The objective of this study was to evaluate the 

antimicrobial resistance profile and to investigate the 

contribution of class 1 and 2 integrons in multidrug 

resistant Salmonella isolates. 


Isolation and identification of bacteria 

A total of eighty four S. enterica isolates attributed to Typhi (n=40), 

Paratyphi A (n=2), Typhymurium (n=12), and non Typhi (n=30) 

serovars were studied. The clinical isolates were recovered from 

stool (n= 69), blood (n= 6), bone marrow (n= 3), synovial fluid (n= 

3), ascites (n= 1), abces (n= 1), urine (n= 1). All isolates were 

identified by standard microbiological techniques as previously 

described (Martin et al., 2008; Ahmed et al., 2009). The serogroup 

was checked with O antisera by the slide agglutination method 

(Difco Laboratories, Detroit, MI). 

Antimicrobial susceptibility test 

The antimicrobial susceptibility test was performed using the 

standard disk diffusion method on Muller-Hinton agar and the 

Minimum Inhibitory Concentrations (MICs) were determined by 

broth microdilution method according to the guidelines of the 

Clinical and Laboratory Standards Institute (CLSI, 2009). Disks 

prepared by MAST company (Mast Co, Merseyside, UK) were 

used to determine the susceptibility of isolates to ampicillin (10 µg), 

tetracycline (30 µg), chloramphenicol (30 µg), trimethoprim (5 µg), 

sulfamethoxazole-trimethoprim (30 µg), streptomycine (10 µg), 

nalidexic acid (30 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), 

levofloxacin (5 µg), norfloxacin (5 µg), gatifloxacin (5 µg), 

moxifloxacin (5 µg), cefotaxime (30 µg), cefixime (5 µg), ceftriaxone 

(30 µg), cefepime (30 µg), ceftazidime (30 µg), amikacin (30 µg), 

azithromycin (15 µg), spectinomycin (100 µg), gentamicin (10 µg), 

colistin-sulfate (10 µg), imipenem (10 µg). The MICs of ampicillin, 

chloramphenicol, streptomycin, nalidixic acid, ciprofloxacin, 

ceftazidime, trimethoprim, sulfamethoxazole and sulfamethoxazoletrimethoprim 

were carried out against all clinical isolates. The 

breakpoints used were those defined by the CLSI for 

Enterobacteriaceae, with the exception of streptomycin (32_g/mL) 

(CLSI, 2009) (Table 1). E. coli ATCC 25922 was used as a quality 

control organism in antimicrobial susceptibility test. 

Detection of class 1 and 2 integrons 

MDR b 

Chromosome and plasmid DNA templates were prepared by 

phenol-chloroform methods as previously described (Sambrook et 

al., 1989). All isolates were screened for detection of class 1 and 2 

integrons by the primers described by Goldstein et al. (2001) 

designed for the intI1 and intI2 genes respectively. The 

amplification program was performed by termocycler (Eppendorf 

Mastercycler®, MA) and started with initial denaturation of 4 min at 

94°C and programmed with 35 cycles of each: 1min at 94°C, 30 s 

at 60°C, 1 min at 72°C. The program finished with the final 

extension of 10min at 72°C. 

RESULTS 

Disk diffusion and minimum inhibitory concentration 

(MIC) 

A total of eighty four Salmonella isolates including S. 

Typhi (n = 40), S.Typhymurium (n=12), S. Paratyphi A (n 

= 2), non-Typhi (n=30) serovars were studied. According 

to the Disk diffusion and minimum inhibitory concentration 

(MIC), antimicrobial resistance patterns were as 

follow: 25 isolates (29.8%) were resistant to 

streptomycin, 25 isolates (29.8%) to sulfamethoxazoletrimethoprim, 

30 isolates (35.7%) to trimethoprim, 23 

isolates (27.4%) to chloramphenicol, 57 isolates (67.9%) 

to tetracycline, 6 isolates (7.1%) to ampicillin, 54 isolates 

(64.3%) to nalidexic acid, 1 isolate (1.2%) to ciprofloxacin, 

6 isolates (7.2%) to cefotaxime, 8 isolates (9.5%) 

to cefexime, 6 isolates (7.2%) to ceftriaxon, 2 isolates 

(2.4%) to ceftazidime, 2 isolates (2.4%) to colistinsulfate, 

3 isolates (3.6%) to gentamicin, 24 isolates 

(28.6%) to spectinomycin, 5 isolates (5.9%) to azithromycin, 

2 isolates (2.4%) to amikacin. All the isolates were 

sensitive to imipenem, ofloxacin, levofloxacin, 

norfloxacin, gatifloxacin, moxifloxacin. Of the 84 isolates 

only 4 isolates (4.7%) were sensitive to the all of the 

tested antimicrobial agents. 

Identification of multidrug resistant (MDR) isolates 

Multi-drug resistance was defined as resistance to at 

least 4 groups of antimicrobial agents. Of the eighty four

Figure 1. PCR Amplification of intl1 Gene. Lane M: 200bp DNA Ladder. Lane 1: int1 PCR product. 

Figure 2. PCR Amplification of intl2 Gene. Lane M: 100bp DNA Ladder. Lane 1: int2 PCR product. 

isolates, eleven isolates (13.1%) were considered as 

MDR Salmonella serovars (1). These MDR isolates were 

attributed to S. Typhi (n=3), S. Typhimurium (n=3), other 

non-Typhi serovars (n=5). One of the MDR isolates were 

recovered from bone marrow and the rest ten MDR 

isolates were originated from stool. 

Detection of class 1 and 2 Integrons 

Rajaei et al. 2251 

Fifty (59.5%) isolates amplified intI1 gene with the amplicon 

size of 280bp and fourteen (16.7%) isolates carried 

intI2 gene with the amplicon size of 232bp (Figures 1 and 

2). Of 50 isolates carrying class 1 integrons, 8 (16%)


Table 2. Information about integron positive isolates among different sources of Salmonella isolates. 

Isolation 

source 

No. of intI1 positive isolates 

(%) 

No. of inti2 positive isolates 

(%) 

No. of intI1 and inti2 positive isolates 

(%) 

Stool(69) 41 13 13 

Blood(6) 3 0 0 

Bone marrow(3) 2 0 0 

Synovial fluid(3) 1 0 0 

Abces(1) 1 0 0 

Ascites(1) 1 0 0 

Urine(1) 1 1 1 

Total(84) 50(59.5) 14(16.7) 14(16.7) 

Table 3. Information about integron positive isolates among serovars of Salmonella spp. 

Serovars (No.) 

No. of intI1 and intI2positive 

isolates (%) c 

No. of intI2-positive 

isolates (%) b 

No. of intI1-positive 

isolates (%) a 

S. Typhi (40) 5 5 21 

Non-typhi serovars (30) 8 8 20 

S. Typhimurium (12) 1 1 7 

S. Paratyphi A (2) 0 0 2 

Total (84) 14(16.7) 14(16.7) 50 (59.5) 

a indicates the number and percentage of intI1-posetive in Salmonellae serovars 

b indicates the number and percentage of intI2-posetive in Salmonellae serovars 

c indicates the number and percentage of serovars carrying both intI1 and intI2 genes. 

isolates amplified class 1 integrons exclusively on 

chromosome, 12 (24%) isolates exclusively on plasmid, 

30 (60%) isolates both on plasmid and chromosome. 

Among fourteen isolates harbored class 2 integrons, 3 

(21.4%) isolates carried class 2 integrons only on 

plasmids and 11 (78.6%) isolates both on plasmid and 

chromosome (Tables 2 and 3). 

DISCUSSION 

The worldwide emergence of multidrug-resistant (MDR) 

phenotypes among Salmonella serotypes is causing an 

increasing concern. Use of antimicrobials in clinical and 

veterinary medicine is a recognized driving force for the 

selection of resistant bacteria. Selective pressure has 

resulted in the development of strains that are resistant 

to more than one antimicrobial agent (Miriagou et al., 

2006; Yang et al., 2009). Integrons are genetic elements 

able to recognize and capture mobile gene cassettes 

carrying antibiotic resistance genes leading to MDR 

distribution and subsequently limitation of treatment 

options for infections (Stokes and Hall, 1989). 

In this study eighty four clinical isolates of Salmonella 

spp. were subjected to molecular investigations to detect 

integron-associated multidrug resistance. 

Of 84 isolates, 11 (13.1%) isolates were resistant to 

more than 4 groups of antimicrobial agents and considered 

as MDR Salmonella serovars (Table 1). Most of 

the MDR isolates were recovered from stool and related 

to the non-Typhi serovars of Salmonella enterica. Fifty 

(59.5%) isolates harbored class 1 integrons and 

amplified intI1 gene more detected in Typhi serovar and 

14 (16.7%) isolates contained class 2 integrons carrying 

intI2 gene as well as intI1 gene which were more 

considered in non–Typhi serovars of Salmonella enterica 

(Table 3). 

Our study indicates that all the MDR isolates harbored 

class 1 integrons and one of the MDR isolates carried 

class 2 in addition to class 1 integrons. This result 

highlights the integron role in MDR distribution (Table 1). 

To confirm the previous studies, according to the 

results of MIC determination, most of the intI-positive 

isolates showed high resistance to nalidixic acid, 

trimethoprim and trimethoprim–sulphamethoxazole 

(Chang et al., 2007) and indicated increased MIC to be 

higher than 512 µg/ml. Limitless use of these antibiotics 

in hospitals and other health care environments may be 

involved in the selection and consequently distribution of 

integron-carrying Salmonella. These antibiotics are at 

risk for future use and would be prescribed with more 

consideration. 

In agreement with other studies, fluoroquinolones, 

third-generation cephalosporins and imipenem are

suggested to be used as frontline therapeutic drugs in 

treatment of Salmonella infections. Carbapenems are the 

main class of drugs used for treatment of infections 

caused by MDR and extended-spectrum -lactamaseproducer 

Gram-negative bacteria such as Salmonella 

(Ahmed et al., 2009). 

In conclusion our results support the hypothesis that 

integron acquisition may lead to dissemination of new 

antibiotic resistance determinants and high levels of 

multidrug resistance. Epidemiological studies on the 

distribution of integrons would be of great use in 

detecting MDR isolates. 

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DOI: 10.5897/AJMR11.580 



The synthesis and role of hydroxyectoine in halophilic 

bacterium Halomonas ventosae DL7 

Daochen Zhu 1,2 *, Chenxiang Wang 3 , Shoko Hosoi-Tanabe 4 , Weimin Zhang 2 and 

Shinichi Nagata 4 

1 School of Environmental Engineering, Jiangsu University, 301 Xuefu Road Zhenjiang 212013, China. 

2 Guangdong Provincial Open Laboratory of Applied Microbiology, Guangdong Provincial, Key Laboratory of Microbial 

Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China. 

3 Environmental Science and Engineering College, Dalian Maritime University, 1 Linghai Road, Dalian 116026, China. 

4 Environmental Biochemistry Division, Research Center for Inland Seas, Kobe University, 5-1-1 Fukae, Higashinada-ku, 

Kobe 658-0022, Japan. 


Hydroxyectoine and ectoine were accumulated in the moderately halophilic bacterium Halomonas 

ventosae DL7 as dominant compatible solutes in response to the external thermo- and osmo-stresses 

(30-42°C, 1-3 M NaCl). As the byproduct of ectoine, hydroxyectoine was directly converted from ectoine 

mediated by the ectoine hydroxylase in DL7, and the hydroxylase gene sequence was obtained by PCR. 

Further, the activity of ectoine hydroxylase of strain DL7 cells in vitro was determined at 30 to 42°C, and 

the thermo-stress conduce the inhibition of this enzyme activity. In addition, DL7 cells showed similar 

growth level when grown in presence of same level ectoine or hydroxyectoine (1 mM) in medium with 

high salinity (2-2.7 M NaCl). However, the presence of hydroxyectoine showed higher growth level than 

ectoine when DL7 cells were incubated at 42°C. The results showed that the alteration of temperature 

instead of salinity stress obviously stimulated the expression of ectoine hydroxylase, but inhibited its 

activity, and hydroxyectoine with ectoine together played an important role as thermo- and osmoprotectants 

in strain DL7 to cope with high temperature and salinity environment. 

Key words: Halomonas ventosae DL7, compatible solutes, hydroxyectoine, ectoine hydroxylase, thermostress, 

osmo-stress. 

INTRODUCTION 

To cope with a variety of high osmotic environments, 

halophilic and halotolerant bacteria developed a strategy 

to accumulate a particular class of highly soluble, small 

molecular organic compounds which named compatible 

solutes (Kunte, 2006; Roberts, 2005). Among compatible 

solutes, ectoine and its hydroxylated derivative, 

hydroxyectoine are one of the most widely produced 

solutes. And ectoines are increasingly concerned for their 

superior function on the enzyme, nucleic acid and cell 

wall protective ability against a variety of stress factors 

such as heating, freeze-thawing and freeze-drying 

*Corresponding author. E-mail: dczhucn@hotmail.com. Tel/fax: 

+86-511-88796122. 

(Lippert and Galinski, 1992; Louis et al., 1994). The OH 

group of hydroxyectoine can partly replace the loss of 

water molecules from hydrate shell to further stabilize the 

native structure of the biopolymers compare to ectoine 

(Graf et al., 2008). Hydroxyectoine is proposed as 

suitable healthy cells protectant during chemotherapy 

and molecular chaperon for Alzheimer's disease treatment, 

and it can protect skin cells against the aging and 

function as the PCR enhancer to improve the yield of 

target production (Furusho et al., 2005; Kanapathipillai et 

al., 2005). It would have a significantly applicable value in 

the field of bio-industry, medical therapy, and cosmetic 

ingredients, etc. Therefore, the synthesis pathway of 

hydroxyectoine and its physiological role in microorganisms 

were interesting to researchers. Studies have 

suggested that the biosynthesis of hydroxyectoine as

thermo-protectant has been proved to parallel with high 

environmental temperature (García-Estepa et al., 2006; 

Prabhu et al., 2004). The biosynthetic pathway of hydroxyectoine 

was directly converted via ectoine hydroxylase 

from ectoine in halophilic or halotolerant bacteria and 

induced by osmotic stress of high salinity, and ectoine 

hydroxylase has been found in strains Streptomyces 

chrysomallus, Streptomyces coelicolor and 

Chromohalobacter salexigens (Bursy et al., 2008; García- 

Estepa et al., 2006; Prabhu et al., 2004). Further study 

revealed that the ectoine hydroxylase is a member of the 

non-heme-containing iron (II) and 2-oxoglutaratedependent 

dioxygenase superfamily, and its in vitro 

activity depends on iron (II), molecular oxygen, and 2oxoglutarate 

(Bursy et al., 2008; Reuter et al., 2010). The 

chemical synthesis of hydroxyectoine, however, is still 

difficult at present. Therefore, the production of hydroxyectoine 

by microorganisms is currently a simple and 

meaningful method. To produce hydroxyectoine using 

halophilic bacteria, the fed-batch and milking method has 

been developed and the effective factors for the 

synthesis of hydroxyectoine in the production process 

were studied. For example, the carbon source xylose can 

significantly improve the conversion of ectoine to 

hydroxyectoine in recombinant E. coli BL21, which 

carries ectoine hydroxylase gene (thpD) (Nagata et al., 

2008b). 

Moderately halophilic bacterium, Halomonas ventosae 

DL7, which has been isolated from Dalian saltern, China, 

displays the ability to synthesize a large amount of 

ectoine as well as minor hydroxyectoine under hyperosmolality 

(Zhu et al., 2007). And the synthesized amount 

of hydroxyectoine significantly increased in strain DL7 

when subjected to heat stimulation (Zhu et al., 2008b). 

Strain DL7 as an excellent ectoine or hydroxyectoine 

producer has a potential application value for producing 

ectoines in factory on a larger scale. But there is still 

unclear for the effect factor of hydroxyectoine synthesize 

and activity of ectoine hydroxylase in DL7. Therefore, to 

elucidate the role and synthesis characteristics of 

hydroxyectoine in DL7 is an interesting work. 

The goal of this study was to investigate the effects of 

hydroxyectoine and ectoine on the growth of strain DL7 

when subjected to high salinity and temperature environments. 

And the in vitro activity of ectoine hydroxylase 

with the fluctuant salinity and temperature were 

determined. We also evaluated hydroxyectoine as a vital 

protectant for strain DL7 cells to defend off the 

environmental thermo-stress. 


Bacteria and growth media 

H. ventosae DL7 was aerobically grown in a nutrient medium (g/l): 

Bacto peptone (Difco Laboratories, Detroit, MI, USA), 5.0 and Bacto 

yeast extract (Difco), 3.0. Glucose mineral medium (MM63) was 

also used in this study: 100 mM KH2PO4, 75 mM KOH, 15 mM 

Zhu et al. 2255 

(NH4)2SO4, 1 mM MgSO4, 3.9 µM FeSO4, and 20 mM glucose as 

carbon source. NaCl was added as described in the text and pH 

was adjusted to 7.2 with NaOH. 

PCR amplification and gene analysis 

To isolate the gene encoding ectoine hydroxylase of H. ventosae 

DL7, we designed degeneration of oligonucleotides for PCR. Based 

on the ectD conservation regions from Virgibacillus salexigens 

(AY935522) and Chromohalobacter salexigens (AM231629), the 

universal degenerate primers (5’-GAMAGCMAKGASATCCGKT-3’ 

and 5’-GARCCATGCAKYGTRTT-3’) were employed. Genomic 

DNA of H. ventosae DL7 and C. salexigens DSM 3043T were 

isolated using Genomic DNA Purification Kit (Promega, Madison, 

WI, USA) according to the manufacturer’s protocol. PCR was 

performed under the following condition: 5 min at 94°C, 30 cycles of 

30 s at 94°C plus 1 min at 55°C plus 30 s at 72°C, and finally 5 min 

at 72°C. Electrophoresis was performed on 1% (w/v) agarose gel in 

TAE buffer at 100 V for 30 min. The PCR product on the gel was 

extracted by using a PCR Purification Kit (QIAGEN, Hilden, 

Germany), cloned into the pGEM-T Vector System (Promega), and 

sequenced using an ABI PRISMA 377 sequencer. The nucleotide 

sequence of the PCR product has been deposited in the GSDB, 

DDBJ, EMBL, and NCBI databases (Accession No. EU732521). 

Enzyme assay and the conversion of ectoine to 

hydroxyectoine 

Ectoine hydroxylase activity in the extracts of DL7 cells were 

assayed as described before (Bursy et al., 2007), 70 µl protein 

fractions were suspended in 100 µl reaction mixture that contained 

10 mM TES buffer, pH 7.5, 10 mM 2-oxoglutarate, 5 mM ectoine, 1 

mM FeSO4 . 7H2O, 1.3 kilo units beef liver catalase (Roche, 

Germany). The reaction mixture was incubated at 30 or 42°C for 20 

min and stopped by adding 100 µl acetonitrile. Supernatant was 

analyzed by HPLC. One unit of ectoine hydroxylase activity is 

defined as the conversion of 1 µM of ectoine to 1 µM of 

hydroxyectoine per min. 

Release of hydroxyectoine and ectoine by downshock 

H. ventosae DL7 was aerobically grown in the nutrient medium 

containing 3 M NaCl until stationary phase of growth and harvested. 

The pellets were washed once by KPi buffer with the same NaCl 

concentration as that of corresponding nutrient medium, and then 

resuspended in deionized water or 2% NaCl solution. The cells 

were incubated for 10-120 min at 30°C. After centrifugation the 

supernatant fractions were analyzed by HPLC. Release rate of 

hydroxyectoine and ectoine from the cells were calculated as 

described previously (Nagata et al., 2008a). 

Extraction and determination of compatible solutes 

Cells were aerobically grown in the nutrient medium containing 1-3 

M NaCl at 30 or 42°C. Cells were harvested by centrifuging 

(10,000×g, 4°C, 10 min), and then the pellets were washed once 

with 50 mM potassium phosphate (KPi) buffer (pH 7.2) containing 

the same concentration of NaCl corresponding to growth medium. 

Extraction of intracellular compatible solutes was carried out as 

described previously (Zhu et al., 2008a). Fraction of 80% (v/v) 

ethanol extraction as well as supernatant after osmotic downshock 

analyzed and quantified by HPLC system (L-5020, Hitachi, Tokyo, 

Japan) with a Bio-Sil C8 90-5S reversed phase column (Bio-Rad


Growth (OD600) 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

Laboratories, Hercules, CA, USA) as described previously (Nagata 

et al., 2006; Zhu et al., 2010). Five microliters of samples were 

eluted with 50 mM KPi buffer at pH 6.0 with a flow rate of 1 ml/min 

at 35°C and detected by UV absorbance at 210 nm. Concentrations 

of ectoine and hydroxyectoine were expressed as milligram per 

gram CDW. 


Effect of temperature on the growth of H. ventosae 

DL7 

The influence of temperature on the cells growth of strain 

DL7 was examined using the nutrient medium containing 

3 M NaCl at 30-42°C. Results showed that the growth of 

cells was severely inhibited with the increasing of 

incubation temperature, and reached stationary phase at 

36 h of incubation at 30°C, which prolonged to 57 and 66 

h with the incubation temperature of 37 and 42°C, 

respectively. Optical density (OD600) was significantly 

reduced from 3.3 at 30°C to 2.8 at 37°C and 2.1 at 42°C, 

respectively (Figure 1). In our previous report, the amount 

of hydroxyectoine synthesized was very low comparing 

with ectoine when cells grown at 30°C and the 

concentration of hydroxyectoine in the cells remarkably 

increased with the increase of temperature and reached 

the maximum of 36.6 mg/g CDW at 42°C, (Zhu et al., 

0 

A 

0 10 20 30 40 50 60 70 80 

Incubation Time time (h) (h) 

Figure 1. The growth of strain DL7 cells at different incubation 

temperatures. Strain DL7 was pre-incubated in a nutrient medium 

containing 3 M NaCl for 18 h at 30°C and then separately incubated 

at 30°C (cycles), 37°C (diamonds), or 42°C (triangles). Values are 

the averages ± SD from three independent experiments. 

2008a). It suggests that the synthesis of hydroxyectoine 

was a strategy of strain DL7 cells to defend off the 

environmental thermo-stress. 

Ectoine hydroxylase in H. ventosae DL7 

Ectoine hydroxylase catalyzing the conversion of ectoine 

into hydroxyectoine had been proved in several halophilic 

bacteria (Bursy et al., 2007; García-Estepa et al., 2006; 

Nagata et al., 2008a). The PCR strategy was carrying out 

to clarify whether this hydroxylase was present in strain 

DL7, and we assumed that a homologous hydroxylase 

gene to those of C. salexigens and V. salexigens was 

existed in this strain to design a pair of degenerated 

primers for PCR amplification, and a 480-bp gene 

fragment was obtained and subsequently sequenced 

(EU732521), the amino acid sequence deduced from the 

DNA sequence of this fragment showed 54.8% identity 

with the ectoine hydroxylases from C. salexigens and V. 

salexigens (Figure 2). These data confirmed the presence 

of hydroxylase in H. ventosae DL7, by which the 

conversion of ectoine should be the important pathway 

for the synthesis of hydroxyectoine. To prove this opinion, 

the activity of ectoine hydroxylase of strain DL7 was 

determined. 

To investigate the activity of ectoine hydroxylase under


Figure 2. Alignment of the sequences of reading frame ectD (EU732521) of H. ventosae DL7 with the sequences of the ectoine 

hydroxylases of V. salexigens (AY935522) and C. salexigens (AM231629). Identical positions and conservative fragments are shown 

against a dark and gray background, respectively. 

Table 1. Ectoine hydroxylase activity (cell free extract) from DL7* 

Reaction temperature (°C) NaCl (M) Activity (Unit) Protein (mg)** 

30 0 43.7 916.5 

42 0 29.5 920.3 

30 1 42.1 918.4 

*Cell free extract was prepared from 20 g cell pellets grown in 3 M NaCl at 30°C. 

** Protein (mg): the protein content of 70 µl protein fractions extract from DL7 cells. 

the heat or salt stress, we assayed the cell-free extracts 

from strain DL7 cells in vitro at 30 or 42°C, without or with 

1 M NaCl. Results showed that the activity of hydroxylase 

was reduced from 43.7 to 29.5 U, when the temperature 

was increased from 30 to 42°C (Table 1). However, the 

activity was stable with the fluctuation of NaCl concentration 

(0-1 M) at 30°C. It revealed that the conversion of 

ectoine to hydroxyectoine was inhibited by the higher 

incubation temperature. On the other hand, the activity of 

hydroxylase had no obviously fluctuation with or without 

salt. In addition, similar result has also been found in our 

previous research, i.e., the in vivo activity of hydroxylase 

was inhibited by high temperature, and hydroxyectoine 

showed similar conversion rate in the presence of 

different NaCl concentrations (0.5-1 M) (Zhu et al., 

2008a). Therefore, these in vivo and in vitro data 

supported our suggestion that the hydroxylase activity 

was sensitive to temperature but was not disturbed by 

salinity. 

Based on these data, we could deduce that the 

increased amount of hydroxyectoine synthesized 

following the enhancing of thermo-stress might be that 

the heat stimulated the cells to produce more amount of 

ectoine hydroxylase instead of induction of the increasing 

of the enzyme activity. Further, to detect whether there is 

a high temperature intermediated operon to trigger the 

synthesis of hydroxylase in DL7 is an interesting work in 

future. 

Release of hydroxyectoine and ectoine by osmotic 

downshock 

To investigate the role of hydroxyectoine and ectoine for


Release rate (%) 

96 

92 

88 

84 

80 

10 20 40 60 120 

Time (min) 

Figure 3. Releases of hydroxyectoine and ectoine by H. ventosae DL7 subjected to 

osmotic downshock. Downshock procedures were handled in deionized water to 

release ectoine (white bars) and hydroxyectoine (hatched bars), or in 2% NaCl to 

release ectoine (black bars) and hydroxyectoine (macula bars) during 10-120 min 

incubation. Values are the averages ± SD from three independent experiments. 

strain DL7 in response to the salt or heat stress, it is 

necessary to use the “clearing cells”, which have no 

intracellularly compatible solutes. Therefore, the release 

of compatible solutes from cells and at the same time to 

keep alive of cells was concerned for preparation of the 

compatible solutes free cells. When halophilic bacteria 

are confronted with a rapid osmotic downshock, most of 

the small components in the cytoplasm were released 

into the environments (Frings et al., 1995; Nagata et al., 

1996; 1998; Sauer et al., 1998). In this consideration, we 

tried to examine the release rates of hydroxyectoine and 

ectoine following a shift in medium osmolarity, that is, 

cells were suspended in 2% (0.34 M) NaCl or deionized 

water from 3 M NaCl medium. When the strain was 

downshocked in deionized water, both intracellular 

ectoine and hydroxyectoine were rapidly released to 

environment. During less than 10 min of downshock 

treatment, 90.8 and 89.9% of release rates were 

achieved for ectoine and hydroxyectoine, respectively 

(Figure 3). However, since strain DL7 is a moderately 

halophilic bacterium and a minimum concentration of Na + 

is essential for growth, the cells would burst by osmotic 

downshock from high salinity environment to deionized 

water and could not be recovered to use for further study. 

To remain the growth of cells after downshock, we tried 

to examine 2% NaCl solution instead of deionized 

water. As a consequence, the cells were kept intact after 

re-incubation in 2% NaCl solution for 1 h (data not 

shown). Release rates of ectoine and hydroxyectoine 

reached maximum at 60 min of treatment, and resulted in 

90.1 and 88.4%, respectively. The survival rates of cells 

treated by downshock (3 M NaCl to 2% NaCl and 2 M 

NaCl to 2% NaCl) were determined as 95.3 and 97.6%, 

respectively. It was suggested that the numbers of 

damaged cells were negligible for the present condition, 

and 2% NaCl solution was chosen to prepare the solutes 

free cells for the following study. 

Thermo- and osmo-protection of compatible solutes 

To gain insight into the role of hydroxyectoine and 

ectoine in strain DL7 when cells were subjected to 

thermo-stress, we tried to examine the behavior of strain 

DL7 under high temperature and salinity in the presence 

or absence of compatible solutes. Cells were osmotic 

downshock treated by 2% NaCl solution to avoid the 

interference of intracellular solutes, and pellets were then 

resuspended in MM63 medium containing 1-2.7 M NaCl 

without or with 1 mM each of ectoine or hydroxyectoine at 

30 or 42°C. We obviously observed that the presences of 

hydroxyectoine and ectoine significantly promoted the


3.5 

3 

2.5 

2 

1.5 

1 

A 

A 

0 10 20 30 40 50 60 70 

Time (h) 


2.5 

2.25 

2 

1.75 

1.5 

1.25 

1 

B 

B 

0 12 24 36 48 60 72 

Incubation Time (h) time (h) 

Figure 4. Supplement effects of hydroxyectoine and ectoine on the growth of H. ventosae DL7 under high 

temperatures or high salinities. A. Cells were grown in MM63 medium containing 1 M NaCl at 42°C (open symbols) 

or 30°C (closed symbols) without (triangles), with 1 mM ectoine (circles) or 1 mM hydroxyectoine (squares). B. 

Cells were grown in MM63 medium containing 2 M (open symbols) or 2.7 M NaCl (closed symbols) at 30°C with 1 

mM ectoine (circles) and 1 mM hydroxyectoine (squares). Values are the averages ± SD from three independent 

experiments. 

Table 2. The accumulation of ectoine and hydroxyectoine by downshocked cells of H. ventosae DL7* 

Compatible solutes NaCl (M) 

Ectoine 

Hydroxyectoine 

Concentrations (µM /g CDW) 

Incubation time (h) 

1 3 5 10 

1 241±14.8 245±10.6 253±3.5 258±7.8 

2 327±10.6 368±12.7 395±5.6 411±4.2 

1 234±12.7 244±2.8 241±7.1 244±10.6 

2 304±9.1 355±4.2 369±5.7 390±4.9 

*Downshock treatment was performed according to the method described in Materials and Methods. The 

downshocked cells were incubated in MM63 medium at 42°C with 1 mM each ectoine or hydroxyectoine. The values 

are the means ± SD from three independent experiments. 

growth of cells at 42°C from the growth curve, and the 

optical density of hydroxyectoine showed 1.5 times 

higher than ectoine after 24 h of incubation (Figure 4A). 

However, there was no obviously difference between the 

growth of DL7 in presence of hydroxyectoine and ectoine 

at 30°C. In addition, the downshocked compatible solutes 

free DL7 cells to accumulate hydroxyectoine and ectoine 

reached almost same level with 1 M or 2 M NaCl 

concentrations (Table 2). It suggests that the presence of 

hydroxyectoine result in the growth of cells higher than 

ectoine were not induced by different concentrations of 

hydroxyectoine and ectoine accumulated in cells. The 

above data elucidated that hydroxyectoine protected DL7 


cells more effectively than ectoine at 42°C. 

To investigate the protection functions of hydroxyectoine 

and ectoine on H. ventosae DL7 to defend off 

high salinity, the compatible solutes free cells were 

incubated in MM63 medium containing 2 or 2.7 M NaCl in 

presence of 1 mM each of hydroxyectoine or ectoine and 

the growth was detected. Results verified that the 

supplementation of ectoine or hydroxyectoine conferred a 

similar cell growth both in presence of 2 and 2.7 M NaCl 

(Figure 4B), that is, ectoine and hydroxyectoine have 

similar protection effect for cells grown under high salinity 

environment. 

It was understood that the accumulation of compatible


solutes was osmo-adapted by environmental stresses for 

halophilic bacteria. In this study, we described the 

synthesis of hydroxyectoine and activity of hydroxylase in 

strain DL7, showed that thermo-stress stimulated the 

yield of hydroxyectoine but inhibited the activity of 

hydroxylase. We also noticed the hydroxylase express 

system might be induced by thermo-stress instead of 

osmotic-stress. The study for charactering the 

hydroxylase in structure and exploring its molecular 

mechanism are interesting work in the near future. 


This work was supported by a grant (Code: 

9451007002003073) from the Natural Science 

Foundation of Guangdong Province, China, and by the 

Project Startup Fundation for Distinguished Scholars of 

Jiangsu University (Code: 10JDG084). 

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evolutionarily conserved ectoine hydroxylase. J. Biol. Chem., 282: 

31147-31155. 

Bursy J, Kuhlmann AU, Pittelkow M, Hartmann H, Jebbar M, Pierik AJ, 

Bremer E (2008). Synthesis and uptake of the compatible solute 

ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2) in 

response to salt and heat stresses. Appl. Environ. Microbiol., 74: 

7286-7296. 

Frings E, Sauer T, Galinski EA (1995). Production of hydroxyectoine: 

high cell-density cultivation and osmotic downshock of Marinococcus 

strain M52. J. Biotechnol., 61: 117-158. 

Furusho K, Yoshizawa T, Shoji S (2005). Ectoine alters subcellular 

localization of inclusions and reduces apoptotic cell death induced by 

the truncated Machado-Joseph disease gene product with an 

expanded polyglutamine stretch. Neurobiol. Dis., 20: 170-178. 

García-Estepa R, Argandoña M, Reina-Bueno M, Capote N, Iglesias- 

Guerra F, Nieto JJ, Vargas C (2006). The ectD gene, which is 

involved in the synthesis of the compatible solute hydroxyectoine, is 

essential for thermoprotection of the halophilic bacterium 

Chromohalobacter salexigens. J. Bacteriol., 188: 3774-3784. 

Graf R, Anzali S, Buenger J, Pfluecker F, Driller H (2008). The 

multifunctional role of ectoine as a natural cell protectant. Clin. 

Dermatol., 26: 326-333. 

Kanapathipillai M, Lentzen G, Sierks M, Park CB (2005). Ectoine and 

hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer’s - 

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Kunte HJ (2006). Osmoregulation in bacteria: compatible solute 

accumulation and osmosensing. Environ. Chem., 3: 94-99. 

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compatible solutes: protection against heating, freezing and drying. 

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after exposure to hyperosmotic shock. Appl. Environ. Microbiol., 64: 

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coexisting glutamate on survival and uptake of ectoine in halotolerant 

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DOI: 10.5897/AJMR11.585 



Virulence and characteristics of a new 

nucleopolyhedrovirus strain of Dendrolimus kikuchii 

(Lepidoptera: Lasiocampidae) 

Miao Miao Yang 1,2,6, Meng Lou Li 1 , Yu Zhu Wang 2 , Liang Jian Qu 2 , Ke Yue Huai 3 , 

Xue Wen Nie 4 , Lu Qin Qiao 5 , Jin Ying Ding 6 and Yong An Zhang 2 * 

1 Forestry Entomology Laboratory of Academic Forestry, Northwest Agriculture and Forest University, 

Yangling 712100 China. 

2 The Key Laboratory of Forest Protection, State Forestry Administration of China, Research Institute of Forest Ecology, 

Environment and Protection, Chinese Academy of Forestry, Beijing 100091 China. 

3 Forestry Research Institute of Yunnan, Yunnan 650000 China. 

4 Forest Protection Station Mile County of Yunnan, Yunnan 652300 China. 

5 College of Plant Protection, Shandong Agricultural University, Tai’an 271018 China. 

6 Ningxia agricultural school, Yinchuan 750000 China. 


In present study, a nucleopolyhedrovirus was isolated from the infected larvae of Dendrolimus kikuchii, 

which is a serious pest for a variety of conifers in China. Ultrastructural observation revealed that this 

virus is a multiple-nucleocapsid nucleopolyhedrovirus (MNPV), which was encapsulated within the 

envelope of each virion. Based on the morphological characteristics, it was identified as a new virus 

and named as DkNPV. It exhibited high potential as a biocontrol agent to protect the forest because of 

the high virulence against the third-instar larvae of D. kikuchii. The virus passage in the host after 4 

generations, toxicity and ultrastructural morphology were stable. 

Key words: Dendrolimus kikuchii, multiple-nucleocapsid nucleopolyhedrovirus, Dendrolimus Kikuchii 

Matsumura nuclear polyhedrosis virus (DkNPV), virulence, ultrastructure, biological control. 

INTRODUCTION 

Dendrolimus kikuchii (Matsumura) (Lepidoptera: 

Lasiocampidae), is a serious pest for a variety of 

conifers, including Pinus kesiya, Pinus yunnanensis, 

Pinus massoniana, Pinus armandi, Pinus fenzeliana, 

Cedrus deodara and Keteleeria evelyniana (Figure 1a). 

In China, D. kikuchii mainly occurs in the south part and 

chemical pesticides were primarily used as the first 

control method (Chen, 1990). However, chemical insecticides 

may poison the non-target organisms (e.g., 

humans, livestock and natural enemy), pollute 

environment, and induce insecticide-resistant pests. 

*Corresponding author. zhangyab@caf.ac.cn. Tel: +86- 

1062889520. Fax: +86-10 62884972. 

These problems proved that the application of chemical 

insecticides to be less than an ideal method to controll D. 

kikuchii (Yasuhisa, 2007). 

The insect virus is one kind of pathogenic 

microorganism in nature and may influence the quantity 

of insect population by causing the epidemic disease of 

insect. It has high host specificity, so that it is safe to 

other non-target species and can maintain dynamic 

balance of insect population for a long term by horizontally 

and vertically spreading epidemic disease in the 

pest population (Fuxa, 1989; Laird et al., 1990; Heinz et 

al., 1995). Moreover, it causes no environmental 

pollution. Therefore, it is realized that the use of virus to 

control pest insects is an ideal and sustainable means. In 

1986, a nucleopolyhedrovirus of D. kikuchiii, named as 

Dendrolimus Kikuchii Matsumura Nuclear Polyhedrosis


Virus (DKMNPV), was isolated and its morphological 

characteristics and toxicity were described, however, D. 

kikuchii larvae were not sensitive to DKMNPV (Yang et 

al., 1986). Therefore, the screening of high toxicity and 

strong stability virus is an imperative work for the 

biological control of D. kikuchii. In present study, a 

nucleopolyhedrovirus was isolated from diseased D. 

kikuchii larvae in Mile county, Yunnan province, China. 

Based on its ultrastructural morphological characteristics 

and the virulence against the third-instar larvae of D. 

kikuchii, it was identified as a new virus and named as 

DkNPV (multiple-nucleocapsid nucleopolyhedrovirus of 

D. kikuchii). This virus exhibited high virulence against 

the third-instar larvae of D. kikuchii, and high potential as 

a biocontrol agent to protect the forest from these insects. 


Virus isolation and purification from D. kikuchii 

Diseased D. kikuchii larvae were collected from P. yunnanensis 

forest in Mile, Yunnan Province, China, and kept frozen at -20°C 

until use. Virus occlusion bodies (OBs) were isolated and purified 

from the larval cadavers according to the method developed by EI- 

Salamouny (1998). Briefly, 200 g larvae cadavers were 

homogenized in 500 ml distilled water and filtered through five 

layers of muslin. The filtrate was subjected to centrifugation 

alternated between low and high speed several times until the clear 

off-white OBs were obtained. For further purification, the obtained 

OBs were centrifuged through sucrose gradient of 40-60% (w/w) at 

10,000 g for 30 min at room temperature. The bands containing 

virus were collected, washed with sterile water, and then 

centrifuged at 12,000 g for 30 min at 4°C with three replications. 

The pellets of purified OBs were stored at 4°C for the next study. 

Ultrastructural studies of virus isolated from D. kikuchii 

As to scanning electron microscopy (SEM), the following method 

was used. Firstly, pellets of purified virus were diluted in distilled 

water and the concentration of virus was adjusted to 10 9 PIB·ml -1 

(polyhedral inclusion bodies/ ml -1 ). Secondly, 20-30 l of viral 

suspensions were mounted on a clear coverslip and dried naturally 

in the ventilate place. After that, the coverslip was placed on an 

aluminous specimen stub and coated with gold. Finally, the 

specimen was observed by using a scanning electron microscope 

(Hitachi 3400N, Japan). One hundred OBs were measured to 

determine their mean size. 

The transmission electron microscopy (TEM) was carried out 

according to the method described by Luft (1961). Briefly, pellets of 

purified OBs were fixed directly in 2.5% glutaraldehyde for 2.5 h, 

dehydrated in a graded ethanol series (30, 50, 70, 80, 90, 100%), 

and then embedded in Epon-Araldite resin. Ultrathin sections were 

cut with ultractome, slice sizes range from 50 - 60 nm, double 

strained with uranyl acetate and lead citrate, and then examined 

with a transmission electron microscope (Hitachi JEM-1230, 

Japan). 

Preparation of D. kikuchii larvae 

Larvae of D. kikuchii were reared from surface-sterilized eggs. 

Briefly, the pupae of D. kikuchii were collected from the P. 

yunnanensis forest and kept in the clear cages (30 × 30 × 40 cm) in 

the laboratory. Once emergence, adults were provided with 50 gl -1 

sugar solution as food source and some fresh shoots of P. armandi 

as an oviposition substrate. Eggs were collected and sterilized with 

10% methanal for 0.5 h. After hatch, larvae were group reared on 

fresh needles of the P. armandi within the clear plastic jars (15 × 15 

× 20 cm) with some holes in the bottom until the third-instars. The 

above experiments were carried out at 20°C, 50-65% relative 

humidity and a 18:6 h (day : night) photoperiod (Guo et al., 2007). 

Infection studies of isolated virus against the D. kikuchii larvae 

The infection activity of isolated virus was tested against the thirdinstars 

larvae of D. kikuchii by following the method developed for 

Cydia pomonella granulovirus (Huber, 1981). The pellets of purified 

virus were diluted in distilled water. Five virus suspensions at 

different concentrations (2.2×10 4 , 2.2×10 5 , 2.2×10 6 , 2.2×10 7 and 

2.2×10 8 PIB·ml -1 ) were prepared respectively and used for this 

infection study. For each concentration, fifty larvae were used and 

300 µl of viral suspensions were sprayed onto pine needles, which 

were used as food of D. kikuchii larvae and filled into a plastic jar 

(20 × 20 × 20 cm) with some holes in the bottom. The tested larvae 

were kept at 20°C, 50-65% relative humidity and a 18:6 h (light : 

night) photoperiod. As soon as the treated needles were eaten, the 

tested larvae were provided with fresh, untreated needles, and 

continuously reared in the plastic jar. Larvae with the typical 

symptoms of NPV infection such as a bloated body, hanging upside 

down from the branches and fragile cuticle were recorded as dying 

of virus infection. Mortality of larvae due to the infection of virus was 

recorded from the third day and up to the 13th day at a one day 

interval. Three repetitions were carried out and as control, pine 

needles were treated with distilled water exactly according to the 

above method. 

Stability studies of isolated virus in toxicity and ultrastructural 

morphology 

The virus has been propagated in the fourth-instars larvae of D. 

kikuchii, for 4 generations. Stability of ultrastructural morphological 

and the virulence of the virus were tested according to the above 

method. 


The median lethal time (LT50), the median lethal concentration 

(LC50) and the toxicity regression equations of D. kikuchii virus were 

estimated by probit analysis software SPSS package 17.0 (Yu and 

He, 2003). Death rate and corrected mortality of insect larvae were 

calculated based on bioassays. 

RESULTS 

Ultrastructural characteristics of D. kikuchii virus 

The SEM results showed that OBs of D. kikuchii are 

mostly polyhedral shape, 0.79~2.31 (1.64 ± 0.1) µm 

[minimum~maximum (mean ±SE)] in diameter (n = 100), 

with a lots of small holes on their surface, 173.00~254.00 

nm × 55.10~116.00 nm in size (Figure 2a). Ultrathin 

sections revealed that each OB contains many virions, 

252.00~359.00 nm × 70.18~200.00 nm in size (Figure

Figure 1. (a) Image showing the conifers damaged by D. kikuchii larvae; (b) OBs in D. kikuchii larvae body fluids, 

and for image (b) the bar represents 20 µm. 

Figure 2. The ultrastucture characteristics of DkNPV. (a) Scanning electron micrograph of the DkNPV OBs; (b) The 

ultrastructural section of OBs showing the virions (V) inside it. Each virion contains multiple nucleocapsids (N); for images a 

and b the bar represents 5.00 µm and 500 nm respectively. 

2b). The virion is rod-shaped shaped with the truncated or obtuse 

ends, consists of multiple nucleocapsids (up to 9) within a 

single viral envelope (Figure 2b). b). The size of nucleo- 

capsid is approximately 25.80 ± 0.86 nm (mean ±SE) in 

width and 265.00 ± 12.66 nm in length (n = 50) (Figure 

2b). 

Yang et al. 2263 

Based on the ultrastructural observation, the 

morphological characteristics aracteristics and the virulence of the D. 

kikuchii virus was identified as a new virus in 

nucleopolyhedrovirus of Baculoviridae, and named as 

DkNPV according to the universal system of virus 

taxonomy in the 8th ICTV report (Hong, 2006).


Table 1. The virulence of DkNPV on 3 rd instars larvae of D. kikuchii. 

Concentration (PIB·mL -1 ) Regression equation Correlation coefficient LT50(d) LC50 (PIB·mL -1 ) 

2.2×10 8 Y=10.457x+9.219 0.976 6.89 1.72×10 5 

2.2×10 7 Y=10.051x+8.156 0.986 7.18 — 

2.2×10 6 Y=8.633x+5.487 0.947 8.16 — 

2.2×10 5 Y=6.536x+2.237 0.980 10.31 — 

2.2×10 4 Y=6.039x+0.890 0.988 11.13 — 

Table 2. The difference between DkNPV and DKMNPV. 

Virus name OBs surface Both ends of rod-shape 

The size of nucleocapsids 

(nm) 

Diameter 

(µm) 

LC50 

(PIB·mL -1 ) 

DkNPV Lots of small holes Truncate or obtuse ends 265.00 ± 12.66 × 25.80 ± 0.86 0.79~2.31 1.72×10 5 

DKMNPV The even surface Obtuse both ends only 410 ± 10 × 40 ± 3 1.3~3.0 1×10 5.9 

Infection studies of DkNPV against the D. kikuchii 

larvae 

The results of infection study showed that DkNPV could 

infect and kill the third-larvae of D. kikuchii (Table 1). LC50 

was 1.72 × 10 5 OBs·ml -1 . At the tested five concentrations 

(from 2.2 × 10 8 to 2.2 × 10 4 PIB·ml -1 ), LT50 were 6.89, 

7.18, 8.16, 10.31 and 11.13 days, respectively (Table 1). 

A large number of D. kikuchii larvae begin to die from the 

seven day of infection, and peak mortality from 7 to 10 

days. A large number of virus was found in D. kikuchii 

larvae with the typical symptoms of NPV infection by 

optical microscope (Figure 1b). Therefore, DkNPV is 

virulent to the third-instar larvae of D. kikuchii. 

Our research demonstrates that the virus has been 

propagated 4 generations and the fourth-instars larvae of 

D. kikuchii has stable ultrastructural morphological and 

virulence. 

DISCUSSION 

In present study, a nucleopolyhedrovirus was isolated 

from D. kikuchii. The main morphological characteristics 

of this virus is consistent with the NPVs. When it was 

compared with the reported D. kikuchii virus DKMNPV, 

several significant differences were observed between 

them (Table 2). It is interesting that lots of small holes 

were observed on the surface of DkNPV. Firstly, we 

thought they were caused by the process of sample 

preparation. Other four samples (HcNPV, BusuNPV, 

DnNPV and CzNPV) therefore were prepared exactly 

according to the same method. However, no holes were 

observed on their OBs surface. One difference lied in 

small holes on the surface of DkNPV and the even 

surface of DKMNPV. The second difference existed on 

the both ends of rod-shape virions. DkNPV had truncate 

or obtuse in both ends, but the DKMNPV had obtuse both 

ends only. The third difference was that the size of 

nucleocapsids of DkNPV was smaller than DKMNPV 

(265.00 ± 12.66 nm × 25.80 ± 0.86 nm of DkNPV, 410 ± 

10 nm × 40 ± 3 nm of DKMNPV). Besides, the diameter 

of OBs differed widely, the diameter of DKMNPV was 

1.3~3 µm, but the diameter of DkNPV was 0.79~2.31 µm. 

The shape and size of OBs, the size of nucleocapsids 

and the features of both ends of visions were the 

important basis for the identification of a baculoviruses 

(Xie and Hu, 2000). The fourth difference shows the 

DkNPV is more virulent than DKMNPV against the larvae 

of D. kikuchii. Based on the morphological characteristics, 

the virulence of this virus and the difference between 

DkNPV and DKMNPV, it was proposed as a new strain 

nuclepoyhedrovirus of D. kikuchii and named as DkNPV. 

In the next step, the structure of its nucleic acid and 

biological characteristics will be further studied to confirm 

its identification. Dendrolimus cytoplasmic polyhedrosis 

virus (CPV) were used to biologically control the pest of 

Dendrolimus in China since the first DpCPV (D. 

punctatus Cytoplasmic Polyhedrosis virus) was isolated 

in China in 1973. However, as is well known the time 

required for CPVs to kill the insect larvae was longer than 

that for NPVs obviously. The new isolated DkNPV was 

virulent for the third-instar larvae of D. kikuchii and 

exhibited high infection activity (Table 1). Therefore, this 

virus was very precious for the biological control of D. 

kikuchii. 

ACKNOWLEDGMENTS 

We sincerely thank Yan-Li Duan and Jian-Jie Xue for 

assistance both in field research and virus propagation; 

Chun-Yan Wang for suggestions of revising the 

manuscript. This work was supported financially by 

National Natural Science Foundation of China (No. 

30671688), and Forestry Industry Special Foundation of

China (No. 200904029; No. 200704003). 

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Characterization of Staphylococcus spp strains 

isolated from hospital, community and environmental in 

Puebla city, Mexico 

Ana Marta de los Ángeles Lobo-Sánchez 1 , Patricia Lozano-Zaraín 2 , 

Ygnacio Martínez-Laguna 1,2 , Zita Gutiérrez-Cázarez 3 , Antonio Rivera-Tapia 2 , Carmen Torres 4 

and Rosa del Carmen Rocha-Gracia 1,2 * 

1 Posgrado en Ciencias Ambientales, Área de la Salud, Centro de Investigaciones en Ciencias Microbiológicas, Instituto 

de Ciencias, Benemérita Universidad Autónoma de Puebla. Complejo de Ciencias, edificio No. 103 J. Ciudad 

Universitaria, colonia San Manuel, Puebla 72570, México. 

2 Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de 

Puebla. Complejo de Ciencias, edificio No. 103 J. Ciudad Universitaria, colonia San Manuel, Puebla 72570, México. 

3 Hospital para el Niño Poblano, Blvd. del Niño Poblano No. 5207 Col. Concepción la Cruz, 72190 Puebla, Pue., México. 

4 Área Bioquímica y Biología Molecular, Universidad de la Rioja, Logroño, Spain. 


We investigated the prevalence of methicillin-resistant staphylococci (MRS) and evaluated the 

antimicrobial resistance patterns of 284 Staphylococcus strains isolated from two hospitals, as well as 

from community and the environment in Puebla City, Mexico. Isolates were identified by Gram-stain and 

biochemical reactions and antimicrobial susceptibility testing was performed by Kirby-Bauer and in 

some cases by determination of minimal inhibitory concentrations of antimicrobial agents. Result 

showed that from 284 strains studied, 32% (90 strains) were multidrug-resistant, of which 82% (74 

strains) were methicillin-resistant. From 154 Staphylococcus aureus isolates tested, 41 strains showed 

methicillin-resistance (27%) and 84 of 130 coagulase-negative staphylococci showed methicillinresistance 

(65%). The mecA gene was detected in 38 of 43 oxacillin-resistant staphylococci tested 

(14/14 S. aureus and 24/29 coagulase negative staphylococci). Nevertheless, this gene was also 

identified in 14 S. aureus that exhibited oxacillin and cefoxitin susceptibility. The percentages of 

resistance detected among S. aureus and coagulase negative staphylococcal isolates were: penicillin 

(79 and 67%, respectively), oxacillin (27 and 65%), erythromycin (18 and 36%), tetracycline (6 and 24%), 

gentamicin (71 and 45%), and trimethoprim sulfamethoxazole (19 and 37%). The �-lactamase production 

was positive in more than 80% of isolates. These results show the presence of multiresistant strains in 

these three sources, which supports the control measures taken by health authorities with respect to 

avoiding the misuse and abuse of antibiotics. 

Key words: Staphylococcus, methicillin resistance Staphylococcus aureus (MRSA), methicillin resistance 

coagulase-negative staphylococci (MRCoNS), emerging infections. 

INTRODUCTION 

In the 60's, Staphylococcus aureus was identified as the 

*Corresponding author. E-mail: rochagra@yahoo.com. Tel: 00 

(52) 2222295500 ext 2546. 

main cause of nosocomial infections in the world (Klimek 

et al., 1976; Crossley et al., 1979; Doebbeling, 1995). At 

present, the methicillin-resistant Staphylococcus (MRS) 

and multiresistant Staphylococcus are the leading cause 

of infections in hospitals and community (Schaberg et al., 

1991; Ponce de León, 1996; Diekema et al., 2000; Wu et

al., 2006; Martins et al., 2007; David and Daum, 2010). 

Resistance to methicillin is due to the presence of 

Staphylococal Chromosomal Cassette mec (SCCmec) 

which in addition to carrying mecA gene that encodes a 

penicillin-binding protein (PBP2a) (Hartman and Tomasz, 

1984; De Lencastre et al., 1994; Pinho et al., 2001), also 

can contains genes of resistance for non-beta-lactam 

agents, causing multidrug resistant strains (Katayama et 

al., 2000; Hiramatsu et al., 2001). 

It is reported that S. aureus is the causal agent of more 

than 20% of bacteremias in USA, Canada and Latin 

America (Pfaller et al., 1998; 1999; Diekema et al., 2000; 

Wisplinghoff et al., 2003; Chen et al., 2010), skin 

infections and soft tissue (Gales et al., 2009; Doern et al., 

1999) and pneumonia (Sader et al., 1998; Gales et al., 

2009). Coagulase-negative staphylococci (CoNS) are the 

third most common type of isolates in bacteremias 

(Schaberg et al., 1991; Edmond et al., 1999; Diekema et 

al., 2001; Wisplinghoff et al., 2003; Chen et al., 2010, 

Rosa et al., 2009). In the environment, staphylococci are 

the most frequently isolated bacteria in both open and 

closed areas (Górny et al., 1999; Górny and Dutkiewicz, 

2002; Tsai and Macher, 2005; Eames et al., 2009; Tang, 

2009). 

The emergence of multidrug-resistant staphylococci, 

prompted a warning from the health centers and 

organizations, because epidemiological changes, clinical 

manifestations and their control can become a significant 

Public Health problem in both developed and developing 

countries. In Latin America, it was reported a prevalence 

of 40% of methicillin resistant Staphylococcus aureus 

(MRSA) and over 80% methicillin resistant coagulase 

negative Staphylococcus (MRCoNS) and most of these 

strains were multidrug resistant (Sader et al., 2009). The 

World Health Organization (WHO) has seen the 

emergence and spread of antimicrobial resistance as a 

priority issue and therefore since September 2001 

established a global measure for the containment of 

antimicrobial resistance, which includes as a fundamental 

measure the surveillance of antimicrobial resistance 

caused among other factors by self-medication (WHO, 

2002). In Mexico, a law was issued in which the 

guidelines to which the sale and dispensing of antibiotics 

as a preventive measure to misuse of these drugs would 

be subjected (Official Gazette, 2010). 

The aim of this study was to determine the methicillin 

resistance and other associated resistances of 

staphylococci strains isolated from two general hospitals, 

the community and the environment of the city of Puebla, 

Mexico. 


Bacterial isolates 

284 staphylococci were included in this study. One-hundred-andseventeen 

strains were obtained in two hospitals of Puebla, México 

(Pediatric Hospital in 2003; University Hospital of Puebla City in 

Lobo-Sánchez et al. 2267 

1998-2000) and they were collected from wound and articulation 

secretions, sepsis, pleural and cerebrospinal fluids, peritoneal 

dialysis fluids, catheter tips, post-mortem and throat swabs. Onehundred-and-twelve 

strains were obtained from community during 

2002-2003 and they were collected from outpatients with clinically 

reported staph infections (vulvar swabs, throat, skin and conjunctiva) 

as well as from hospital personnel and community carriers by 

means of throat swabs. By other side, 55 strains from the environment 

were collected in 2004, through the Gravity Sedimentation 

Technique (Frankland and Hart, 1887); specifically, blood agar and 

mannitol salt agar plates, were exposed for a period of approximately 

15 and 45 minutes respectively, at a height of between 1.5 

and 2.0 m (Lighthart and Shaffer, 1995; De la Rosa and Ullán, 

2002; Rosas et al., 2004). Staphylococcus strains were identified by 

standard methods, such as colonial morphology, Gram-stain, 

catalase, coagulase, clumping factor, urease, DNase, hemolysis on 

blood agar and bacitracin test (Finegold and Baron, 1996). 

Antimicrobial susceptibility test and �-lactamase production 

All obtained staphylococci were tested by Kirby-Bauer test (CLSI, 

2008) for susceptibility to the following antimicrobials oxacillin (OX, 

(1 �g/disk), penicillin (PE, 10 U), erythromycin (E, 15 �g), 

tetracycline (TE, 30 �g), gentamicin (GE, 10 �g), vancomycin (VA, 

30 �g) and trimethoprim sulfamethoxazole (SXT, 25 �g) (Sanofi 

Diagnostics Pasteur, SA, Mexico City, Mexico). Mueller-Hinton agar 

(Oxoid, Cambridge, England) supplemented with 4% NaCl was 

used in these assays. The susceptibility of cefoxitin (FOX, 30 

�g/disk) (Difco Laboratories, Detroit, MI, USA) was checked in a 

group of mecA-positive strains (n=14) that appeared as oxacillinsusceptible. 

Additionally, a minimum inhibitory concentration (MIC) 

test of oxacillin using the agar dilution method with Steers 

Replicator was performed for 90 staphylococci isolated from 

different origins (30 from each source). The results of disk diffusion 

tests and MICs were interpreted according to CLSI criteria (CLSI, 

2008). 

The �-lactamase production was performed by Cefinase disks 

(Becton Dickinson) according to manufacturer's instructions. 

mecA gene amplification by polymerase chain reaction (PCR) 

Specific PCR of the mecA gene was performed in 90 staphylococci 

from different origins that were also analyzed by MIC. Because the 

content of A + T in mecA gene is high (70%) and in order to 

minimize the amplification of DNA regions not related to the gene 

(Ubukata et al., 1990; Ryffel et al., 1990; Unal et al., 1992), we 

chose two sets of primers combined in two reactions, the sense 

primer P1, 5'-(911) GGTCCCATTAACTCTGAAG (929)-3' and 

antisense P3, 5'-(1956) AGTTCTGCAGTACCGGATTTGC (1935)-3' 

(Petinaki et al., 2001) and a pair designed in this study, MMecAF 

sense primer, 5'-(539) TCCAGAATGCAGAAAGACC (558)-3'and 

antisense MMecAR, 5'-(1076) TGTATGTGCGATTGTATTGCTATT 

(1053)-3', giving rise to PCR products of 1046 and 538 bp 

respectively. 

RESULTS 

From 284 isolates studied, 154 of them were identified as 

S. aureus (54%), isolated mostly from hospital and 

community sources and 130 isolates (46%) were CoNS, 

mainly obtained from environment and hospital sources. 

Methicillin-resistance in this study was firstly checked by 

the study of the susceptibility to oxacillin. Table 1 shows


Table 1. Species distribution and methicillin resistance (MR) in Staphylococcus strains from three sources analyzed. 

Origin 

(n isolates) 

Studied isolates 

a Methicillin-resistance 

OXA/FOX-susceptible 

mecA-positive S. aureus 

S. aureus CoSCN S. aureus (%) CoSCN (%) 

Hospital (117) 71 46 24 (34) 36 (78) 5 

Community (112) 80 32 17 (21) 15 (47) 6 

Environmental (55) 3 52 0 33 (63) 3 

Total: 284 154 (54%) 130 (46%) 41 (27) 84 (65) 14 

CoSCN: coagulase negative staphylococci; OXA: oxacillin; FOX: cefoxitin 

a Isolates included were those that presented oxacillin resistance (by Kirby Bauer or MIC). 

Figure 1. mecA gene detection by PCR, amplified in agarose gel and subjected to electrophoresis. A. P1F-P3R 

primers (Petinaki et al., 2001) to amplify 1046 bp. Line 1: Marker �X174; 2, 3-5, 7: mecA positive strains; 6: mecA 

negative strain and 8: ATCC 25923 (negative control). B. MmecAF-MmecAR primers to amplify 538 bp. Line 1: Marker 

�X174; 2, 3-8, 10 and 11: mecA positive strains; 9, 12 and 13: mecA negative strains and 14: ATCC 25923 (negative 

control). 

the percentage of methicillin resistance among our 

isolates using this criterium. Forty-one S. aureus isolates 

of the 154 tested showed methicillin-resistance (27%), 

and isolates of hospital origin showed higher percentage 

of resistance than those of the community (34 and 21%, 

respectively). On the other hand, 84 of 130 coagulasenegative 

staphylococci showed methicillin-resistance 

(65%) and percentages were higher among hospital and 

environmental isolates (78 and 63%, respectively) (Table 

1). 

The presence of mecA gene was analyzed in a group 

of 90 staphylococci that included isolates of the three 

sources and also isolates with different oxacillin susceptibilities 

(Figure 1). The mecA gene was detected in 38 of 

43 oxacillin-resistant staphylococci tested (14/14 S. 

aureus and 24/29 coagulase negative staphylococci, data 

not showed). Nevertheless, this gene was also identified 

in 14 S. aureus that exhibited oxacillin and cefoxitin 

susceptibility, observing a phenotypic-genotypic discrepancy 

(Table 1). From 90 selected strains and tested by 

�-lactamase production it was found that 100% hospital 

isolates, 83% community isolates and 93% environmental 

isolates were positive. 

Figure 2 shows the percentages of resistance to 

different antimicrobial agents of S. aureus and 

coagulase-negative staphylococci. All strains tested were 

resistant to two or more agents and it was considered as 

multidrug-resistant strains those that were resistant to 

more than four antibiotics. From 284 strains studied, 32% 

(90 strains) were multidrug-resistant, of which 82% (74 

strains) were methicillin-resistant. The percentages of 

resistance detected among S. aureus and coagulase 

negative staphylococcal isolates were: penicillin (79 and 

67%, respectively), oxacillin (27 and 65%), erythromycin 

(18 and 36%), tetracycline (6 and 24%), gentamicin (71 

and 45%), and trimethoprim sulfamethoxazole (19 and 

37%). 

DISCUSSION 

As expected, the percentages of methicillin resistance 

detected among S. aureus and CoSCN in this study were 

higher in the hospital isolates in relation to those of the

Figure 2. Resistance staphylococci strains isolated from Hospital, Community and 

Environmental by Kirby-Bauer test. PE: penicillin; OX: oxacillin; E: erythromycin; TE: 

tetracycline; GE: gentamicin; SXT: trimethoprim-sulfamethoxazole and VA: vancomycin. 

RSA, Resistant Staphylococcus aureus strains; RCoNS, Resistant Coagulase Negative 

Staphylococcus strains. Intermediate Resistance: a GE SA 8% and CoNS 6%; b TE CoNS 2% 

and c SXT 1%. 

community. Hospital isolates presented high percentages 

of resistance (34 and 78% for S. aureus and CoSCN, 

respectively), what indicate that this type of resistant 

microorganisms can be a problem in the hospitals tested. 

These percentages are similar to other data previously 

published for Mexican hospitals and in other Latin 

American countries (Guzmán-Blanco et al., 2009). 

The results of this study show that in our community 

Staphylococcus is a major concern for public health as a 

cause of emerging infections, due to the presence of 

multiresistant strains with ability to survive and spread. 

Among 284 strains collected in this study, the highest 

percentage was S. aureus isolated from community, 

which is consistent with that reported by the Centers for 

disease control and prevention (CDC) in USA (Kuehnert 

et al., 2006) and the Instituto Mexicano del Seguro Social 

(IMSS) in México (García-Contreras et al., 2000). The 

SCoN were isolated mostly from environmental sources, 

probably because they are found in greater numbers in 

skin and mucous membranes of both human animals as 

compared with S. aureus (Bischoff et al., 2004; 2007). 

The resistance rates observed in this study agree with 

those reported in other regions (Diekema et al., 2001; 

Cuevas et al., 2004; Gales et al., 2009; Sader et al., 

2009) and may be related to selective pressure to which 


bacteria are subjected in our community, because in the 

hospitals the committee epidemiological surveillance sets 

that the treatment of staphylococcal infections begins 

with �-lactam antibiotics, which in some cases, antimicrobial 

therapy of choice is subject to the standard antibiotic 

treatments available or unfinished treatments due to lack 

of resources of both health institutions and patients. In 

community, easy access to this type of antibiotics until 

2010 year (Official Gazette, 2010) allowed the selfmedication, 

resulting in inadequate doses or unfinished 

treatments (Dreser et al., 2008), favoring the spread of 

multiresistant strains in the community. 

The GE resistance above 45%, consistent with that 

reported in 2005 by Public Health in Mexico (Benavides- 

Plascencia et al., 2005), where GE is among the seven 

antibiotics account for 80% of the observed resistance. In 

recent years, glycopeptides have been the mainstay of 

treatment of infections due to MRS. However, the isolation 

of CoNS with decreased susceptibility (Schwalbe et 

al., 1987), the report heteroresistant S. aureus strains 

(Hiramatsu et al., 1997) and the presence of strains 

resistant to VA (CDC, 2002; Palazzo et al., 2005; Tiwari 

and Sen, 2006), led that the treatment options for 

infections by MRS are committed and required of new 

antimicrobial agents (Appelbaum, 2006; Menezes et al.,


2008). In this paper, strains with intermediate susceptibility 

to VA were not identified however; the results 

emphasize the need for continuous monitoring of the 

levels of MIC to this antibiotic in the MRS, since these 

results alert of the risks and implications of the indiscriminate 

use of this drug. The E and SXT has been widely 

used in the treatment of staphylococcal infections 

(Huovinen et al., 1995; Schlegelová et al., 2002), 

however, clinical experience has shown that resistance to 

both drugs has developed worldwide (Huovinen, 2001; 

Gales et al., 2009; Sader et al., 2009). The TE is a 

relatively cheap antibiotic, has therefore been widely 

used in prophylaxis, treatment of infections and as animal 

growth promoter, so the selective pressure for its use, 

has led to resistant bacterial strains (Committee on Drug 

Use in Food Animals, 1999; Chopra and Roberts, 2001). 

The worldwide prevalence of resistance to TE in S. 

aureus is higher than CoNS (Diekema et al., 2001; Gales 

et al., 2009). However, in Spain was reported a 

resistance less than 5% in S. aureus and between 5% 

and 15% in CoNS (Pérez-Trallero and Iglesias, 2003). 

The TE resistance observed in this study is relatively low 6% 

for S. aureus and 24% for CoNS, which could be related 

to the TE is one of the lowest drug consumption in 

Mexico (Benavides-Plascencia et al., 2005) and is not 

considered a treatment of choice for serious 

staphylococcal infections (WHO, 1999). It is interesting 

intermediate susceptibility to GE, TE and SXT, while not 

reaching high levels, confirming the need for selection 

and rational use of these antibiotics. 

The MRSA and MRCoNS mecA positive showed 

uniformity in the expression of resistance (Chambers, 

1988), so that might be considered strains with homogeneous 

resistant (Weller, 1999). In the MSSA-mecA 

positive, the results confirmed with FOX could coincide 

with that reported by Hososaka et al. (2007), so the 

absence of the phenotypic expression of resistance in 

these strains, suggests new molecular targets that could 

be related to susceptibility to ME (Rohrer et al., 2003), or 

the presence of mutations in the fem genes, which are 

essential factors for ME resistance expression 

(Chambers, 1997), contributing to low levels of resistant 

to this antibiotic, without any alteration in the mecA gene 

(Giannouli et al., 2010). In MRCoNS-mecA negative, the 

expression of resistance to ME, could be related to 

different mechanisms for the production of PBP2a 

(McDougal and Thornsberry, 1986; Tomasz et al., 1989; 

Suzuki et al., 1993; Weller, 1999), resulting in the 

presence of extremely heteroresistant strains (Chambers, 

1988). The MSCoNS-mecA positive strains reported as 

MR by test MIC, could be pre-MRCoNS (Hiramatsu, 

1995), because mutations in regulatory genes of mecA 

would originate phenotypically methicillin resistant strains 

(Suzuki et al., 1993; Kobayashi et al., 1996; 1998). 

In most of the strains tested the �-lactamase production 

was detected, suggesting that the regulatory genes blaR1 

and blaI could also be related to the phenotypic 

expression of resistance (Cohen et al., 1972; Boyce et 

al., 1990; Hiramatsu et al., 1990; Ryffel et al., 1992; 

Hackbarth and Chambers, 1993; Hackbarth et al., 1994; 

Chambers, 1997; Rosato et al., 2003). 

Finally, the results of this study confirm the presence of 

MRS strains, which are also multiresistant, whose 

mechanisms of resistance may not only be related to the 

interaction of the mec genes, but with much more 

complex regulatory mechanisms, so its worth conducting 

more detailed studies and further epidemiological 

surveillance of Staphylococcus strains isolated from 

different sources, to show the behavior over time of 

resistance in these strains, based on the measure 

introduced from August 2010 on the sale of antibiotics in 

pharmacies throughout Mexico through prescription 

(Official Gazette, 2010). 


Ana Marta de los Ángeles Lobo Sánchez was supported 

by a student scholarship from Consejo Nacional de 

Ciencia y Tecnología, México (CONACYT 174306). 

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DOI: 10.5897/AJMR11.590 



Interaction of severe acute respiratory syndrome 

(SARS) nucleocapsid protein with macrophage 

migration inhibitory factor protein (MIF) 

Yang Lin 1,2 *, Baohua Wang 3 *, Yijun Liu 1 , Zhigang Yu 1 , Manhua Cui 2 and Haichun Ma 1 

1 The First Hospital of Jilin University, Changchun130021, China. 

2 Department of Gynaecology and Obstetrics, the Second Hospital of Jilin University, Changchun 130041 China. 

3 Department of Anesthesia, the Affiliated Hospital of Changchun University of Traditional Chinese Medicine, 

Changchun 130021, China. 


The nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) is a major structural component of 

virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. 

However, how N protein interacts with host protein remains largely elusive. To identify cellular proteins 

that interact with the N protein and to elucidate the possible involvement of N protein in SARS-Cov 

replication and translation, a human lung cDNA library was screened using a yeast two-hybrid system 

assay. In this study, we have identified Macrophage migration inhibitory factor protein (MIF) as a novel 

interaction partner of N protein by yeast two-hybrid system. The direct interaction and co-localization of 

N protein with MIF were confirmed by immunoprecipitation and confocal microscopy analysis, 

respectively. The mapping studies localized the critical N sequences for this interaction to amino acid 

140-210 including SR motif by yeast two-hybrid system. To the best of our knowledge, this is the first 

report that SARS-N protein interacts with the MIF within host cells, which enhance our understanding of 

the molecular mechanisms of SARS replication. 

Key words: Severe acute respiratory syndrome (SARS) coronavirus, nucleocapsid protein, Macro|phage 

migration inhibitory factor protein, yeast two-hybrid. 

INTRODUCTION 

Severe acute respiratory syndrome (SARS) is a novel 

worldwide infectious disease, which is caused by a newly 

identified member of the coronaviridae family, SARS 

coronavirus (SARS-CoV) (Ksiazek et al., 2003; Marra et 

al., 2003; Peiris et al., 2003; Rota et al., 2003). Like other 

known coronaviruses, SARS-CoV is an enveloped virus 

containing three outer structural proteins, namely the 

membrane (M), nucleocapsid (N), and spike (S) proteins 

(Lai et al., 1997; Versteeg et al., 2007). The N protein is 

the most abundant viral protein in coronaviruses which is 

produced throughout infection and is an important 

multifunctional protein. Several functions have been postulated 

for the coronavirus N protein throughout the 

*Corresponding author. E-mail: Haichun514@yahoo.cn. Fax: 

+86-0431-845-14166. 

virus life cycle, including viral packaging, viral core 

formation, and signal transduction (He et al., 2004; 

Hiscox et al., 2001). 

Mapping virus–host protein interactions can provide 

important clues on the initial stages of infection. For coronaviruses, 

the N protein plays an important role during 

host cell entry and virus particle assembly and release 

(Hatakeyama et al., 2008; Narayanan et al., 2001). It has 

been reported that the N protein of SARS interacts with 

human cellular heterogeneous nuclear ribonucleoprotein 

A1 (hnRNPA1) (Wang et al., 1999), which shown that N 

protein plays a crucial role in viral RNP assembly and 

replication by interaction with host proteins. However, 

how N protein interacts with host protein is still unclear. 

To obtain a more detailed insight into the N protein and 

host relationship, a yeast two-hybrid system was 

employed in the present study to identify host proteins 

which bind to N protein of SARS.



Strains and general techniques 

The strain of Saccharomyces cerevisiae used in this study was 

AH109 from Clontech (USA). Yeast cells were cultured at 30°C 

either in a complete YPD medium (1% yeast extract, 1% peptone, 

2% glucose) or in a synthetic defined (SD) medium supplemented 

with required essential nutrients. Plates contained 2% agar. 

Transformation of yeast cells was performed by the lithium acetate. 

Escherichia coli KC8 were used for general cloning. DNA manipulation 

was performed according to established protocol. Betagalactosidase 

assays were carried out according to the 

CLONTECH Matchmaker manual (PT3024-1, Clontech, USA). 

Plasmids and construction of recombinant vectors 

For bait construction, the full-length N gene of the SARS was PCRamplified 

from a genomic construct of clone, and cloned into the 

pMD18-T vector (Takara, China). The full-length N gene was 

subjected to DNA sequencing, and the inserts were verified against 

the corresponding region of the SARS coronavirus. The full-length 

N gene was excised from the pMD18-T –N construct using the 

restriction enzymes EcoRI and BamHI, and ligated into the pGBKT7 

vector to generate an N-terminal in frame fusion with the GAL4 

activation domain (BD), and the resultant plasmid was named as 

pGBKT7-N. To identify the putative domain of amino acid sequence 

required for MIF/N interaction. The truncated mutants N1–139, 

N140–280, N281–422, N140–210 and N211–280 were subcloned 

into the yeast two-hybrid vector pGBKT7. The MIF gene was 

obtained by PCR from the rat liver cDNA library, and subcloned into 

the yeast vector pGADT7. For mammalian cell expression, the fulllength 

N gene and MIF were subcloned into the pCMV-Myc vector 

(Clontech, USA) and pCMV–HA, and fluorescence vector pEGFP- 

N1 and pDsRed-N1, respectively. All DNA manipulations were 

performed as described by Sambrook et al. (2001). All constructs 

were verified by restriction digestion and sequencing. 

Yeast two-hybrid screening 

Yeast two-hybrid experiments were performed as described in the 

Clontech manual for the MATCHMAKER GAL4 two-hybrid system 

and in the Clontech yeast protocols handbook (Clontech, USA). 

The rat liver Matchmaker cDNA library (Clontech, USA) cloned in 

frame with the GAL4 activation domain in the PGAD vector was 

used as a prey. The bait pGBKT7-N was transformed into yeast 

strain AH109. The bait construct did not show any toxic effect and 

autonomous transcriptional activation on the host strain. The prey 

PGAD-library was then transformed into the bait-transformed 

AH109 cells, and the cells were incubated on minimal synthetic 

dropout medium for yeast (SD)/-His/-Leu/-Trp at 30°C. The fresh 

growing clones were assayed for -galactosidase activity by replica 

plating the yeast transformants onto Whatman filter papers; the 

filters were snap-frozen in liquid nitrogen for 10 sec twice and 

incubated in a buffer containing 5-bromo-4-chloro 3-indolyl- -Dgalactopyranoside 

solution at 30°C for 1–8 h. Positive interactions 

were detected by the appearance of blue clones. The liquid - 

galactosidase activity was determined using the substrate ONPG as 

described standard Protocols Handbook (PT3024-1, Clontech, 

USA). Data for quantitative assays were collected for yeast cell 

number and are the mean ± S.E.M. of triplicate assays. Appropriate 

positive/negative controls and buffer blanks were used. The positive 

pGAD-cDNA plasmids were isolated from positive yeast transformants 

by culture in leucine-deficient medium, which resulted in 

spontaneous loss of the plasmid pGBK-N and transformed into E. 

coli KC8 for sequence analysis. Auto sequencing assay was 

performed in Takara Company (China) and the resulting sequence 

was analyzed in the database of EMBL\Gene Bank by the BLAST 

program. 

In vivo co-immunoprecipitation and western blotting 

To reaffirm the results observed from yeast two-hybrid assays, another 

independent assay, co-immunoprecipitation was carried out. 

Vero cells were co-transfected with the plasmids expressing pCMV- 

Myc-MIF and pCMA-HA-N using the Iipofectamine TM transfaction 

reagent (Invitrogen, USA). At 48 h post-transfection, cells were 

washed with PBS and then lysed in lysis buffer. Cell lysate was 

then mixed with anti-HA magnetic microbeads for 30 min on ice. 

100 l of 10% suspension of protein A–Sepharose was then added 

to the samples. The mixture was allowed to shake for 1 h at 4°C, 

after following which the beads were washed four times in lysis 

buffer, and protein was eluted in 2×SDS dye by boiling the sample 

for 5 - 10 min. Samples were resolved by 10% SDS-PAGE and 

transferred to a nitrocellulose membrane. The membrane was 

blocked using 0.5% BSA in PBST for 1 h, and incubated overnight 

with anti-Myc antibodies (1:1000; Clontech, USA). The blot was 

then washed three times in PBST, incubated with anti-mouse IgG 

HRPO for 1 h and washed three times in PBST and the proteins 

were visualized with 5-bromo-4-chloro-3-indolyl phosphate and 

nitroblue tetrazolium. The Vero cells transected with the pCMV-Myc 

and pCMV-HA vectors were used as negative controls. 

Confocal microscopy 

Hela cells were grown on coverslips in a 6-well chamber and 

simultaneously transfected with the recombinant fluorescence 

plasmids pEGFP-N and pDsRed-MIF. After 24 h transfection, the 

cells were washed with PBS three times and fixed in 4% paraformaldehyde 

for 20 min at room temperature. The coverslips were 

then washed with PBS and mounted. Intracellular localization of the 

N protein and MIF was observed under a Leica confocal 

microscope (Germany). 

RESULTS 

Yeast two-hybrid identified MIF as an N interacting 

protein 

A yeast two-hybrid approach was used to identify host 

proteins that interact with N protein. For this purpose, the 

complete N gene was cloned into a two-hybrid bait 

expression vector. The resulting Gal4-N fusion protein 

was used to screen a human lung cDNA library. Approximate 

2.2 × 10 6 transformants were screened for His-Ade- 

Trp-Leu independent growth and blue colony formation in 

the -galactosidase assay. Thirty-six positive clones were 

obtained. As some AD-cDNA fusion products can activate 

reporter gene transcription without interacting with 

the BD-N fusion protein, this false-positive clone can be 

identified using the technique of segregation analysis. 

Only 4 of 36 clones survived all genetic tests and were 

considered to be genuine positive clone, DNA sequence 

analysis of the fragment revealed that the four cDNA 

fragments inserted have a high identity with four genes in 

the GeneBank database. One of these clones was identified 

as MIF, which encodes a 158-amino acid protein.

Figure 1. MIF protein was immunoprecipitated with the MHV-N protein. Indicated 

plasmids were simultaneously transfected into Vero cells. Twenty-four hours after 

transfection, coimmunoprecipitation was per-formed using anti-HA magnetic 

microbeads, the proteins immuno-precipitated (IP) were assayed with an anti-myc 

monoclonal antibody. Cell lysates were immunoblotted (IB) with anti-Myc to confirm 

the expression of the object proteins. M: MIF protein; N : N protein. 

Lin et al. 2275 

Figure 2. Co-localization of the N protein and MIF. pEGFP-N (green) and pDsRed-RACK1 (red) were co-transfected into 

HeLo cells. After 24 h, cells were fixed, mounted, and the localization of the proteins was observed with a Leica confocal 

microscope. As shown, the N protein and MIF were colocalized in the cytoplasm. 

As shown in Figure 3B, the protein encoded by the 

pGAD-MIF clones interacted specifically with the N 

protein and did not interact with the unfused GAL4-BD 

protein expressed from the parental pGBKT7 vector. 

Co-immunoprecipitation determined the interaction 

of the N protein and MIF 

To confirm the specific interaction between N protein and 

MIF, co-immunoprecipitation was performed. The N protein 

was fused at the amino terminus with a Myc-tag, and 

MIF was fused at the carboxyl terminus with a HA tag. 

The two plasmids were cotransfected into Vero cells and 

immunoprecipitated. The immunoprecipitated complexes 

were separated on SDS–PAGE, and analyzed by Western 

blot with anti-Myc monoclonal antibodies. As shown 

in Figure1, the Myc-fused N protein immunoprecipitates 

with HA-MIF. However, it does not immunoprecipitate 

with HA alone. These findings indicate that N protein 

interacts with MIF in mammalian cells 

Confocal microscopy assay the N protein and MIF 

To further confirm the interaction between N and MIF 

proteins, the localization patterns of the N protein and 

MIF were investigated in Vero cells using confocal microscopy 

technology. pEGFP-N and pDsRed-MIF were 

transfected simultaneously into Vero cells. As shown in 

Figure 2, MIF and N protein mainly were localized in the 

cytoplasm. The merged image revealed that the N protein 

and MIF co-localized in the cytoplasm of Vero cells indicating 

that N protein interacts with MIF in cells.


MIF+N281-422 

MIF+N211-280 

MIF+N140-210 

MIF+N140-280 

MIF+N1-139 

MIF+N 

Negative 

Postive 

0 20 40 60 80 100 

Figure 3. Mapping the interaction domain of N protein. The 

empty vectors pGBKT7 and pGADT7 co-transformed were used 

as the negative control and the pGBKT7-53 and pGADT7- T cotransformed 

were used as the postive con-trol. Every 

experiment was repeated for at least three times and the data 

were obtained by average. The error bars represent standard 

error of the mean. -galactosidase activity assay. 

Mapping the MIF binding region of the N Protein by 

yeast two-hybrid assays 

To map the involved regions of N protein in the N/MIF 

interaction, the full-length N protein was divided into three 

domains to study the binding domain of the N protein: the 

N-terminal domain N1–139, the middle domain N140– 

280 containing the SR-rich motif and the C-terminal 

domain N281–422 (Figure 3). These three truncated 

proteins were tested for MIF binding using yeast twohybrid 

assay. The results indicated that N140–280, the 

middle domain of the N protein, is responsible for the 

interaction (Figure 3). To determine further the region of 

the N protein involved in MIF binding, the middle domain 

was divided into two parts, N140–280 containing the SRrich 

motif and N140–210, In the yeast two-hybrid assay, 

the -galactosidase activity of N140–210 fragment is 

much higher than the activity of N211–280 fragment 

(Figure 3). These results implied that the SR-rich motif of 

the N protein is responsible for the majority of the binding 

to MIF. 

DISCUSSION 

In the present study, by employing a series of biochemical 

and biophysical methods, we have firstly reported 

that SARS-N protein has a specific binding to human MIF 

protein, and the further yeast two-hybrid assay demonstrated 

that the fragment amino acid 140–210 of SARS-N 

probably contribute to the N/MIF interaction. MIF was first 

discovered as a lymphokine involved in delayed hypersensitivity 

and various macrophage functions, including 

phagocytosis, spreading, and tumoricidal activity. It has 

been reported that MIF is associated with the pathogenesis 

of a variety of diseases (Crichlow et al. , 2009; 

Oddo et al., 2005). Multiple clinical studies have indicated 

the utility of MIF as a biomarker for different diseases that 

have an inflammatory component; these include systemic 

infections and sepsis, autoimmune diseases, cancer, and 

metabolic disorders such as type 2 diabetes and obesity 

(Shen et al., 2003; Wistow et al., 2005). Over the past 

few years, the significance of the role of MIF in a variety 

of both solid and hematologic tumors has been 

established. Both circulating and intracellular MIF protein 

levels are elevated in cancer patients and MIF expression 

reportedly correlates with stage, metastatic spread and 

disease-free survival. 

To our knowledge, the fragment amino acid 140–210 

contains SR-rich motif (containing rich serine and 

arginine) that is multifunctional and conserved in N 

protein of coronaviruses (Toney et al., 2004). SR-related 

proteins are often involved in protein-RNA and protein– 

protein interactions (Blencowe et al., 1999), and the SRrich 

motif is conserved in the N protein of coronavirus. It 

has been reported that SR-rich motif is indispensable for 

SARS N oligomerization and for N protein interaction with 

SARS-CoV membrane protein. The SR-rich motif is also 

responsible for the interaction with hnRNPA1 in SARS 

(Haibin et al., 2005). In addition, this motif is also involved 

in interaction with Hubc9 in SARS (Fan et al., 2006). All 

of these facts indicate that the SR-rich motif of the N 

protein might play a crucial role in SARS infection. 

In conclusion, our data have shown for the first time 

both in vitro and in vivo that the nucleocapsid protein of 

SARS has a high binding affinity to human MIF and such 

a protein–protein interaction involves the region amino 

acid 140–210 of MHV-N. However, the pathophysiological 

significance of the interaction between N and MIF 

is largely unknown; elucidation of these questions will 

depend on further studies. Moreover, the disruption of 

interaction between N and MIF proteins using RNA 

interference technology may provide further clues to the 

specific function of N and MIF protein. This current 

research contributes useful data that will shed light on the 

molecular mechanism of N phosphorylation and provide 

valuable clues for mutagenic studies in disrupting virion 

assembly and replication and developing antiviral agents. 


The authors gratefully acknowledge the financial support 

provided by Jilin administration of traditional Chinese 

medicine TCM science project (20090461042). 

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DOI: 10.5897/AJMR11.611 



Isolation and characterization of high caffeine-tolerant 

bacterium strains from the soil of tea garden 

Fang-Yuan Fan 1 , Yan Xu 1 , Yue-Rong Liang 1 , Xin-Qiang Zheng 2 , Devajit Borthakur 1,3 and 

Jian-Liang Lu 1 * 

1 Tea Research Institute, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China. 

2 Key Laboratory of Horticultural Plants Growth, Development and Biotechnology, Agricultural Ministry of China, 

Hangzhou 310058, China. 

3 Tocklai Experimental Station, Tea Research Association, Jorhat, Assam 785001, India. 


Microbial biodegradation is an important and promising decaffeination approach because of its low 

cost and high security. The efficiency of this approach mainly depends on the characteristics of the 

strain. Twenty bacterium strains were isolated from soil of tea garden and exhibited high caffeinetolerance, 

and these strains could grow on the medium supplemented with 20 g L -1 caffeine as the sole 

source for nitrogen and carbon. According to the physiological-biochemical characteristics and 16S 

rRNA gene sequence blast, two of them (CT25 and CT75) were identified as Pseudomonas putida. The 

results also showed that caffeine was not the preferential nutrition source for growth of strain CT25, 

and high level caffeine inhibited the bacterium amplification although caffeine could be metabolized by 

this strain. The CT25 grew well in agitated liquid medium when the incubation temperature was around 

30°C. High initial concentration of strain inoculums would improve the efficiency of caffeine 

degradation. 

Key words: Caffeine-tolerant bacterium, identification, 16S rRNA gene, caffeine biodegradation, growth 

condition. 

INTRODUCTION 

Caffeine (1,3,7-trimethylxanthine), a member of purine 

alkaloids, was isolated first from tea and coffee in the 

early 1820s and then reported in the beans, leaves and 

fruits of more than 60 plants, such as coffee, tea, mate, 

guarana, cola and cocoa (Heckman et al., 2010). 

Medicinally, caffeine is used as a cardiac, cerebral and 

respiratory stimulant (Buerge et al., 2003). However, it 

can also produce adverse effects on human health 

unavoidably. Caffeine consumption during pregnancy is 

associated with an increased risk of fetal growth 

restriction (CARE Study Group, 2008). Excessive intake 

of caffeine can increase the risk of miscarriage, 

independent of pregnancy-related symptoms (Weng et 

*Corresponding author. E-mail: jllu@zju.edu.cn. Tel: 86-571- 

88982704. 

al., 2008). Statistically significantly higher risk of ovarian 

cancer among women who reported drinking five or more 

cups of caffeinated coffee per day compared to nonconsumers 

of coffee (Lueth et al., 2008). The ingestion of 

either a high or low glycemic index meal with caffeinated 

coffee significantly impairs acute insulin sensitivity 

compared with the effect of decaffeinated coffee (Moisey 

et al., 2008). Large sample and population-based studies 

indicated that regular daily dietary caffeine intake is 

associated with disturbed sleep and associated daytime 

sleepiness (Roehrs and Roth, 2008). For older women, 

blood pressure response is augmented, and subjective 

feelings of behavioral mood state are attenuated to a 

greater degree following acute caffeine ingestion (Arciero 

and Ormsbee, 2009). Higher level of caffeine also leads 

to higher risk of developing bone problems, such as 

osteoporosis (Hallstrom et al., 2006), alveolar bone loss 

in ligature-induced periodontitis (Bezerra et al., 2008), an

d disturbing the early stages of bone healing (Duarte et 

al., 2009). Tea polyphenols, an excellent antioxidant 

extracted from tea (Panza et al., 2008), is always contaminated 

with amount of caffeine because of the technical 

constraints of decaffeination, which hampers their 

application in the field of pharmaceutical and functional 

food. 

Environmentally, caffeine has been suggested as a 

chemical indicator of ecosystem since it is difficultly 

metabolized (Ogunseitan, 2002). When the exposure 

dosage of caffeine in water was higher than 300 mg L -1 , 

no zebrafish embryos could survive, and caffeine-treated 

embryos exhibited significantly reduced tactile sensitivity 

frequencies of touch-induced movement even when 

exposure dosages were very low (Chen et al., 2008). 

Reports also showed that caffeine is toxic to most of the 

aquatic organism such as Ceriodaphnia dubia (Moore et 

al., 2008) and coral algae (Pollack et al., 2009). Therefore, 

release of some caffeine-containing wastes, such as 

coffee pulp/husk from coffee producing factory, infused 

tea leaves and sewage from the factory of tea 

polyphenols preparation, leads serious ecological 

pollutions and becomes a big disposal problem (Pandey 

et al., 2000). 

To date, four major approaches for reducing caffeine 

content from caffeine-containing products are conventional 

breeding, physicochemical methods, genetic 

engineering and microbial degradation, whilst approach 

for wastes decaffeination is rarely concerned. Decaffeination 

through conventional breeding is difficult to achieve 

because of non-availability of the low-caffeine containing 

germplasms. Genetically-modified (GM) plants with low 

caffeine can be directly obtained either by down-regulating 

the caffeine synthesis pathway or by up-regulating 

the caffeine degradation pathway (Yadav and Ahuja, 

2007). However, this approach can not be applied 

recently because of their instability and ecological risk of 

GM plants, even though successful study in laboratory 

scale was reported (Kato et al., 2000). Decaffeination 

with special reagents extraction (Sun et al., 2010) and 

chromatograph separation is usually adopted in practices 

(Dong et al., 2011; Lu et al., 2010; Ye et al., 2009) 

although these techniques need to be optimized furthermore 

since their lack of security or/and yield. Comparatively, 

decaffeination through microbial degradation is more 

beneficial and attractive than other methods since it can 

be conducted safely and at a low budgetary requirement 

(Gummadi and Santhosh, 2006; Ramarethinam and 

Rajalakshmi, 2004). 

Microbial decaffeination, mainly including bacteria and 

fungi, was reported first in the early 1970’s (Kurtzman 

and Schwimmer, 1971). Reports showed that Aspergillus 

and Penicillium (Hakil et al., 1998), Rhizopus delemar 

(Tagliari et al., 2003) and Aspergillus tamari (Gutierrez- 

Sanchez et al., 2004) can effectively degrade caffeine. 

Major bacteria with capability of caffeine degradation 

belong to Pseudomonas (Gokulakrishnan et al., 2005; 

Mazzafera, 2004; Sarath Babu et al., 2005; Dash and 

Fan et al. 2279 

Gummadi, 2006a), beside some other species within the 

genera Alcaligenes, Rhodococcus, Klebsiella (Dash and 

Gummadi, 2006b), Acinetobacter, Enterobacter, 

Stenotrophomonas, Pantoea spp. (Padmanabhan et al., 

2003) and Serratia marcescens (Mazzafera et al., 1996). 

The characteristic and mechanism of caffeine biodegradation 

were investigated in many Pseudomonas strains 

which were isolated from the wastewater (Ogunseitan, 

1996, 2002) and from the soils of coffee plantation area 

(Dash and Gummadi, 2007, 2008; Gokulakrishnan and 

Gummadi, 2006; Gokulakrishnan et al., 2007; Gummadi 

et al., 2009b) and normal agricultural land (Topp et al., 

2006; Yu et al., 2008, 2009). Comparatively, the 

Pseudomonas isolated from coffee plantation area 

exhibits the highest caffeine tolerance, indicating that 

caffeine content in the growth environment might affect 

the caffeine tolerance capability of the strains. Recently, 

bioreactor for decaffeination was also developed 

(Gummadi et al., 2009a, 2009b) and optimized 

(Gummadi and Santhosh, 2010) by using Pseudomonas 

strains isolated from coffee plantation area. However, 

researchers never stop exploring the new strains with 

high caffeine tolerance and efficient caffeine degradation 

since the strain is the one of most important factors for 

developing caffeine biodegradation technique. In this 

study, twenty strains with high caffeine-tolerant capability 

were isolated from the caffeine-containing soil of tea 

garden, and two of them were identified as Pseudomonas 

putida based on the physiological-biochemical tests and 

16S rRNA gene analysis. Effect of caffeine concentration 

and other culture conditions on the strain growth was also 

investigated. 


Preparation mediums for screening and amplifying the 

caffeine-tolerant bacteria 

Solution with mineral substances, including KCl (0.370 g L -1 ), 

MgSO4 (0.205 g L -1 ), Fe2(SO4)3 (0.710 g L -1 ), Na2HPO4·12H2O 

(0.205 g L -1 ), CaCl2 (80 mg L -1 ), ZnSO4·7H2O (15 g L -1 ), 

NaMoO4·2H2O (12 g L -1 ), MnSO4 (11 g L -1 ), CuSO4 (10 g L -1 ) 

and H3BO4 (10 g L -1 ), was prepared and centrifuged (Beckman J2 

HS; Beckman Instruments Inc., Fullerton, CA, USA) at 5000 ×g for 

10 min after its pH value was adjusted to 7.0. The supernatant was 

collected and used for preparation of different culture mediums. 

Solid screening medium (SSM) for isolating the caffeine-tolerant 

bacteria was prepared by mixing the mineral solution with caffeine 

(2.5 g L -1 ) and agar (1.5%) and autoclaved at 121°C for 10 min. 

Solid purifying medium (SPM) was also prepared as SSM except 

20.0 g L -1 caffeine was supplemented. Liquid amplifying medium 

(LAM) was obtained after addition of caffeine (0.5 g L -1 ) and 

sucrose (5.0 g L -1 ) in the mineral solution and disinfection. 

Isolation, purification and amplification of caffeine-tolerant 

bacteria 

Soils were collected randomly from experimental tea garden of 

Zhejiang University. Some of them (100 g) were soaked in 1 L 

distilled water for 30 min. Solution containing microorganisms were


obtained by filtration and diluted 10 3 -10 6 times, and spread over the 

SSM surface in Petri dishes (0.5 ml solution for each dish). After 

incubation at 30°C for 2-3 d, the fast-growing single colony was 

picked up with tooth tip and inoculated on SPM surface for further 

purification. Twenty colonies, which could grow normally on the 

SPM, were separately picked up again and transferred to the tubes 

containing 1.0 ml LAM medium (each colony for one tube), then 

incubated at 150 r min -1 and 30°C in a shaking incubator (Taicang 

Experimental Instruments Factory, Jiangsu, China) for amplification. 

Three days later, 0.3 ml of bacterial solution was mixed with 0.1 ml 

dimethyl sulfoxide and stored at -70°C, and the remaining solution 

was used for further tests. 

Physiological-biochemical identification of caffeine-tolerant 

bacteria 

Two of the above twenty strains with high-caffeine tolerant 

capability, recorded as CT25 and CT75, were randomly selected for 

physiochemical identification. The capability to metabolize carboncontaining 

compounds was assessed by glycolysis test, methyl red 

test (MR) and Voges-Proskauer test (VP), citrate utilization test, 

oxidase activity and starch hydrolysis test. The activity of 

metabolizing nitrogen-containing compounds was assessed by 

indole production, hydrogen sulfide production, urease production 

tests and gelatin liquefaction test. In order to make further 

determination of the objective strains, the arginine dihydrolase 

activity test was also performed. The identification was carried out 

in detail according to the “Bergey's Manual of Determinative 

Bacteriology” (Buchanan and Gibbons, 2000). 

16S rRNA gene identification of caffeine-tolerant bacteria 

Amplified caffeine-tolerant bacteria solution (0.5 ml) of strain CT25 

and CT75 was centrifuged at 2000 ×g for 5 min and washed with 

PBS buffer, and suspended in distilled water (0.1 ml). Degenerate 

primers (forward primer: 5’-agagtttgatcmtggctcag; reverse primer: 

5’-ggytaccttgttacgactt, predicted length 1498 bp) were used to 

amplify the partial 16S rRNA gene fragments. PCRs were carried 

out in a 25 l mixture containing 17.3 l double-distilled water, 2.5 

l 10×PCR buffer, 1.5 l 25 mM MgCl2, 0.5 l 10 mM dNTPs, 0.5 l 

each primer, 0.2 l Taq DNA polymerase (2.5 U l -1 ) and 2 l strain 

resuspension. Amplification was performed on Veriti 96-well Fast 

Thermal Cycler (Applied Biosystems, Inc., Carlsbad, CA, USA) 

according to the following time program: predenaturation at 94°C 

for 5 min, 1 cycle; denaturation at 94°C for 45 s, annealing at 50°C 

for 60 s and extension at 72°C for 90 s, 30 cycles; and final 

extension at 72°C for 10 min, 1 cycle. Partial amplified solutions (10 

l) were subjected to electrophoresis for checking presence of the 

target band, the remaining was sent to Invitrogen (Shanghai) Life 

Science Corporation for sequencing. The obtained sequences were 

deposited in GenBank (www.ncbi.nlm.nih.gov) after blasting with 

known sequences by using the tool of “blastn”. 

Effect of culture conditions on the growth of isolated strain 

For evaluating the effect of oxygen, the amplified bacteria (50 l) of 

strain CT25 was inoculated in a conical flask containing 50 ml 

mineral solution supplemented with 2.0 g L -1 caffeine, and 

incubated in a shaking incubator at 150 r min -1 and 25ºC for 30-72 

h. Static culture (without shaking) was also carried out as control. 

For evaluating the effect of nitrogen source, the CT25 strain was 

inoculated in a mineral medium supplemented with 2.0 g L -1 

peptone instead of 2.0 g L -1 caffeine, and incubated at 150 r min -1 

and 25°C for 14 to 42 h. 

For evaluating the effect of caffeine concentration, the CT25 

strain was incubated in mineral medium with supplement of different 

concentration caffeine (0.1, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 g L -1 ) at 

150 r min -1 and 25°C for 30 h. 

For evaluating the effect of temperature, the CT25 strain was 

inoculated in a caffeine-supplemented (2.0 g L -1 ) medium and 

shook (150 r min -1 ) at different temperature (20, 25, 30 and 35°C) 

for 12-72 h. 

After incubation, the optical density of bacterium solutions was 

measured at 600 nm (OD600) with a HP8453 UV-visible 

spectrophotometer (Hewlett Packard, CA, USA), and the residue 

level of caffeine in caffeine supplemented medium was also 

monitored by HPLC (LC20A, Shimadzu Corporation, Tokyo, Japan) 

method as described previously (Liang et al., 2007). 

Data analysis 

Observations were made in triplicates and the mean ± standard 

deviation (SD) was presented. Statistics was carried out on Version 

8.1 SAS System for Windows (SAS Institute Inc, Cary, NC, USA) 

software. 

RESULTS 

The isolated high caffeine-tolerant bacteria 

After screening, a total of 103 colonies were obtained 

based on rapid proliferation on the SSM medium 

containing 2.5 g L -1 caffeine. These colonies exhibited a 

similar appearance, circular in shape and ivory-white in 

colour. Twenty strains of them could grow normally but 

somewhat slowly on the SPM medium supplemented with 

20.0 g L -1 caffeine after further purification, and were 

recorded as CT03, CT12, CT25, CT26, CT33, CT38, 

CT39, CT42, CT45, CT47, CT55, CT57, CT66, CT70, 

CT75, CT78, CT79, CT80, CT94 and CT97 respectively. 

These strains can be barely differentiated from each 

other according to their appearances and growths, 

suggesting they might belong to a same genus. 

The characteristics of high caffeine-tolerant bacteria 

A series of determinative tests were carried out for 

identifying the randomly selected caffeine-tolerant strains 

(CT25 and CT75) because different genera have specific 

metabolic characteristics and different capacities to utilize 

amino acids, carbohydrates and proteins (Harley, 2008). 

Table 1 summarized the physiological-biochemical 

characteristics of the strains CT25 and CT75. Both CT25 

and CT75 were preliminary identified as Pseudomonas 

putida after comparison with Bergey’s Manual of 

Determinative Bacteriology (Buchanan and Gibbons, 

2000). 

The partial gene (1498bp) of 16S rRNA genes from 

strains of CT25 and CT75 were amplified and sequenced 

(Figures 1 and 2), then deposited in GenBank 

(GU828029 for CT25 and GU828030 for CT75).

Table 1. Physiological-biochemical characteristics of caffeine-tolerant bacteria*. 

Items CT25 CT75 

Glucose fermentation + + 

Lactose fermentation + + 

VP & MR - - 

Citrate utilization test - - 

Starch hydrolysis - - 

Oxidase + + 

Indole production - - 

Hydrogen sulfide production - - 

Gelatin hydrolysis - - 

Urease + + 

Nitrate reduction + + 

Arginine dihydrolase + + 

“+” for positive and “-” for negative. 

Figure 1. Partial sequence of 16S rRNA gene from CT25 strains (GU828029). 

Homology search with the tool of “blastn” showed that the 

sequences of 16S rRNA gene from CT25 and CT75 were 

99% identical with corresponding sequences of 

Pseudomonas putida (Table 2). The result confirmed the 

previous identification on the basis of physiologicalbiochemical 

characteristics, therefore, the isolated twenty 

strains, including randomly selected CT25 and CT75, 

were the Pseudomonas putida strains with high caffeinetolerant 

capability. 


The growth of high caffeine-tolerant bacteria 

Incubation results showed that the OD600 value of CT25 

strain under shaking culture condition was significantly 

higher than that undern static culture condition (Table 3), 

suggesting the strain CT25 is an aerobe and its growth 

depends obviously on oxygen concentration in medium. 

Different source of N and C test showed that the OD600 

value of CT25 cultured in mineral medium with addition of


Figure 2. Partial sequence of 16S rRNA gene from CT75 strains (GU828030). 

Table 2. Blast result of 16S rRNA gene from CT25 and CT75. 

Accession Description E-Value Consistency (%) 

GU828029 Pseudomonas putida strain 25 16S ribosomal RNA gene, partial sequence Sequence from CT25 

GU828030 Pseudomonas putida strain 75 16S ribosomal RNA gene, partial sequence Sequence from CT75 

CP002290 Pseudomonas putida BIRD-1, complete genome 0.0 99 

CP000926 Pseudomonas putida GB-1, complete genome 0.0 99 

AE015451 Pseudomonas putida KT2440 complete genome 0.0 99 

CP000712 Pseudomonas putida F1, complete genome 0.0 99 

AM411058 Pseudomonas putida partial 16S rRNA gene, strain 5zhy 0.0 99 

DQ657850 Pseudomonas sp. ND9 16S ribosomal RNA gene, partial sequence 0.0 99 

Table 3. Effect of oxygen on growth of CT25 in the medium with 

2.0gL -1 caffeine * . 

Items 30 h 72 h 

Static culture 0.0633±0.0034 a 0.0634±0.0067 a 

Shaking culture 0.1461±0.0032 b 0.2827±0.0029 b 

* The OD600 was used as growth indicator of bacterium, and the 

different letters in column indicated significant difference at p

600 

OD 

Table 4. Comparison of CT25 growth cultured with different sources for nitrogen and carbon * . 

Nitrogen source 14 h 30 h 42 h 

Caffeine (2.0g L -1 ) 0.0305±0.0012a 0.1461±0.0032a 0.2514±0.0033a 

Peptone (2.0g L -1 ) 0.8911±0.0045b 1.0084±0.0049b 1.3896±0.0174b 

* The OD600 was used as growth indicator of bacterium, and the different letters in column indicated significant 

difference at p


Percentage of residual caffeine in %) 

110 

100 

90 

80 

70 

60 

50 

40 

%) 

30 C 

35°C 

0 20 40 60 80 

Time (h) 

Figure 5. Caffeine-degrading efficiency of CT25 cultured in medium 

supplemented with 2.0 g L-1 initial caffeine at different temperature. 

The bar shows the standard deviation. 

DISCUSSION 

The isolated twenty strains could grow normally on the 

medium supplemented with 20 g L -1 caffeine, indicating 

these strains can tolerate high concentration of caffeine 

and metabolize it as sole nitrogen (N) and carbon (C) for 

their growth demand since no other source of N and C 

was present in that medium. Previous reports showed 

that some Pseudomonas sp. strains isolated from coffee 

plantation area could survive in the mediums 

supplemented with caffeine at initial concentration of 10 g 

L -1 (Gokulakrishnan and Gummadi, 2006; Gummadi and 

Santhosh, 2006; Dash and Gummadi, 2008), 15 g L -1 

(Dash and Gummadi, 2007; Dash et al., 2008) and 20 g 

L -1 (Gummadi et al., 2009b; Gummadi and Santhosh, 

2010) and degrade the caffeine sufficiently in short 

period. From this view, the isolated strains in this study 

might possess similar high caffeine-tolerant capability to 

the reported ones although some investigation should be 

further conducted with these strains. It was also 

suggested that soil microorganisms have the ability for 

natural enrichment under different ecological condition 

according to their chemotaxis (Mazzafera, 2002). High 

caffeine-tolerant bacteria can be isolated from caffeinecontaining 

soils such as tea and coffee garden where 

caffeine is continuously released from the litters of these 

plants. 

Series physiological-biochemical reactions (Buchanan 

and Gibbons, 2000) and the sequence of 16S rRNA gene 

(Garrity et al., 2004) are widely used for bacterial 

identification. The randomly selected strains (CT25 and 

CT75) with high caffeine-tolerant capability were 

identified as Pseudomonas putida according to the 

similarity of physiochemical characteristics and 16S rRNA 

gene sequences. It seemed that all of the isolated twenty 

strains might belong to Pseudomonas putida since the 

appearance of the colonies was quite similar, and this 

species might be a dominant bacterium in soil of tea 

garden because special metabolic pathway(s) related to 

caffeine degradation were reported to be present 

inherently in many Pseudomonas strains (Dash and 

Gummadi, 2006b; Mazzafera, 2004; Yamaoka-Yano and 

Mazzafera, 1999; Yu et al., 2009). 

The growth of strain CT25 was highly depended on 

oxygen in liquid medium, which is consistent with other 

reports on the growth of Pseudomonas strains (Beltrán et 

al., 2006; Gummadi and Santhosh, 2006; Gummadi et 

al., 2009b; Middelhoven and Lommen, 1984; Woolfolk, 

1975). Although caffeine could be used as sole source for 

N and C, the strain CT25 amplified more slowly in 

medium supplemented with caffeine than that with 

peptone, and its growth was significantly inhibited especially 

when high level caffeine was supplemented. Similar 

results were reported previously in the studies about 

other caffeine-degrading strains (Sarath Babu et al., 

2005; Gokulakrishnan and Gummadi, 2006; Dash and 

Gummadi, 2007, 2008; Gummadi et al., 2009b). It was 

suggested that the isolated bacteria can utilize the 

caffeine as their nonpreferential N and C sources and 

may take time to activate their metabolic pathway(s) to 

transform the caffeine into some components directly 

used for growth. This result agrees with some findings 

that the caffeine-degrading enzymes of bacteria were 

induced (Dash and Gummadi, 2007; Gummadi and 

Santhosh, 2006; Sarath Babu et al., 2005; Ogunseitan, 

2002; Woolfolk, 1975). The fastest amplification was 

observed when the strain CT25 was incubated at around 

30°C, while amplification would dramatically decrease 

when the strain was incubated at 35°C, which coincides 

with the previous observations on the growth of other 

Pseudomonas strains (Dash and Gummadi, 2007; Sarath 

Babu et al., 2005) and the optimum catalytic temperature 

of caffeine-degrading enzyme (Beltrán et al., 2006; 

Yamaoka-Yano and Mazzafera, 1999). However, this 

result is not in accordance with another report in which 

the optimum temperature for the growth of Pseudomonas 

sp. strain was 35°C (Gummadi and Santhosh, 2006). The 

difference might be explained as different strain or other 

culture parameters such as caffeine concentration. 

Caffeine was metabolized rapidly when the CT25 was 

incubated at 30°C, while slowly at 35°C (Figures 4 and 

5), indicating that the caffeine degradation was positively 

correlated with the bacterial growth as more bacterial 

cells would metabolize much more caffeine into nutrition 

for further bacterial growth. Thus, in order to improve the 

decaffeination efficiency, the initial inoculums concentration 

should be optimized. Compared with the caffeine 

residue, metabolites of caffeine were barely detected in 

the incubated medium (Figure 4), suggesting that initial 

degradation of caffeine might be a rate-limiting step and

the metabolites might be instantly broken down 

furthermore. 

The bacteria isolated from soil of tea garden (such as 

CT25) have high capability for caffeine-tolerance and 

caffeine-degradation, and can be used as an alternative 

for decaffeination of tea products or caffeine-containing 

wastes. Further studies like usage method 

(immobilization or not), optimization of decaffeination 

efficiency and assessment of products quality and 

security, should be conducted prior to exploitation of the 

bacterial strains for caffeine degradation purpose. 


This study was financed by the National Natural Science 

Foundation of China and “R&D Project” of Education 

Office of Zhejiang Province. 

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DOI: 10.5897/AJMR11.613 



Biopreservative potential of marine lactobacillus spp 

K. Indira*, S. Jayalakshmi, A. Gopalakrishnan and M. Srinivasan 

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, 

Annamalai University Parangipettai – 608 502, Tamil Nadu – India. 


Bacteriocins are of special interest due to their potential value as natural preservative. The present 

study is a trial for production of such bacteriocin from a marine Lactobacillus sp. Lactic acid bacteria 

(LBA) was isolated from fish gut (Mugil cephalus) and prawn muscle (Peneaus monodon) samples and 

their density were found to be 5.2 x 10 7 and 6.4 x 10 7 CFU/g respectively. Various pathogens were 

isolated from ready to eat pickle samples (bottled). The LAB strains were tested against 10 different 

commercial antibiotics. Among them Vancomycin was the only antibiotic that showed a minimum of 

40% resistance to the LAB strains tested. LAB strains were optimized at different parameters and 

maximum bacteriocin production was at pH 6, temperature of 35°C, 3.5% of salt concentration, 24th h of 

incubation period. Bacteriocin produced by these strains were precipitated from the culture filtrate 

using methanol and TCA which was further dialyzed, centrifuged and lyophilized. Lactobacillus 

fermentum was selected as the most potential strain for both bacteriocin production as well as 

antimicrobial activity. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis 

proved that the obtained protein is homologous with a molecular weight of 18 kDa. FT-IR spectrum also 

confirmed the obtained protein as a bacteriocin. The study revealed that Lactobacillus strains of marine 

origin are having the potential to be used as biopreservatives especially in seafood industries. The 

production of bacteriocin from L. fermentum was found to be ideal for industrial scale production and 

commercial utilization. 

Key words: Bacteriocin, Lactobacillus fermentum, biopreservative, fourier transform infrared (FT-IR). 

INTRODUCTION 

Food fermentation has a great economic value and the 

products obtained through this process put in improving 

human health. In this way LAB has contributed in a lot to 

fermented foods worldwide. The major genera of LAB is 

of importance in the food industry are Bifidobacterium, 

Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, 

Oenococcus, Pediococcus, Streptococcus and 

Tetragenococcus. They are significant in production and 

preservation of milk and dairy products, meat and meat 

products, vegetable products and as forage crops for 

animals (Lee, 2004). 

Mankind had exploited lactic acid bacteria (LAB) for the 

production of fermented foods because of their ability to 

produce desirable changes in taste, flavor and texture as 

well as to inhibit pathogenic and spoilage microbes. 

Since they are involved in numerous food fermentations 

*Corresponding author. E. mail: microind03@yahoo.co.in. Tel: 

04144 – 243070 – 243071. Fax: 04144-243555. 

for millennia, it is assumed that most representatives of 

this group do not pose any health risk to man and are 

designated as GRAS (generally recognized as safe) 

organisms (Holzapfel et al., 1995). Different antimicrobials, 

such as lactic acid, acetic acid, hydrogen peroxide, 

carbon dioxide and bacteriocins produced by these 

bacteria, can inhibit pathogenic and spoilage microorganisms, 

extending the shelf-life and enhancing the 

safety of food products (Aymerich et al., 2000). 

One important attribute of LAB is their ability to produce 

antimicrobial compound called bacteriocin. Bacteriocins 

are proteinaceous compound, inhibitory effects towards 

sensitive strains produced by both Gram-positive and 

Gram-negative bacteria (Tagg et al., 1976). Bacteriocins 

producing lactic acid bacteria are used in food fermentations 

especially in dairy products. In USA, only nisin 

produced by Lactobacillus lactis has been permitted as a 

food preservative (FDA, 1988). It has also been used in 

health care products and cosmetics for treatment of acne. 

They are also being used in toothpaste and mouthwash 

for the inhibition of dental caries and periodontal diseases


(Harlander, 1993). Bacteriocins can be exploited to inhibit 

undesirable microorganisms in the fermentation of wine 

(Navarro et al., 2000), beer (Ogden et al., 1985), vegetables 

(Daeschel and Fleming, 1984) and dairy products 

(Castla et al., 1996; Ross et al., 1999). The present study 

is on bacteriocin production by LAB of marine environment 

and its use as a biopreservatives in sea foods. 


The fin fish and prawn samples were collected from the Annan koil 

landing centre (Latitude 11° 29 ’N; Longitude 79° 46’ E) for the 

isolation of lactic acid bacteria. The pickle samples were collected 

from Tuticorin. Tuna and Prawn pickles were analyzed in the 

present study. Homogenized extract of the fish and prawn samples 

were serially diluted up to 10 -5 dilutions and were plated on MRS 

agar (HI-MEDIA) by spread plate technique and incubated at 28 ± 

2°C for 24 h. The isolated potential strains were identified based on 

Bergey’s manual of systematic bacteriology (Buchanan et al., 

1974). Approximately 10 g of sample (pickle) was homogenized in a 

sterile mortar and pestle using 90 ml of sterile 50% sea water and 

then serially diluted. About 0.1ml of the serially diluted samples was 

inoculated into the selective media to isolate the specific 

pathogens. The isolated pathogens were identified based on 

Bergey’s manual of systematic bacteriology (Buchanan et al., 

1974). 

Primary screening for antibacterial activity 

A preliminary test for inhibitory assay of LAB against food borne 

pathogens, isolated from the seafood pickles was done. 

Well diffusion method (Reinheimer et al., 1990) 

After swabbing the pathogens on the Muller Hinton agar plates, 0.1 

ml of cell free culture broth of Lactic acid bacteria (LAB) centrifuged 

at 10,000 rpm for 20 min, poured into the wells and plates were 

incubated at 37°C for 24 h. The bacterial culture filtrate inhibiting 

the growth of pathogen was assessed based on the inhibition zone 

around the well and the results were recorded. 

Test for antibiotic resistance of LAB 

Muller Hinton Agar was used to check the antibiotic resistance of 

LAB against antibiotics like Ampicillin (A), Bacitracin (B), 

Cephotaxime (Ce), Ciprofloxacin (Cf), Erythromycin(E), Nalidixic 

acid (Na), Novobiocin (Nv), Penicillin-G(P), Tetracyclin (T) and 

Vancomycin (Va). After swabbing the LAB on MHA plates, the 

selected antibiotic discs were placed onto the agar medium and the 

incubated plates were observed for antibiotic assay. 

Screening of bacteriocin producing bacteria 

Screening was done by well diffusion method using the crude 

extract. The inhibitory activity was tested against the seafood borne 

pathogens. 

Optimization of cultural condition for bacteriocin production 

The effect of incubation period on growth and bacteriocin 

production was studied for 0 - 52h with 12 h intervals. The pH 

ranges (3, 4, 5, 6 and 7) were tested. A temperature range 20, 25, 

30, 35 and 40°C were optimized to find out the optimum 

temperature. The range of 3-5% of salt concentration was 

optimized. Based on the results observed in the optimization the 

mass scale culture was carried out with the ideal parameters. 

Extraction and partial purification of bacteriocins 

The precipitation was made by the solvent extraction method. To 

the filtrate obtained 70% Methanol and 10% of Tri chloroacetic acid 

(TCA) were added in equal proportion and kept for 48 h at room 

temperature. This precipitate was dissolved in deionized water and 

dialyzed through a 1000 molecular weight cut of dialysis membrane 

against deionized water. The dialysis was done for 24 h and then 

centrifuged. The precipitate was lyophilized and used for further 

analysis. 

Protein estimation 

The Folin-Ciocalteu phenol method (Lowry et al., 1951) was used 

for the estimation of the total protein content in the sample. 

FT-IR analysis 

In the study of molecular vibrations, Infrared spectroscopy has 

contributed more to this field than Raman due to the rapid 

developments in Infrared instrumentation (Merritt et al., 1986). The 

vibrational spectra can be utilized directly and simply as molecular 

“finger prints” to characterize and identify the molecule (Roberts et 

al., 1985). The lyophilized bacteriocin sample from LAB6 was 

subjected to FT-IR analysis. The IR spectrum of the bacteriocin was 

recorded with a perkin-Elmer model 297 IR spectrophotometer. One 

part of the extract was mixed with 99 part of dried potassium 

bromide and it was scanned between 600-4000 wave number (cm - 

1 ) at a speed of 1 micron. and with a programmed slit opening 2x 

and air as reference. 

RESULTS 

The total heterotrophic and lactic acid bacterial count in 

fish gut was found to be 2.14 x 10 9 and 5.2 x 10 7 CFU/g 

respectively. Similarly, the total heterotrophic and lactic 

acid bacterial count in prawn muscle was found to be 2 x 

10 9 and 6.4 x 10 7 CFU/g respectively. A total of 116 

morphologically distinct strains were isolated from the 

serially diluted fish gut and prawn muscle samples. 4 

strains from fish gut and 6 strains from prawn muscle 

samples were identified as potent strains for bacteriocin 

production. After the screening procedures the potential 

strains were identified. They are LAB1 Corynebacterium 

bovis, LAB2 Corynebacterium xerosis, LAB3 

Lactobacillus alimentarius, LAB4 Lactobacillus animalis, 

LAB5 Lactobacillus casei, LAB6 Lactobacillus fermentum, 

LAB7 Lactobacillus plantarum, LAB8 Micrococcus 

varians, LAB9 Staphylococcus epidermidiis, and LAB10 

Streptococcus mitis. 

Selective media used for the isolation of seafood 

pathogens are EMB agar, MRS agar, Listeria isolation 

agar, SS agar, TCBS agar, Yersinia identification agar

Figure 1. Antibiotic activity against LAB strains. 

Table 1. Antibiotic activity of lactic acid bacteria. 

LAB A B Ce Cf E Na Nv P T Va 

LAB1 R R R R R R R R R - 




LAB5 R R R R R R R R R R 

LAB6 R R R I R R R R R R 





*I- intermediate; R- resistant. 

Figure 2. Antibacterial activity of bacteriocin 

against Vibrio parahaemolyticus 

where in the density of E. coli, Lactobacillus spp, Listeria 

spp, Salmonella, Vibrio spp and Yersinia spp were 

enumerated to be in the fish pickle 1.9x10 4 , 2.7x10 4 , 

2.1x10 4 , 3.2x10 4 , 1.5x10 4 CFU/g. Likewise in the prawn 

pickle the pathogens were enumerated to be 1.3x10 4 , 

1.7x10 4 , 2.2 x10 4 , 1.5 x10 4 , 2.8 x10 4 and 1.0 x10 4 CFU/g. 

Isolated pathogens were identified as FBP1 E.coli, FBP2 

Indira et al. 2289 

Lactobacillus vulgaris, FBP3 Listeria monocytogenes, 

FBP4 Listeria spp, FBP5 Salmonella spp, FBP6 Shigella 

spp, FBP7 Staphylococcus aureus, FBP8 Vibrio cholera, 

FBP9 Vibrio parahaemolyticus and FBP10 Yersinia spp. 

All the 10 LAB strains isolated were found to be 

resistant to Ampicillin (A), Bacitracin (B), Cephotaxime 

(Ce), Erythromycin (E), Nalidixic acid (Na), Novobiocin 

(Nv), Penicillin-G(P), and Tetracyclin (T) (Figure 1). The 

resistance against Ciproflaxacin (Cf) was shown by all 

the LAB strains except LAB6, which showed an intermediate 

activity. Resistance to Vancomycin (Va) was shown 

by LAB 5, LAB 6, LAB 9 and LAB 10, these results are 

given in Table 1. 

The bacteriocin obtained from LAB6 showed the 

maximum zone of inhibition compared to all the other 

strains. It showed a zone of inhibition of 7 mm against V. 

parahaemolyticus (Figure 2), 6 mm against L. 

monocytogenes, Listeria spp. 5 mm against E. coli, 

Salmonella spp. S. aureus and Yersinia spp and 4mm 

against Shigella spp. Vibrio cholerae and Lactobacillus 

vulgaris, whereas the supernatant of the same strain 

showed zone of inhibition of 5 mm against Vibrio 

paraheamolyticus (Figure 3), 4 mm against L. 

monocytogenes, S. aureus and Yersinia spp and 3 mm 

against E. coli, Listeria spp, Salmonella spp, Vibrio


Figure 3. Antibacterial activity of LAB strains against Vibrio parahaemolyticus. 

Figure 4. Antibacterial activity of LAB cell free extracts (Zone of clearance in mm). 

cholerae and Lactobacillus vulgaris. These results are 

given in Figures 4 and 5 respectively. 

The bacteriocin production was found maximum at an 

incubation period of 24 h. The results were depicted in 

(Figure 6). Among the various pH ranges studied, the 

growth of the potential strain was found to be highest at 

pH 6 (Figure 7). The optimum temperature for bacteriocin 

production was found to be 30 - 35°C (Figure 8). The 

optimum salt concentration for bacteriocin production was 

found to be 3.5% salt concentration (Figure 9). 

The amount of protein was estimated to be 0.36 mg/ml. 

The FT-IR Spectrum of the protein revealed the presence 

of peaks at the wave numbers of 3288, 3090, 2958, 2924 

and 2849 cm -1 which indicated the presence of NH, NH3, 

CH3, CH2 and CH2 groups respectively. The wave 

numbers 1660, 1632, 1589 and 1540 cm -1 indicated the 

presence of their bending mode of amide, methyl, and 

amide groups respectively. The asymmetric mode of the 

NH band occurred at the wave number 827 cm -1 . The 

molecular weight of bacteriocin from L. fermentum was 

found to be 18 kDa. 

DISCUSSION 

In the present study the isolation, partial characterization 

and activity of bacteriocin produced by L. fermentum was 

done. The bacteriocin producing lactic acid bacteria 

(LAB) were isolated from the fresh meat of marine fin fish 

and shell fish. It is interesting to note that majority of the 

Lactobacillus spp. that have been isolated from fresh and 

frozen fish/prawns were those species which were commonly 

found in animals and human beings (Kandler and 

Weiss, 1986). There are only a few reports available on 

isolation of LAB from fresh and seawater fish (Cone, 

1982; Okafor and Nzeako, 1985). The LAB strains were 

evaluated for the production of inhibitory substances 

against various food borne pathogens. The pathogens 

used in the present were isolated from fish pickle. All the 

LAB strains showed a moderate inhibitory activity against 

the pathogens isolated from pickle samples. The use of 

bacteriocinogenic starter/protective cultures could 

improve the quality and increase safety by inhibiting the 

food-borne pathogens and spoilage microorganisms.

Figure 5. Activity of bacteriocins against pathogens (Zone of clearance in mm). 

Figures 6. Bacteriocin activity at (a)12th h (b) 24th h (c) 36th h (d) 48th h (e) 60th h of incubation period. 

Indira et al. 2291


Figures 7. Bacteriocin activity between pH range of (a) 3, (b) 4, (c) 5, (d) 6 and 7 (e). 

Recent outbreaks of emerging pathogens such as L. 

monocytogenes that has caused severe illness through 

food ingestion have prompted the scientific community to 

focus their studies on the anti-Listeria activity of 

bacteriocins produced by Lactobacillus and Pediococcus 

strains (Todorov et al., 1999; Aymerich et al., 2000; 

Messens et al., 2002). 

In the present investigation 100% of the isolated LAB 

strains were resistance to Ampicillin, Bacitracin, 

Cephotaxime, Erythromycin, Nalidixic acid, Novobiocin, 

Penicillin-G and T etracyclin. However only 40% of the 

strains showed resistance to Vancomycin. Generally 

many LABs are resistant to antibiotics. This resistance 

attributes are often intrinsic and non transmissible 

(Curragh and Collins, 1992). Among antibiotics, 

Vancomycin is one of the last antibiotics broadly 

efficacious against clinical infections caused by multidrug 

resistant pathogens (Johnson et al., 1990). 

The effect of incubation period, pH, temperature and 

salinity of medium on the production of bacteriocin was 

also investigated in all LAB strains. The pH 6, temperature 

of 35°C, salinity of 35 ppt, 24 h of incubation was


Figures 8. Bacteriocin activity between (a) 20°C, (b) 25°C, (c) 30°C, (d) 35°C, and (e) 40°C of temperature ranges with 5°C interval. 

found to be the optimal parameters for the most potential 

producers of bacteriocin (that is) L. fermentum. According 

to Ogunbanwo et al. (2003) the use of constituted 

medium at 30°C of incubation temperature, initial pH of 

5.5 and 48 to 60 h fostered the best production of 

bacteriocin by Lactobacillus brevis OG1 which seemed to 

differ from the results of the present study. 

L. fermentum produced bacteriocin in high level and the 

strain showed the maximum inhibitory activity against 

Vibrio parahaemolyticus, L. monocytogenes , Listeria sp. 

and S. aureus. Besides, the productions of bacteriocins 

having a wide spectrum of antibacterial activity against 

seafood borne pathogens like Listeria, Clostridium and 

even Gram-negative pathogens like Pseudomonas and 

E. coli to employ as biopreservatives. Accordingly L. 

acidophilus and L. casei (Stiles and Holzapfel, 1997) may 

be of great interest as probiotics strains because of their 

ability to adhere to intestinal epithelial cells and being of 

human origin. 

The protein purification was done by methanol and 

trichloroacetic acid precipitation followed by dialysis 

against deionised water. Extraction of bacteriocin using 

organic solvents indicated that bacteriocin was removed 

from the aqueous phase and could be recovered from the 

organic phase. This suggested that part of the bacteriocinmolecule 

has a hydrophobic character, and shares this 

property with most other bacteriocins (Klaenhammer, 

1993). In the present study also Trichloroacetic acid


Figures 9. Bacteriocin activity between (a) 3%, (b) 3.5%, (c) 4%, (d) 4.5% and to (e) 5% of salt concentration with 0.5% of interval. 

precipitation at the rate of 70% methanol and 10% tri 

chloroacetic acid aggregated bacteriocin from cell free 

broth and the antibacterial activity was also observed 

which the same in the bacteriocin R from L. fermentum. 

In the present study FT-IR Spectrum of the protein 

revealed the presence of peaks at the wave numbers of 

3288, 3090, 2958, 2924 and 2849 cm -1 which indicated 

the presence of NH, NH3, CH3, CH2 and CH2 groups 

respectively. The wave numbers 1660, 1632, 1589 and 

1540 cm -1 indicated the presence of their bending mode 

of amide, methyl, and amide groups respectively. 

Comparing the results with the nisin standard, the protein 

in the sample was confirmed as a bacteriocin. The peak 

at 1546 cm -1 indicated a secondary amide was reported 

(Silverstein et al., 1991; Yakimov et al., 1995). The FT-IR 

spectrum offered concrete evidence that the substance 

contained a peptide in its structure. Acidocin 8912 

(Tahara et al., 1992) and lactacin B (Barefoot and 

Klaenhammer, 1984) were reported to be 5.4 and 6.5 

kDa, respectively. In the present study the molecular 

weight determination thorough SDS-PAGE showed that 

the molecular weight of bacteriocin as 18 kDa protein. 

Compared to many other studies the molecular weight 

obtained in the present study seemed to be high.

Conclusion 

Figure 10. The FT-IR spectrum of Bacteriocin from L. fermentum. 

Lane 1 Lane 2 

Figure 11. SDS-PAGE of partially 

purified protein Lane 1-Marker, 

Lane 2-Partial purified Bacteriocin. 

The present study showed that the bacteriocin of L. fer 

effects on some clinically important food borne 


pathogens. This revealed the potential application of 

bacteriocin produced by L. fermentum as a biopreservatives 

for the improvement of the microbial safety of 

fermented foods and reduction in food contamination 

which causes illness to human beings. The study 

revealed that Lactobacillus strains of marine origin are 

having the potential to use as biopreservatives especially 

in seafoods. The production of bacteriocin from L. 

fermentum, seems to be ideal for industrial scale 

production and commercial utilization. 


We thank Dr. T. Balasubramanian, Dean, Faculty of 

Marine Sciences, Annamalai University, Parangipettai for 

providing facilities. 

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DOI: 10.5897/AJMR11.616 



Ephedra alata as biologically-based strategy inhibit 

aflatoxigenic seedborne mold 

Al-Qarawi, A. A. 1 , Abd_Allah, E. F. 1 * and Hashem Abeer 2 

1 Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Saudi Arabia. 

2 Department of Botany and Microbiology, Faculty of Science, King Saud University, Saudi Arabia. 


Investigation has been carried out to evaluate the effect of range plant Ephedra alata as biologicallybased 

strategy inhibit growth characters and aflatoxin production (in vitro and in vivo) of Aspergillus 

flavus. The aqueous extract of E. alata had significant inhibitory potential against growth as well as 

aflatoxin production by aflatoxigenic seedborne mold (Aspergillus flavus). Moreover, it has been found 

that, the addition of 1 and 2% (w/w) of plant powder material of E. alata to corn grains and soybean 

seeds respectively decreased the aflatoxin contamination and improve their nutritional value (total 

nitrogen content, fiber content, total lipids content and ash content) under storage conditions. The 

results observed here suggest employment of E. alata as an alternative non-chemical mean to control 

aflatoxin contamination of fodder in Saudi Arabia. 

Key words: Biocontrol, Aflatoxins, Aspergillus flavus, Ephedra alata, nutritional value, Saudi Arabia. 

INTRODUCTION 

Aflatoxins are secondary metabolites produced by many 

strains of Aspergillus flavus, known to be potent 

carcinogens as well as hepatotoxic agents and pose 

serious hazards to human, animal health and milk 

production in many countries including Saudi Arabia. 

Aspergillus flavus are widely distributed in agricultural 

commodities like maize (Giorni et al., 2007) and soybean 

(Gupta and Venkitasubramanian, 1975). The death of 

thousands of camels in Saudi Arabia may have been 

caused by the consumption of these aflatoxinscontaminated 

fodders (Bokhari, 2010). Bioremediation of 

mycotoxins has been carried out by bacteria (Abd_allah 

and Ezzat, 2004), fungi (Abd_allah and Ezzat, 2005) and 

friendly chemicals such as chitosan (Abd_Allah and 

Hashem, 2006). Recently, impacts of natural products of 

plant origin have been introduced as non-chemical mean 

for biocontrol and bioremediation of mycotoxins in 

agricultural commodities (Parsaeimehr et al., 2010). 

Ephedra, a medicinal plant belonging to the Ephedraceae 

is a genus of non-flowering seed plants belonging to the 

Gnetales, the closest living relative of the angiosperm 

(Friedman, 1996). The antimicrobial potential of some 

*Corresponding author. E-mail: eabdallah@ksu.edu.sa. 

Epedhra species such as E. altissima Defs (Tricker et al., 

1987), E. transitorai (Al-Khalil, 1998), E. nebrodensis 

(Cottiglia et al., 2005), E. major (Bagheri et al., 2009) and 

E. breana (Feresin et al., 2001) has been recognized. 

Ephedra alata is a range plant with medicinal application, 

belonging to ephedra native of Asia including Saudi 

Arabia (Abourashed et al., 2003). Additionally, the foliage 

of E. alata has acceptable aroma and used as food stuff 

during animal grazing in Saudi Arabia (Al-Taisan et al., 

2010). 

The objective of the current study was to describe the 

antifungal activity of E. alata (aqueous extract of shoot 

system) grown in Saudi Arabia, against growth 

characters and aflatoxin production of local seedborne 

isolate of Aspergillus flavus in vitro and on both maize 

grains and soybean seeds under storage conditions. 

Also, the relevance of nutritional value of stored 

agricultural commodities (maize grains and soybean 

seeds) was studied in relation to storage treatments. 


Experimental mold 

Local aflatoxin producing isolate similar to Aspergillus flavus link 

was isolated from soybean seed samples collected from Derab


Agriculture Research Station, Riyadh, Saudi Arabia. The mold was 

identified using current universal keys of identification (Domsch et 

al., 1993) by Dr. Abeer Hashem, Botany and Microbiology 

Department, Faculty of Science, King Saud University, Riyadh, 

Saudi Arabia. 

Plant extract and antifungal evaluation 

Fresh aerial parts (shoot system) of E. alata Decne were collected 

from wildlife Research and Development at Thumama, Riyadh, 

Saudi Arabia. Plant materials were dried in dark at room 

temperature (25±1°C) and powdered. Known weight of powdered 

material was extracted with aqueous ethanol (10:90, v/v) at 30°C 

for overnight and filtered through double layers of Whatman no. 1 

filter paper. The extract was completed to known volume using 

aqueous ethanol as above. The extract was filtered using 0.22 

micron cellulose acetate filter units (Whatman, Dassel, Germany) 

for sterilization. The antifungal activity of plant extract was 

determined by disc method (Roberts and Selitrenikoff, 1988), agar 

plate method (Bobbarala et al., 2009) and broth culture flask using 

glucose-ammonium nitrate salt medium (Brain et al., 1961). The 

production and germination of conidia were estimated according to 

the methods described by Wilson et al. (2004) and Ghaly et al. 

(1998) respectively. 

Mycelial dry weight 

At the end of incubation period, the culture broths were filtered 

through pre-weighed filter papers (Whatman no. 1). The filter 

papers with mycelial growth washed carefully with distilled water 

followed by drying at 80°C up to two successive constant weights 

were obtained. The net dry weight of mycelia was then determined. 

Aflatoxin analysis 

Replicated samples (each 50 ml of culture growth medium or 100 g 

of both maize grains and soybean seeds) were extracted using 

chloroform and cleaned up as described by Dutton and Westlake 

(1985). Column chromatography (AOAC, 1970) was carried out on 

silica gel G-60 mesh 0.02- 0.2 (BDH Chemicals, Poole, UK). 

Chemical and biological confirmatory tests to aflatoxin B1 were 

carried out according to AOAC (1970) and Madhyastha et al. 

(1994), respectively. The sensitive strain of Bacillus subtilis was 

used throughout for biological confirmatory test (Abd_Allah and 

Ezzat (2004). Thin layer chromatography (TLC) plates (20 x 20 cm) 

coated with 0.5-mm-thick silica gel DG (Kieselgel-DG, Riedel- De 

Haen, Seelze-Hannover, Germany) were used for chromatographic 

separation of aflatoxin B1 followed with elusion of aflatoxin B1 spots 

using benzene: acetonitrile (98:2 [v/v]). Quantitative determination 

of aflatoxin B1 was done spectrophotometrically according to 

Nabney and Nesbitt (1965). Standard aflatoxin B1 (Sigma) was 

used as reference in all experiments. 

Storage experiments 

Storage experiment was carried out in polyethylene pouches (200 x 

150 mm) each containing 100 g of blend consisted of powdered E. 

alata, mixed well with powdered maize grains or soybean seeds to 

give final concentration of 0.5, 1.0 and 2.0% (w/w). The pouches 

were inoculated with A. flavus (10 6 spores/pouch). Control pouches 

were used as reference. The moisture content was expressed as 

percentage of net weight (Abd_Allah and Hashem, 2006). The 

storage experiment continued for six months at room temperature 

(25 ± 1°C) in dark. 

Estimation of nutritional values 

The nutritional values (total lipid content, ash content, fiber content, 

total nitrogen content) of all plant materials were estimated 

according to AOAC (1995). 


All experiments were repeated at least three times and treatment 

means were compared using Least Significant Difference (LSD) 

analysis according to Daniel (1987). 

RESULTS 

Thin layer chromatographic examinations indicated that, 

Rf -values and color of spots developed from both 

chloroform extract of A. flavus and standard aflatoxin B1 

were the same before and after chemical confirmatory 

tests. The first conformity test (addition of HCl) indicated 

the presence of one blue spot (under UV light 366 mm) 

with Rf -value 0.66. The second conformity (addition of 

conc. HCl + Acetanhydride) test indicated the presence of 

two spots with Rf -values were 0.10 and 0.42 

respectively. Bioautographic examination (for both 

chloroform extract and standard aflatoxin B1) against 

Bacillus subtilis (Biological indicator) indicated the 

presence of one inhibitor zone with Rf value was 0.78. 

Preliminary experiment was carried out (in vitro) to 

demonstrate the antifungal potential of the plant (E. alata) 

extract against A. flavus using disc diffusion plate 

bioassay (Figure 1). The results in Table 1 revealed that 

the gradual concentrations of plant extract caused 

significant inhibition in both radial growth rate by percent 

of 41.55, 67.98, 88.83 and conidial production (Figure 2) 

by percent of 13.82, 50.00 and 90.42 of A. flavus, 

respectively . 

Data in Table 2 shows the inhibitory effect of plant 

extract on the number of germinated conidia especially 

after 6 h (absolutely inhibition was observed). It was 

noted that, the prolongation of incubation period 

decrease the inhibitory effect potential of plant extract at 

all concentrations used (Table 2). 

The results in Table 3 indicated clearly that all concentrations 

of plant extract induced significant inhibition in 

both mycelial growth (Figure 3) and aflatoxin B1 

production by percent of 33.49, 58.90, 79.76 and 65.75, 

83.90, 100 respectively. It is worthy to mention that such 

inhibitory effect of plant extract was more intense towards 

aflatoxin B1 production compared with mycelial growth at 

the same concentrations. 

A storage experiment was carried out to investigate the 

antagonistic potential of E. alata against aflatoxin 

production by A. flavus on maize grains and soybean 

seeds (in vivo) with special reference to their nutritional 

value. The obtained data indicated that employment of E. 

alata at concentrations 0.5 and 1.0% caused significant 

inhibition in the accumulation of aflatoxin B1 on maize 

grains and soybean seeds (Table 4) by percent of 63.81,

Figure 1. The antifungal activity of plant (E. alata) extract (arraw) 

against radial growth of A. flavus (A) using disc assay method. 

Table 1. Effect of different concentrations of E. alata (w/v) on radial growth rate (cm day -1 ) and conidial 

production ([conidia mm -2 ] X 10 4 ) of A. flavus grown at 28±°C for 96 h in dark. 

Treatment 

(E. alata w/v) 

Radial growth rate 

(cm day -1 ) 

Conidial production 

(conidia mm -2 X 10 4 ) 

Control 1.362 9.382 

E. alata 0.5% 0.796 8.102 

E. alata 1.0% 0.436 4.708 

E. alata 2.0% 0.152 0.584 

LSD at: 05 0.066 0.475 

Figure 2. Effect of different concentrations (A: control; B: 0.5%; C: 1.0%; D: 

2.0%) of plant (E. alata) extract on conidial production of A. flavus. 

Al-Qarawi et al. 2299


Table 2. Effect of different concentrations of E. alata (w/v) on number of germinated conidia (out of 50 conidia) after different 

incubation periods (hours). 

Treatment 


Number of germinated conidia (out of 50 conidia) after different incubation periods (h) 

6 h 12 h 24 h 

Control 20.67 49.33 50.00 

E. alata 0.5% 11.00 42.83 49.17 

E. alata 1.0% 6.17 36.67 48.67 

E. alata 2.0% 0.00 32.17 47.17 

LSD at: 05 4.7884 3.4099 2.4237 

Table 3. Effect of different concentrations of E. alata (w/v) on mycelial dry weight (g 50 ml -1 culture medium) and 

aflatoxin B1 production (µg 50 ml -1 culture medium). 

Treatment 


Mycelial dry weight 

(g 50 ml -1 culture medium) 

Aflatoxin B1 production 

µg 50 ml -1 culture medium µg g -1 dry weight 

Control 0.50154 351.6 701.04 

E. alata 0.5% 0.33354 120.4 360.97 

E. alata 1.0% 0.21616 56.6 261.84 

E. alata 2.0% 0.1015 0.0 0.0 

LSD at: 05 0.0331 22.192 

Figure 3. Effect of different concentrations [w/v] (A, control, B, 0.5%, C, 0.1%, D, 0.2%) of plant (E. alata) extract on 

mycelial growth of A. flavus. 

84.89 and 59.57, 89.72, respectively. Absolute inhibition 

of aflatoxin B1 accumulated in both maize and soybean 

grains was observed accompany with employment of 

2.0% (w/w) E. alata (Table 4). 

The results show that A. flavus caused significant 

deterioration in the nutritional value of maize grains 

(Table 5) and soybean seeds (Table 6). Such deterioration 

in the nutritional value of agricultural commodities 

was aware as significant decrease in their total lipids, 

crude fibre and crude protein accompanied with a 

significant increase in ash content (Table 5 and 6). The 

employment of powdered E. alata succeeded to

Al-Qarawi et al. 2301 

Table 4. Effect of different concentrations of E. alata (w/w) on aflatoxin B1 production (µg kg -1 ) on both maize 

grains and soyabean seeds stored under biological stress of A. flavus. 

Treatment 

Aflatoxin B1 production (µg /kg 

(E. alata w/w) 

-1 ) 

Maize grains Soyabean seeds 

control 212.22 131.20 

E. alata 0.5% 76.79 53.04 

E. alata 1.0% 32.06 13.48 

E. alata 2.0% 0.0 0.0 

LSD at: 05 23.096 17.137 

Table 5. Effect of different concentrations of E. alata (w/w) on nutritional value of maize grains stored under biological stress of 

A. flavus. 

Treatment 

( E. alata w/w) 

Total lipids* 

Nutritional value of maize grains Z 

Crude fiber* 

Ash content* Crude protein** 

Control (maize before storage) 4.7752 6.3021 4.2205 2.8240 

Maize + A. flavus 2.2577 1.0217 6.3914 1.8387 

E. alata 0.5% 3.6204 2.4314 5.3093 2.1400 

E. alata 1.0% 4.1111 4.7984 5.4991 2.7304 

E. alata 2.0% 4.1139 5.3666 5.6747 3.0224 

LSD at: 05 0.3725 0.5438 0.1567 0.3331 

*= Total lipids, crude fiber and ash content expressed as percentage (%). 

**= Crude protein = total nitrogen (mg/g dry wieght) X 6.25. 

Z= Total lipids, crude fiber, ash content and crude protein of E. alata (shoot system) were 1.0425, 4.2463, 7.7599 and 3.1341, 

respectively. 

Table 6. Effect of different concentrations of E. alata (w/w) on nutritional value of soyabean seeds stored under biological stress 

of A. flavus. 

Treatment 

(E. alata w/w) 

Total lipids* 

Nutritional value of maize grains Z 

Crude fiber* 

Ash content* Crude protein** 

Control (maize before storage) 18.5986 4.3731 6.4366 34.1012 

Maize + A. flavus 15.4252 1.9727 7.5799 12.2231 

E. alata 0.5% 16.3723 2.5918 6.8635 18.0028 

E. alata 1.0% 17.7983 3.5118 6.4536 24.6835 

E. alata 2.0% 18.2606 4.0598 6.3337 30.7081 

LSD at: 05 0.7713 0.2725 0.6477 11.735 

*= Total lipids, crude fiber and ash content expressed as percentage (%). 

**= Crude protein = total nitrogen (mg/g dry wieght) X 6.25. 

Z = Total lipids, crude fiber, ash content and crude protein of E. alata (shoot system) were 1.0425, 4.2463, 7.7599 and 3.1341, 

respectively. 

protect the maize grains (Table 5) and soybean seeds 

(Table 6) against the destroyer effect of A. flavus. The 

constrictive role of E. alata was directly proportional with 

its employed concentrations. 

DISCUSSION 

Aflatoxins are highly toxic secondary metabolites 

produced by certain strains of Aspergillus flavus caused 

many diseases for animals as well as humans. In our 

investigation, the chemical and biological confirmatory 

tests demonstrated the production of aflatoxins B1 by our 

experimental mold (A. flavus). Aflatoxins contamination of 

foodstuffs and agricultural commodities is a worldwide 

problem in tropical and subtropical regions where climatic 

conditions and storage practices are conducive to fungal 

growth and mycotoxin production (Thompson and Henke, 

2000). Many strategies including employment of biological 

products from plant origin are being investigated to


manage deterioration of agricultural commodities 

including animal fodder. In the same connection, 

investigations of antifungal potential of Ephedra were 

carried out recently (Bagheri et al., 2009; Parsaeimehr et 

al., 2010). In our results, the aqueous extract of E. alata 

was found to be effective in controlling the growth rate 

and conidial production by A. flavus. The antifungal 

potential of E. alata (aqueous extract) was extended to 

inhibit number of germinated conidia, mycelial growth 

(mycelial dry weight) and aflatoxin B1 production by A. 

flavus in variable percentages. In the same concept, the 

results of storage experiment indicated that application of 

plant (E. alata) powder was accompanied with significant 

decrease in contamination of both maize grains and 

soybean seeds with aflatoxin. The antimicrobial potential 

of E. alata noted here in the present study has been attributed 

to presence of Cis-314-methanoproline (Caveney 

et al., 2001), Citronellol (Rosato et al., 2007) and 

Heptadecane (Bagheri et al., 2009) which are recorded 

as photochemicals possess antimicrobial activity found in 

Ephedra. 

In geneal, oil seeds introduced into animal feeds as 

source of fiber, lipids and fat soluble vitamins, however 

the fungal contamination lead to significant deterioration 

in their nutritional value due to substrate utilization by 

molds. Previous studies indicated the decrease in fat, 

protein and fiber contents of many seeds and grains 

involved sorghum and soybean infected with different 

seedborne fungi and this was accompanied with an 

increase in ash content (Fapohunda and Olajuyiabe, 

2006; Ezekiel et al., 2010). In this regard, fiber content 

with available protein and fat formed ready substrate for 

fungal activities including production of hydrolytic 

enzymes (Mellon et al., 2007). The results of our study 

recorded similar significant decrease in protein, fiber, and 

lipids accompanied with an increase in ash content of 

both maize grains and soybean seeds due to the 

inoculation with A. flavus. The application of plant (E. 

alata) powder succeeded to decrease the deterioration of 

the nutritional value (fat, protein, fiber, ash contents) of 

both maize grains and soybean seeds due to infection 

with A. flavus. Regarding the justification manner, our 

results showed E. alata possess highly antagonistic 

potential against growth and aflatoxin production of A. 

flavus in agree with Bagheri et al. (2009) and 

Parsacimehr et al. (2010). 

Furthermore, with respect to E. alata, it has been 

shown that range plant contain suitable contents of 

protein and fiber (Nawwar et al., 1985) able to 

compensate the decrease in nutritional value of maize 

grain and soybean seeds due to mold contamination. Our 

results provide evidence for the first time to application of 

E. alata as naturally additive control aflatoxin contamination 

of fodder in addition to improve its nutritional 

value. This may be a novel non-chemical strategy to 

protect thousands of camels in Saudi Arabia against the 

mystery of fodder contamination with aflatoxins, the main 

reason for camels' death in our country. 


The authors wish to acknowledge Research Centers in 

both college of Science and college of food sciences and 

agriculture, King Saud University, Riyadh, Saudi Arabia. 

At the same time, we acknowledge Deanship of Scientific 

Research, King Saud University, Riyadh, Saudi Arabia. 

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DOI: 10.5897/AJMR11.619 



Primary resistance rates of Mycobacterium 

tuberculosis complex strains isolated from new 

tuberculosis cases: A 6-year observation 

Servet Kayhan*, Alper Akgüne , Hikmet Tereci and Ümit Tutar 

Samsun Chest Diseases and Thoracic Surgery Hospital, Turkey. 


The aim of the study was to evaluate the automated mycobacteria growth ındicator tube (MGIT) for drug 

susceptibility testing of Mycobacterium tuberculosis and to determine resistance patterns. We used 

BACTEC MGIT 960 System to determine the susceptibility of M. tuberculosis complex isolates to major 

anti tuberculous agents. Patients with single and first positive isolates were enrolled in the study. We 

have performed our drug susceptibility study between January 2005 and December 2010 for monitoring 

of drug resistance patterns in six years. A total of 1240 (77.16%) of the 1607 isolates were susceptible to 

all four of the antimycobacterial agents while 369 (22.96%) were found to be resistant to one or more of 

the drugs. The rate of isolates resistant to streptomycin (SM) was 6.84%, the other rates were 17.17% to 

isoniazid (INH), 5.28% to rifampicin (RIF) and 4.10% to ethambutol (ETM). Single drug resistance rates 

were found to be 12.13% for INH, 0.99% for RIF, 5.6% for SM, and 1.74% for ETM. The ratio of resistant 

isolates to all four drugs was 0.74% (n=12) and the prevalence of multidrug resistant isolates was 3.92% 

(n=63). It was concluded that resistance to INH and RIF continues almost in a straight line in the present 

study. Monitoring of drug resistance patterns is essential for accurate drug regimen in management of 

tuberculosis. 

Key words: Mycobacterium tuberculosis complex, primary resistance, ısoniazid, rifampicin. 

INTRODUCTION 

The World Health Organisation estimated the global 

burden of tuberculosis disease in 2009 as 9.4 million 

incident patients, 14 million prevalent cases and 2.38 

million deaths in the 2010 TB report 

(WHO/HTM/TB/2010-3; WHO/HTM/STB/2010-2). An 

estimated 11–13% of incident cases were HIV-positive. 

Among TB patients notified in 2009, an estimated 250, 

000 had multidrugresistant TB (MDR-TB). There were an 

estimated 440,000 cases of MDR-TB in 2008. The four 

countries that had the largest number of estimated cases 

*Corresponding author. E-mail: servet-kayhan@hotmail.com.tr. 

Tel: +903624400038. Fax: +903624400042. 

Abbrevations: MGIT, Mycobacterium growth indicator tube; 

MTC, Mycobacterium tuberculosis complex; TB, tuberculosis; 

INH, ısoniazid; RIF, rifampicin; ETM, ethambutol; SM, 

streptomycin; MDR-TB, multi drug resistant tuberculosis; XDR- 

TB, extensively drug-resistant tuberculosis. 

of MDR-TB in absolute terms in 2008 were China, India, 

the Russian Federation and South Africa. Primary 

resistance is the resistance pattern seen in new patients 

who have not previously been exposed to anti-TB drugs. 

Secondary resistance is the resistance pattern in patients 

with a previous history of anti-TB treatment and is due to 

ineffective chemotherapy. Fifty eight countries and 

territories have reported at least one case of extensively 

drug-resistant TB (XDR-TB). XDR-TB is defined as 

resistance to isoniazid and rifampicin (that is, MDR-TB) 

plus resistance to a fluoroquinolone and, at least, one 

second-line injectable agent (amikacin, kanamycin and/or 

capreomycin) (Bozkurt et al., 2010; EuroTB, 2007). Surveillance 

of primary and secondary resistance patterns is 

important in assessing the quality of chemo-therapy 

programs over several years and detecting errors in past 

treatments respectively. The resistant strains are 

characterised by a unique, lipid- containing and rigid core 

of the cell wall. The mycobacterial cell wall is less 

permeable to hydrophilic molecules than other

Table 1. Concentrations of drugs used in this study. 

Drug 

Concentration of drug after 

reconstitution (µg/mL) 

Volume added to MGIT 

Tubes for test (µL) 

Kayhan et al. 2305 

Final concentration in 

MGIT tubes (µg/mL) 

MGIT Streptomycin 83 100 1.0 

MGIT Isoniazid 8.3 100 0.1 

MGIT Rifampicin 83 100 1.0 

MGIT Ethambutol 415 100 5.0 

bacteria. That is why mycobacteria are resistant against 

the majority of drugs commonly used against bacteria 

(De Rossi et al., 2006). Anti tuberculous agents interfere 

with enzymes involved in cell wall biosynthesis (isoniazid, 

ethambutol, ethionamide), protein synthesis 

(streptomycin, other aminoglycosides, macrolides), 

transcription (rifampin), or DNA replication (quinolones). 

In M. tuberculosis spontaneous mutations occur at a 

frequency of approximately 10 -5 to 10 -8 (Inderlied and 

Nash, 1996). Since resistances to various drugs arise 

independently, the likelihood of spontaneous mutation to 

isoniazid and rifampin, for instance, is one in 10 -14 . The 

threat of dual mutations is one of the rationales for why 

an anti-tuberculosis therapy should consist of a 

combination of drugs (Brennan and Draper, 1994). To be 

successful in the management of tuberculosis, rapidly 

detecting the susceptibility of MTC strains to first line 

drugs is very important (Aydın et al., 2011). 

Many studies performed in Turkey confirmed that the 

resistance rates and patterns of MTC may show different 

characters in different regions (Talay et al., 2003; Ta ova 

et al., 1997). We aimed in this study to observe the 

regional and current rates of primary drug susceptibilities 

for MTC strains in active pulmonary tuberculosis by using 

clinical specimens (bronchial fluid and sputum). 


American Center for Disease Control (CDC) recommends 

performing susceptibility tests on specimens, within, 28 to 30 days 

after they are submitted to a laboratory, as we have done in this 

study (CDCP, 1993). 

Strains 

1607 MTC strains isolated from sputum and bronchial fluid samples 

of active pulmonary tuberculous patients who were diagnosed and 

treated in Samsun Pulmonary Disease and Chest Surgery Hospital 

from 2005 to 2010. We have reproduced positive samples by 

Lowenstein- Jehnsen (LJ) culture examination in a microbiology 

laboratory; then the single and first positive isolates from each 

patient enrolled in the study were used. 

Homogenisation and decontamination 

The bronchioloalveolar lavage fluids (BAL) and sputum samples of 

suspected tuberculosis patients were subjected to homogenisation 

and decontamination process by N-Acetyl-L-Cistein (NALC) and 

sodium hydroxide (NaOH) mixture and then to LJ culture . 

Culture and identification 

The resistance characteristics related to the primary antituberculous 

drugs (SM, INH, RIF, ETM) of 1607 units of MTC strains which 

were produced in Solid (Lowenstein-Jensen) and liquid (BACTEC 

12B and the Mycobacteria Growth IndicatorTube (MGIT)) nutrient 

media were detected by the BACTEC 960 (Becton Dickinson, USA) 

system. After the reproduction process, MTC and nontuberculous 

mycobacteria (MOTT) separation was made by a p-nitro- - - 

hydroxy-asetilamino-propiofen (NAP) test. 

Susceptibility test against primary antituberculous drugs 

We used BACTEC MGIT 960 System to determine the susceptibility 

of M. tuberculosis Complex isolates to major anti tuberculous 

agents. The susceptibility test was performed according to the 

manufacturer's recommendations (Becton, Dickinson and Company 

USA). This research is the result of several years study but we used 

same method (Trade mark; Becton, Dickinson and Company USA) 

and same drug concentrations in six years overall susceptibility 

tests. So there was not any change in the brand of drugs. Dilutions 

of drugs used in this study are shown in Table 1. 

Quality control 

For quality control of susceptibility tests, the ATCC 27294 (H37Rv) 

strain was used. The Rome Supranational Tuberculosis Reference 

Laboratory of World Health Organization and Turkey’s Refik 

Saydam Hygiene Center Tuberculosis Research Laboratories, 

cooperated in the preparation of a quality control program which 

was implemented in this study. 


We used SSPS.15 and minitab.16 programme for statistic analysis 

on INH and MDR ratios by years. 


We introduced the state of primary resistance in MTC to 

four major antituberculous agents in the last six years in 

Samsun province (the central city of Turkey’s Middle 

Black sea region, with a population of 1.3 million) and we 

also retrospectively compared these values with the other 

regional and national determinants on drug resistance. In 

this study, 16,932 patient specimens were sent to our


Table 2. The primary resistance rates of Mycobacterium tuberculosis complex isolates according to years. 

Type of resistance 2005 2006 2007 2008 2009 2010 Total (n) 

Single drug resistance 

SM (%) 1(0.32) 5(1.67) 12(5) 7(2.99) 5(2.02) 13(4.69) 43(2.67) 

INH (%) 27(8.64) 25(8.38) 32(13.33) 26(11.11) 21(8.46) 33(11.91) 164(10.20) 

RIF (%) 1(0.32) 5(1.67) 6(2.50) - 2(0.80) 2(0.72) 16(0.99) 

ETM (%) - 7(2.34) 7(2.91) 11(4.7) 2(0.80) 1(0.36) 28(1.74) 

Any (total) monodrug resistance 

SM(%) 7(2.25) 14(4.70) 28(11.67) 14(5.98) 23(9.27) 24(8.66) 110(6.84) 

INH(%) 49(15.80) 39(13.08) 58(24.17) 39(16.66) 41(16.53) 50(18.05) 276(17.17) 

RIF(%) 19(6.12) 11(3.69) 20(8.33) 5(2.13) 15(6.04) 15(5.41) 85(5.28) 

ETM(%) 5(1.61) 9(3.02) 20(8.33) 23(9.82) 4(1.61) 5(1.80) 66(4.10) 

Resistance to 2 drugs 

INH-RIF(%) 13(4.19) 3(1.01) 5(2.08) 1(0.42) 4(1.61) 5(1.80) 31(1.93) 

INH-SM(%) 3(0.96) 6(2.01) 5(2.08) 1(0.42) 7(2.82) 5(1.80) 27(1.68) 

INH-ETM(%) 1(0.32) 2(0.67) 6(2.50) 5(2.13) 1(0.40) 0 15(0.93) 

RIF-SM(%) 0 0 0 0 3(1.20) 0 3(0.18) 

RIF-ETM(%) 0 0 0 1(0.42) 0 1(0.36) 2(0.12) 

SM-ETM(%) 0 0 1(0.42) 0 0 0 1(0.06) 


INH-RIF-SM(%) 1(0.32) 3(1.01) 4(1.67) 0 5(2.02) 4(1.44) 17(1.05) 

INH-RIF-ETM(%) 2(0.64) 0 0 0 0 1(0.36) 3(0.18) 

INH-SM-ETM(%) 0 0 1(0.42) 3(1.28) 1(0.40) 0 5(0.31) 

RIF-SM-ETM(%) 0 0 0 0 0 0 0 


INH-RIF-SM-ETM(%) 2(0.64) 0 5(2.08) 3(1.28) 0 2(0.72) 12(0.74) 

MDR strains 

MDR(%) 21(6.77) 6(2.01) 14(5.83) 4(1.70) 9(3.62) 12(4.33) 63(3.92) 

Total(n) n:310 n:298 n:240 n:234 n:248 n:277 n:1607 

microbiology laboratory. Only the single and first isolates 

of each new patient enrolled in the drug susceptibility 

study were used. The test was performed on a total of 

1607 isolates from January 2005 to December 2010. 

Three hundred and eighty nine (24.20%) isolates were 

obtained from females and 1218 (75.80%) were obtained 

from male patients. All of the isolates were obtained from 

respiratory samples consisting of 113 (7.03%) 

bronchioloalveolar lavage (BAL) and 1494 (92.7%) 

sputum samples. 1240 (77.16%) of isolates showed sensitivity 

to all four drugs. The distribution of the samples 

studied over the years, and resistance profiles are 

presented completely in Table 2. 

Because of global increasing in multidrug resistance 

rates we used statistical analysis on type of drug and 

years to see the changes in our province. Statistic results 

for INH and MDR ratios were analysed by SSPS.15 and 

minitab.16 programmes (Table 3). INH drug resistance 

rates between 2005-2007 differed a 5% significance level 

according to the dual rate comparison over the years. 

INH drug resistance rates differed between 2005-2008, 

2006-2007 and 2007-2008, and these were statistically 

significant according to the dual rate comparison over the 

years. The relationship between INH drug sensitivity and 

years was determined (chi-square, p=0.025). MDR drug 

resistance rates differed between 2005-2008, 2006-2007 

and 2007-2008 and these were statistically significant 

according to the dual rate comparison over the years. 

Between 2005 and 2006, MDR resistance rates differed 

at a 5% significance level according to the same method. 

Due to previously predicted MDR-TB rates of 20% fort 

he present year(2011) in the world, resistance testing in 

all high risk cases for drug resistance is recommended by 

WHO (WHO/HTM/TB/2010,3). MDR-TB diagnosis and

Table 3. Statistical analysis of isoniazid and multidrug resistance of the study (with SSPS.15 and 

minitab.16). 

Years 

INH resistance 

MDR resistance 

Z account P Z account P 

2005-2006 0.94 0.348 2.34 0.019 

2005-2007 -2.44 0.015 -0.25 0.806 

2005-2008 -0.29 0.775 2.44 0.015 

2005-2009 0.01 0.992 1.11 0.268 

2005-2010 -0.76 0.447 0.69 0.491 

2006-2007 -3.27 0.001 -2.27 0.023 

2006-2008 -1.15 0.252 0.20 0.841 

2006-2009 -0.87 0.383 -1.13 0.259 

2006-2010 -1.66 0.097 -1.59 0.113 

2007-2008 2.04 0.042 2.37 0.018 

2007-2009 2.34 0.019 1.23 0.219 

2007-2010 1.68 0.093 0.86 0.390 

2008-2009 0.28 0.779 -1.26 0.207 

2008-2010 -0.43 0.667 -1.71 0.088 

2009-2010 -0.73 0.465 -0.42 0.676 

treatment must be done according to international 

guidelines (WHO/HTM/TB/2010-3; WHO/HTM/STB/2010- 

2). The World Report 2008 on antituberculosis drug 

resistance’ reported global risks for one or more drug 

resistance rates 0-56.3% in new cases, 0- 85.9% in 

patients treated previously, and 0-68.9% in all cases 

(WHO, 2009). According to the Turkish Ministry of 

Health’s 2008 report, 18,452 tuberculous patients were 

recorded. The incidence of tuberculosis in Samsun 

province in 2008 was 25.8 and the case speed was 28.0 

in Samsun. 414 new tuberculosis cases were diagnosed 

in 2005, 384 in 2006, 327 in 2007 and 346 in 2008 

(Bozkurt et al., 2010). 

According to the results in the Turkey (2008) TB report, 

a total of 4,963 drug susceptibility patient test were 

examined and 19.1% were found to be resistant to at 

least one drug. The highest rate of resistance among 

drugs belonged to isoniazid. Drug susceptibility tests 

detected the total multidrug resistant (MDR-TB) ratio to 

be 5.3% (263 people), 3% in new cases and 18.6% in 

cases having been previously treated (Table 4) (Bozkurt 

et al., 2010). We found 15.74% primary resistance to at 

least one drug in this study. Mono drug primary 

resistance rates for SM, INH, RIF, ETM were 6.84, 17.17, 

5.28 and 4.10% respectively. INH primary resistance 

rates are higher than the other major drugs in the present 

study and much greater than Turkey’s rates. MDR-TB 

has been identified as 3.92%. This rate is lower than the 

global estimated average ratio (20%) and Turkey’s ratio 

(5.3%) but higher than national primaries (3.0%) 

(WHO/HTM/STB/2010-2; Bozkurt et al., 2010). Due to a 

low ratio of susceptibility testing in the area (4963 tests in 

18452 cases 26.89% for 2008), these results for the 

country may not reflect the real resistance state in 


Turkey; however, this study reflects the real and nearly 

exact drug resistance rates of provincial tuberculosis. The 

limitation of our study is not having the secondary 

resistance rates at the same time. 

In recent years, a variety of resistance test studies 

against major drugs have been performed in our country 

and the comparative results are shown in Table 4. 

We have also analysed the rates of primary, secondary 

and total resistance in Turkey in Table 5, by using the 

2005-2008 data of Turkish Ministry of Health (Bozkurt et 

al., 2010). Secondary resistance rates were extremely 

higher than primaries. The ratio of drug resistance to INH 

was high compared with the other major drugs. In 

addition, multiple and single drug resistances in these 

years were close to each other on some level in our 

country and in our study. 

Regional differences in the resistance prevalence 

studies were published by authors. Arseven and his 

colleagues reported results from the provinces of the 

Eastern Black Sea region between the years 1985 and 

1990. A total of 564 (40.6%) of the 1388 culture-positive 

TB patients were determined to be resistant to at least 

one of the following: INH, RIF, SM, and ETM. Drug 

resistance rates against INH, RIF, SM and ETM were 

29.6, 17.1, 23.3 and 8.8% respectively. They measured 

the poly (more than one) drug-resistance rate to be 

22.4%, and the rate of MDR strains to be 13% (Arseven 

et al., 1995). Saral and his colleagues found the rates of 

resistance to INH, RIF, SM and ETM in a study as 24.6, 

15.8, 9.9 and 18.8% respectively. In the same study, the 

rate of MDR-MTC was reported as 14.7% (Saral et al., 

2007). Aydın et al. (2011) performed a similar study in 

Trabzon and found the single drug resistance to INH to 

be 6.1%, RIF 0.5% , SM 5.2% and ETM 2.4%. In the


Table 4. Some study results about primary resistance in Turkey. 

Reference City/Region Date 

Number of isolates 

(susceptibility test studied) 

Primary resistance rates of major anti-TB drugs 

SM(%) RIF(%) INH(%) EMB(%) 

Turkey (2008) Whole country 2005-2008 15735 8.37 6.77 13.37 4.23 

Present study Samsun 2005-2010 1607 6.84 5.28 17.17 4.10 

Yolsal et al Regional metaanalysis 1984-1989 368 8.8 5.7 14.4 2.2 

Yolsal et al Regional metaanalysis 1990-1995 2848 10.1 8.9 8.8 3.0 

Do an et al Sivas 1999-2004 316 15.2 4.1 19.9 2.5 

Güneri et al Aegean region 1999-2001 387 0 5.7 12.4 0 

Çetinkaya et al Elazı 1989-1994 125 16.6 7.6 11.5 1.2 

Sürücüo lu et al Manisa 1997-2003 285 13.3 6.0 14.4 8.4 

Talay et al. (2003) stanbul 1997-2000 135 13.3 3.0 8.8 2.2 

Otkun et al (38) Edirne 1996 44 32.0 11.0 30.0 9.0 

Karada et al. (2004) Samsun 2004 50 4.0 4.0 8.0 2.0 

Aydın et al. (2011) Zonguldak 2003-2005 99 13.1 2.0 18.2 3.0 

Aydın et al. (2011) Trabzon 2005-2010 212 13.7 5.7 17.5 5.7 

Korkmaz et al. (2002) Gaziantep 2002-2003 104 2.89 10.58 25 18.27 

Korkmaz et al. (2002) Gaziantep 2002-2003 104 2.89 10.58 25.04 25.96 

same study, of the 212 isolates, 25 (11.8%) 

strains were multiple drug resistant 10(4,7%) 

(Aydın et al., 2011). Many studies have been 

performed on relevant city analysis of TB for 

mono drug and multidrug resistance ( enol et al., 

2004; Uçar et al., 2010). For example Aydın et al. 

(2011) analysed 125 strains in the province of 

Zonguldak and reported the sensitivity rate to all 

drugs as 69.6%, resistance rate to INH as 23.2% 

and the rate of multidrug resistance as 8% in 2005 

(Aydın et al., 2008). In our country, it has been 

reported that INH+RIF resistant strains are 2.7% 

in Isparta, 7 and 3% (two studies done at different 

times) in Edirne, and 12.8 and 19.6% in 

Gaziantep (two different studies) (Yaylı et al., 

2003; Tansel et al., 2003; Balcı et al., 1999; Gani 

et al., 2002). Another study reported resistance 

rates in our city (Samsun) as 8% to INH, 4% to 

RIF, 4% to SM, 4% to ETM, 2% to pyrazinamide 

and 4% to MDR-TB strains in 2004 (Karada et 

al., 2004). 

The prevalence of MTC drug resistance varies 

from one part of the world to another (Jaffar et al., 

2005). In the United States, drug-resistant 

tuberculosis was detected in 14.2% in 1991 and 

10% in 1997 (Bloch et al., 1994; Espinal et al., 

2001). In the United States, isoniazid resistance 

was the most prevalant and accounted for 8%. 

Isoniazid resistance has ranged from 0% in New 

Caledonia to 7.9% in Mozambique, and was 10% 

in India (Pereira et al., 2005). An article from 

Saudi Arabia reported the fact that the rate of 

resistance to isoniazid varied from one part of the 

country to another such as: 4.2-7.2% in Riyadh, 

6% in Dammam and Taif, 10.3- 28.7% in Jeddah 

and 41% in Gizan. A 15-year study found rates of 

resistance to INH in different drug concentrations 

as follows: 12.5% resistance in INH (1 g/mL), 

and 2.9% resistance in INH (5 g/mL) in Dhahran 

(Jaffar et al., 2005). The prevalence of MDR-TB 

among new TB cases may differ in diverse 

geographies such as 14% in Estonia, 9% in 

China, Henan province, 9% in Lithuania, 9% in 

Russia, Ivanovo province, 5% in Iran and 4.5% in 

China, Zhejiang province (Espinal et al., 2001). In 

the early stages of tuberculosis treatment, the 

most potent bactericidal drug is isoniazid. INH 

resistance may also be an indicator of success in 

the treatment of tuberculosis. Storla and 

colleagues reported a high treatment failure in 

areas with high resistance to INH in Bangladesh 

(Storla et al., 2007). 

Directly observed therapy (DOT) in TB patients 

has been successfully completed in some 

countries such as the United States and a significant 

fall in TB and MDR-TB cases was provided 

contrary to the global rising in the number of

Table 5. The rates of primary, secondary and total drug resistance types in Turkey in 2005-2008. 

Drugs Resistance type 2005 (%) 2006 (%) 2007 (%) 2008 (%) 

INH Primary 9.0 10.7 11.9 11.3 

secondary 27.4 23.8 27.6 27.9 

Total 11.5 12.6 14.4 13.8 

RIF Primary 4.4 4.5 4.9 3.9 

Secondary 21.1 19.8 18.7 21.8 

Total 6.7 6.7 7.1 6.6 

ETM Primary 3.0 3.6 2.8 3.4 

Secondary 10.0 13.2 8.3 9.6 

Total 4.0 5.0 3.6 4.3 

SM Primary 7.0 8.4 7.1 6.5 

Secondary 15.2 17.0 13.8 12.9 

Total 8.1 9.7 8.2 7.5 

MDR-TB Primary 3.1 3.2 2.9 3.0 

Secondary 17.7 16.6 15.5 18.6 

Total 5.1 5.1 4.9 5.3 

multidrug-resistant tuberculosis. WHO declared the fact 

that between 1995 and 2009, a total of 41 millionTB 

patients were successfully treated in DOT, and up to six 

million lives were saved, including two million women and 

children. 

In conclusion, resistance to INH and RIF continues, 

almost in a straight line in Samsun Province. DOT must 

not be disregarded due to global rises in drug resistant 

MTC strains. Surveillance of the primary resistance 

patterns is important in assessing the quality of 

chemotherapy programs over several years. 

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DOI: 10.5897/AJMR11.622 



Cytokine genes expression in mice hepatocytes during 

malaria infection 

Saad Alkahtani 1 *, AL-Farraj S. A. 1 , Saud A. Alarifi 2 , AL-Eissa Mohammed saad 3 and 

Al-Dahmash B. 4 

1 Department of Biology, Teachers College, King Saud University, Riyadh, Saudi Arabia. 

2 Department of Zoology, Science College, King Saud University, Riyadh, Saudi Arabia. 

3 Department of Biology, Science College, Hail University, Hail, Saudi Arabia. 

4 Department of Medical Laboratory, College of Health Sciences, King Saud University, Riyadh, Saudi Arabia. 


The aim of this study was to investigate the inflammation genes (IL-1 , IL-6, TNF- and iNos) responses 

to Plasmodium chabaudi malaria in the liver of female C57BL/6 hepatocytes via mRNA expression due 

to infection with P. chabaudi at different time points. Mice were injected intraperitoneally (ip) with 10 6 P. 

chabaudi-infected erythrocytes and then scarified at days (0, 1, 4 and 8 respectively). RT-PCR was used 

to quantify liver inflammation genes. The levels of IL-1 , IL-6 and TNF were significantly increased at 

days 1 and 8. The total iNos were significantly increased at all days after infection. In conclusion, 

present data has shown that infection with P. chabaudi stimulated infalammation genes in the liver. 

Thus, we suggest the implication of oxidative stress due to outcome of malaria in mice hepatocytes 

according to its natural function need to be confirmed with a larger number of samples to be used as a 

reliable inflammation detection method. 

Key words: Inflammation, malaria, mice, liver. 

INTRODUCTION 

Despite decades of intense research, malaria remains a 

major global health problem with an estimated mortality 

of 1-2 million (Alkahtani, 2011). Due to increasing the 

resistance of parasites no effective vaccine is available 

and all efforts to develop an effective anti-malaria vaccine 

have failed to date (Bergmann-Leitner et al., 2009; 

Alkahtani, 2010). However, several vaccine candidates 

and vaccine platforms have yielded encouraging results 

in animal models of malaria, but have not been 

sufficiently evaluated in clinical trials. A rather convenient 

malaria model system is the Plasmodium chabaudi blood 

stage infection in mice, since P. chabaudi shares several 

characteristics with P. falciparum, the most dangerous 

human malaria species (Hernandez-Valladares et al., 

2005). 

Liver plays a very important role in malaria: It is the site 

*Corresponding author. E-mail: salkahtani@ksu.edu.sa. 

of pre-erythrocytic development of Plasmodium parasites, 

and also acts as an effector against malarial blood stages 

as recently shown (Krücken et al., 2005b; Wunderlich et 

al., 2005). Kupffer cells are able to eliminate parasitederived 

hemozoin and even Plasmodium-infected 

erythrocytes. As a result of this process, Kupffer cells 

become increasingly activated, which manifests itself as 

a production of NO (nitrogen oxide), diverse ROS (radical 

oxygen species), and the cytokines IL-1 and TNF . In 

addition, cytokines have primary roles in physiology, 

including neurophysiology, innate and acquired immune 

responses and wound healing, as well as pathogenesis. 

Some cytokines, such as TNF, LTs and IL-1 are proinflammatory, 

whereas others such as IL-4, IL-10 and the 

TGF family, are anti-inflammatory, being subsequently 

induced to inhibit these changes (Clark et al., 2010). NO, 

ROS and the cytokines result in enormous pathological 

complications in the liver. Indeed, IL-1 and TNF are 

thought to induce the acute phase response and a 

dramatic decrease in liver metabolism, both majorly


based on liver cells (Krücken et al., 2005a; Guha et al., 

2006). Recent results with deficient mice in the 

expression of those cytokines indicate that they are in 

general more sensible to insults resulting in neural 

damage. Some of the actions induced by TNF- and IFNincluding 

both beneficial and detrimental, are mediated 

by inducible nitric oxide synthase (iNOS) -derived nitric 

oxide (NO) production (Munoz-Fernandez and Fresno., 

1998). 

The present study was undertaken to investigate if the 

inflammation genes responses to Plasmodium chabaudi 

malaria in the liver of mice and to detect if these genes 

could be a good marker for inflammation and metabolism 

instability due to infection with Plasmodium chabaudi in 

mice hepatocytes. 


Animals and infection 

Normal female C57BL/6 mice 10-14 weeks old were obtained from 

the central animal facilities of Heinrich Heine University, Düsseldorf, 

Germany, and housed in plastic cages. Mice were bred and 

maintained under specified pathogen free conditions. Mice were fed 

with standard diet (Wohrlin, Bad Salzuflen, Germany) and water ad 

libitum. The experiments were approved by the State authorities 

and followed German law on animal protection. 

Blood stage malaria 

A non-clonal line of P. chabaudi was used (Wunderlich et al., 1988) 

exhibiting a very similar, but not identical restriction length 

polymorphism pattern to P. chabaudi (Kruecken et al., 2005b). 

Erythrocytic stages of P. chabaudi were passaged weekly in NMRI 

mice. From these mice, blood was taken and 10 6 P. chabaudiinfected 

erythrocytes were injected i.p. in the mice. Parasitemia was 

evaluated in Giemsa-stained blood smears. The total number of 

erythrocytes was determined in a Neubauer chamber. A total of five 

mice were scarified at each day (0, 1, 4 and 8 respectively) by 

cervical dislocation. Livers were removed and cut into smaller 

pieces and kept at -80°C. 

RNA-Isolation 

Approximately 250 mg frozen liver was homogenized with an 

ultraturrax in 5 ml Trizol (Peqlab Biotechnology, Erlangen, 

Germany) for one minute. After mixing with 1 ml chloroform for 15 s, 

the suspension was incubated for 15 min at room temperature and 

centrifugated at 3.000 x g for 45 min. After isopropanol precipitation 

of the supernatant, the pellet was washed twice with 80% ethanol 

and air-dried and dissolved in 200 µl RNase-free water. RNA 

concentrations were determined at 260 nm, and the purity of RNA 

was checked in 1% agarose gel. 

Quantitative real-time PCR 

All RNA samples were treated with DNase (Applied Biosystems, 

Darmstadt, Germany) for at least 1 h and then converted into cDNA 

following the manufacturer’s protocol using the Reverse 

Transcription Kit (Qiagen, Hilden, Germany). Quantitative real-time 

polymerase chain reaction (qRT-PCR) was performed using the ABI 

Prism ® 7500HT Sequence Detection System (Applied Biosystems, 

Darmstadt, Germany) with SYBR Green PCR Mastermix from 

Qiagen (Hilden, Germany). We investigated the genes encoding the 

mRNA for following proteins: 18S, Interleukin-1beta (IL-1 ), Tumor 

necrosis factor alpha (TNF ), Interleukin-6 (IL-6), Inducible nitric 

oxide synthase iNOS. All primers used for qRT-PCR were 

commercially obtained from Qiagen. PCR reactions were conducted 

as follows: 2 min at 50°C to activate uracil-N-gylcosylase, 95°C for 

10 min to deactivate UNG, 40 cycles at 94°C for 15 s, at 60°C for 

35 s and at 72°C for 30 s. Reaction specificity was checked by 

performing dissociation curves after PCR. For quantification, mRNA 

was normalized to 18S rRNA. The threshold Ct value is the cycle 

number selected from the logarithmic phase of the PCR curve in 

which an increase in fluorescence can be detected above 

background. The Ct is determined by subtracting the Ct of 18S 

rRNA from the Ct of the target ( Ct = Ct-target – Ct-18S rRNA). 

The relative mRNA levels of non-infected mice are described as a 

ratio of target mRNA copy to 18S rRNA copy = 2 – Ct . The fold 

induction of mRNA expression on days (0, 1, 4 and 8 p.i. was 

determined using the 2 - ct -method (- ct = ct day 0 p.i. - ct day 

8 p.i.). 

Blood analysis 

In sera, IL-1 , IL-6, TNF were measured using ELISA kits (R&D 

Systems, Minneapolis, USA) according to the manufacturer's 

protocols. Total iNOS was analyzed using a commercially available 

kit (R&D Systems). 


Two-tailed Student’s t-test and Fisher’s exact test were used for 

statistical analysis. 

RESULTS 

Characteristics of P. chabaudi infection 

The parasitemia of mice with 10 6 P. chabaudi-infected 

erythrocytes became evident on day 4 and rocketed to 

reach its peak (48%) on day 8 and then reduced rapidly 

to about 0.4% on day 12 (Figure 1). 

Inflammation genes expression 

Quantitative real-time PCR was used to detect changes 

in mRNA levels of different inflammation genes. The 

levels of IL-1 , IL-6 and TNF were significantly (P

-Fold change in mRNA expression 

Alkahtani et al. 2313 

Figure 1. Parasitemia (48%) of female C57BL/6 mice (n=20) infected with 10 6 P. chabaudi-infected erythrocytes. 

Figure 2. RT-PCR of IL-1 , IL-6, TNF and iNOS genes expression in the liver of Plasmodium chabaudi infected 

mice. The expression of these genes was measured at different times. The data present are the mean ±SE (n=5). 

*: significant value at (P


Table 1. Serum parameters of control (day 0) and infected (day 8) mice with 10 6 P. chabaudi. 

Parameters in blood sera 0 day 8 days p.i. p-value 8d p. i. vs. 0d 

Parasitemia 0.00 % 48 % < 0.01 

IL-1 [ng/l] 1.17 ± 0.2 4.5 ± 1.3

Clark I, Alleva L, Vissel B (2010). The roles of TNF in brain dysfunction 

and disease. Pharmacol. Therap., 128(3): 519-548. 

Clarka I, Allevaa L, Cowdenb W (2008). Understanding the role of 

inflammatory cytokines in malaria and related diseases. Travel Med. 

Infect. Dis., 6: 67-81. 

Guha M, Kumar A, Choubey V, Maity P, Bandyopadhyay U (2006). 

Apoptosis in liver during malaria: role of oxidative stress and 

implication of mitochondrial pathway. The FASEB J., 20: E439-E449. 

Guha M, Maity P, Choubey V, Mitra K, Reiter R, Bandyopadhyay U 

(2007). Melatonin inhibits free radical-mediated mitochondriadependent 

hepatocyte apoptosis and liver damage induced during 

malaria infection. J. Pineal Rea., 43(4):372-381. 

Häussinger D, Schliess F (2008). Pathogenetic mechanisms of hepatic 

encephalopathy. Gut, 57: 1156-1165. 

Hernandez-Valladares M, Naessens J, Iraqi FA (2005). Genetic 

resistance to malaria in mouse models. Trends Parasitol., 21: 352- 

355. 

Krücken J, Dkhil MA, Braun JV, Schroetel RMU, El-Khadragy M, 

Carmeliet P, Mossmann H, Wunderlich F (2005)b. Testosterone 

suppresses protective responses of the liver to blood-stage malaria. 

Infect. Immun., 73: 436-443. 

Alkahtani et al. 2315 

Krücken J, Mehnert LI, Dkhil MA, El-Khadragy M, Benten WPM, 

Mossmann H, Wunderlich F (2005)a. Massive destruction of malariaparasitized 

red blood cells despite spleen closure. Infect. Immun., 73: 

6390-6398. 

Munoz-Fernandez M, Fresno M (1998). The role of tumour necrosis 

factor, interleukin 6, interferon- and inducible nitric oxide synthase in 

the development and pathology of the nervous system. Progress 

Neurobiol., 56(3): 307-340. 

Nussler A, Silvio M, Liu Z, Geller D, Freewick P, Dorko K, Bartoli F, 

Billiar T (1995). Further characterization and comparison of inducible 

nitric oxide synthase in mouse, rat, and human hepatocytes. 

Hepatology, 21(6): 1552-1560. 

Wunderlich F, Dkhil MA, Mehnert LI, El-Khadragy M, Borsch E, 

Hermsen D, Benten WPM, Pfeffer K, Mossmann H, Krücken J 

(2005).Testosterone-responsiveness of spleen and liver in female 

lymphotoxin receptor-deficient mice resistant to blood stage 

malaria. Microbes Infect., 7: 399-409. 

Wunderlich F, Mossmann H, Helwig M, Schillinger G (1988). 

Resistance to Plasmodium chabaudi in B10 mice: influence of the 

H2-complex and testosterone. Infect. Immun., 9: 2400-2406.



DOI: 10.5897/AJMR11.625 



Phylogenetic analysis of the nematicidal actinobacteria 

from agricultural soil of China 

Xu Chuan Kun 1 , Lou Xiao Jun 1 , Xi Jia Qin 2 , Gu Lei 3 , Duan Chang Qun 1 , Mo Ming He 1 *, 

Zhang Ke Qin 1 , Yang Fa Xiang 4 and Fang Dun Huang 5 

1 Laboratory for Conservation and Utilization of Bio-Resources and Key Laboratory for Microbial Resources of the 

Ministry of Education, Yunnan University, Kunming, 650091, Yunnan Province, P. R. China. 

2 Zhengzhou Tobacco Research Institute of China Netherlands Trade Company (CNTC), Zhengzhou, 45000, P. R. China. 

3 Yunnan University Secondary School, Kunming, 650091, Yunnan Province, P. R. China. 

4 Yunnan Yunye Fertilizer Company Limited, Kunming, 650217, P. R. China. 

5 Yunnan Academy of Tobacco Agricultural Science, Yuxi 653100, P. R. China. 


The purpose of this study was to assess the diversity and biocontrol potential of nematicidal 

actinobacteria from agricultural soils. Two hundred soil samples were collected from 20 provinces 

(autonomous regions or municipalities) of China, and 4000 actinobacteria isolates were obtained. Of the 

4000 isolates evaluated, 533 (13.3% of total) and 488 (12.2%) respectively showed nematicidal 

properties to target nematodes Panagrellus redivivus and Bursaphelenchus xylophilus with nematicidal 

activities (NA) of more than 30%. The sum of strains with NAs of 90-100%, 80-90%, 60-80% and 30-60% 

was 55, 100, 127 and 251 to P. redivivus, while 37, 85, 111 and 255 to B. xylophilus, respectively. The 

most active isolates showed high host selectivity, in which only 101 isolates (5.1% of the total) were 

toxic to the both targets with NAs>30%. Additionally, 69 of the 101 isolates were randomly selected for 

species diversity analysis. Phylogenetic analysis placed the 69 actinobacteria in three families 

(Streptomycetaceae, Pseudonocardiaceae and Nocardiaceae) of the Actinobacteria with sequence 

similarity of 97.4-100%. The largest group was Streptomycetaceae, containing 58 isolates (84.1% of the 

total) that showed 97% to 100% sequence identity to 28 species of the genera Streptomyces (57 

isolates, 27 species) and Kitasatospora (1, 1). The Pseudonocardiaceae group contained 7 isolates 

(10.1% of the total) showing 98.5-99.7% homology to Amycolatopsis lurida (4 isolates) and A. 

niigatensis (3). The Nocardiaceae group included 4 isolates (5.8% of the total) with 99-99.6% sequence 

identity to one species N. fluminea. 

Key words: Nematicidal actinobacteria, nematode, biocontrol, phylogenetic analysis. 

INTRODUCTION 

Plant parasitic nematodes cause damages to a variety of 

agricultural crops throughout the world. Only the rootknot 

nematodes (Meloidogyne spp.) cause about US$100 

billion loss annually to a wide variety of crops worldwide 

(Oka et al., 2009). Currently, the application of chemical 

nematicides and fumigants is still the main strategy to 

*Corresponding author. E-mail: minghemo@yahoo.com.cn. Tel: 

+86871 5031396. Fax: +86871 5034838. 

control these pathogens. Although chemical nematicides 

are effective, easy to apply, and show rapid effects, they 

have begun to be withdrawn from the market in some 

developed countries owing to concerns about public 

health and environmental safety problems (Schneider et 

al., 2003). At present, several successful biocontrol 

agents mainly using nematophagous fungi have been 

used widely for nematode diseases (Tikhonov et al., 

2002). As antagonists of parasitic nematodes, 

nematophagous fungi exhibited their biocontrol traits by

predation, parasitism or poisonous effects. 

Extracellular enzymes of nematophagous fungi, especially 

the serine protease, served as the vital virulence 

factor in the process of infection (Huang et al., 2004). A 

few species of antagonistic bacteria have been 

developed into agents against plant parasitic nematodes, 

e.g. Bacillus thuringiensis and Pasteuria penetrans (Chen 

and Dickson, 1998; Javed et al., 2008). B. thuringiensis 

exhibited toxic effects against invertebrate by its 

parasporal inclusion and has been used widely (Wei et 

al., 2003; Meadows et al., 1989). P. penetrans is an 

obligate parasite of root knot nematodes (Meloidogyne 

spp.), but its fastidious life cycle and the obligate nature 

of parasitism have inhibited its mass culturing and 

deployment in field conditions. Actinobacteria, however, 

have so far received relatively little attention as potential 

biocontrol agents against plant-parasitic nematodes. 

Actinobacteria is a group of gram positive bacteria with 

a G+C content of over 55%. More than 70% of bioactive 

compounds are produced from these microorganisms 

and they have shown significant applications in pharmacy, 

industry, agriculture and environmental protection. 

Members of this group are best known for their ability to 

produce lytic enzymes, various secondary metabolites, 

including antibiotics. For example, avermectins includes a 

series of macrocyclic lactone derivatives produced by 

Streptomyces avermitilis that belongs to a new family of 

potent antihelminthic agents (Burg et al., 1979). Since the 

early 1980s, the avermectins and their derivatives have 

been used widely in the world to control parasitic 

nematodes and pests. The discovery and the huge commercial 

market of avermectins promoted an increasing 

number of researchers to study the biocontrol potential of 

actinobacteria. For example, Mishra et al. (1987) found 

that metabolites from 15 isolates of aerobic 

actinobacteria (screened from 502 actinobacteria) were 

toxic to the free-living nematode Panagrellus redivivus 

(Mishra et al., 1987). 

A species of the genus Streptomyces isolated from 

nematode suppressive soil inhibited the reproduction of 

Caenorhabditis elegans in the laboratory test and 

reduced tomato root galling caused by M. incognita in the 

greenhouse experiment (Dicklow et al., 1993). Some 

nematicidal actinobacteria were observed in eggs and 

cysts of Heterodera glycines (Nour et al., 2003) and H. 

trifolii (Hay and Skipp, 1993). Sun et al. (2006) isolated 

30 actinobacteria from root-knot samples and found that 

47% of them were virulent to eggs and juveniles of M. 

hapla. 

These investigations indicated that nematicidal 

actinobacteria are abundant in agricultural environments. 

However, no study has been performed to analyze the 

diversity and phylogenetic relationships of nematicidal 

actinobacteria from a wide range of agricultural soils. The 

objective of this study was to estimate the relative 

biocontrol potential of actinobacteria isolated from 20 

provinces (municipalities or autonomous regions) of 

China. We also presented a phylogenetic analysis to 

Kun et al. 2317 

characterize these nematode-antagonistic microorganisms. 


Soil sampling 

A total of 200 agricultural soil samples were collected from 20 

provinces (municipality or autonomous regions) of China (Figure 1). 

In each province (municipality or autonomous region), 10 samples 

were collected. For each sample, approximately 1 kg of soil was 

sampled randomly from the top layer (2-15 cm) over an area of 

more than 5 m 2 per field. Soil samples were spread out to air dry at 

room temperature for 2-3 days and sieved through a 2 mm sieve, 

then stored in glass bottles at 4°C until used. 

Isolation of actinobacteria 

To isolate the soil actinobacteria, 1 g of each dry sample was 

suspended in 10 m1 sterile distilled water and diluted 1000-fold. 0. l 

ml of the dilutions was spread on ISP5 medium (ISP5 medium: Lasparagine 

1g, glycerol 10g, K2HPO4 1 g, trace salts 1 ml, agar 18 

g, pH 7.2, added to 1000 ml with ddH2O, where the trace salts 

including FeSO4•7H2O 0.2 g, MnCl2•2H2O 0.1 g, ZnSO4•7H2O 0.1 g, 

added to 100 ml with ddH2O). The plates were incubated at 28°C 

for 2 weeks and actinobacteria were purified from single colonies 

using the same medium. 

Nematode inocula 

In this study, a free-living nematode, Panagrellus redivivus, and a 

pinewood nematode, Bursaphelenchus xylophilus, were used as 

target hosts for assaying the in vitro nematicidal activity of the 

actinobacteria. P. redivivus was cultured on autoclaved oatmeal as 

previously described (Walker and Barrett, 1991) and B. xylophilus 

was fed with a fungus as described by Dong et al. (2004). The 

juveniles were separated from media through the Baermann funnel 

technique (Gray, 1984), and suspended in the water for use. 

Nematicidal activity assay of actinobacteria 

Each purified isolate was inoculated into 50 ml of ISP5 liquid 

medium in a 150 ml flask. After incubation of 2 weeks at 28°C, 200 

rpm, the culture suspension without actinobacteria was obtained by 

sterile filtration and used for nematicidal activity assay. Briefly, 1 ml 

of the culture suspension was added into a well of a 24-well cell 

culture plate, and 0.1 ml nematode suspension (approximate 150 

juveniles) was added and mixed. After incubation at 28°C for 24 h, 

the mobile (live) and immobile juveniles were recorded by counting 

>150 individuals under a microscope. Those immobile juveniles 

were taken as dead when they could not revive within 12 h after 

being transferred to fresh WA and subsequently to tap water. Each 

treatment was replicated three times, and the experiment was run 

three times. 

Sequence generation and phylogenetic analysis 

Genomic DNA of actinobacteria was extracted using a bacterial 

genomic DNA extraction kit (BioTeke Corporation, China, 

Cat#:DP2001) and their 16S rRNA genes were amplified by PCR 

using the universal primer combination 27f (5 AGA GTT TGA TCC 

TGG CTC AG 3 ) and 1492r (5 GGT TAC CTT GTT ACG ACT T 3 ). 

The amplified products were purified using the Agarose gel DNA


Figure 1. Location of the 20 sampling sites (∗) in this study distributed on the map of China, which including two 

municipalities (BJ and SH), six autonomous regions (GX, NM, NX, XJ and XZ), and twelve provinces 

(Abbreviation meanings on the map, BJ: Beijing, GS: Gansu, GD: Guangdong, GX: Guangxi, GZ: Guizhou, HN: 

Hainan, HeB: Hebai, HeN: Henan, HLJ: Heilongjiang, HuN: Hunan, JL: Jilin, JS: Jiangsu, LN: Liaoning, NM: 

Neimenggu, NX: Ningxia, SD: Shandong, SX: Shanxi, SH: Shanghai, SC: Sichuan, XZ: Xizang). 

purification kit (TakaRa, code DV805A) and submitted to Beijing 

Genomics Institute for sequencing. The resulting sequences of 16S 

rRNA gene were compared with those available in the GenBank 

using the BLAST network service to determine their phylogenetic 

affiliation. Multiple alignments and sequence evolutionary distance 

calculations were carried out using CLUSTAL X version 2.0 

(Thompson et al., 1997). Phylogenetic analysis was performed 

using the MEGA software packages (Kumar et al., 2004), with gaps 

treated as missing data. Clustering was performed using the 

Neighbour-Joining method (Saitou and Nei, 1987). Bootstrap 

analysis was used to evaluate the tree topology of the Neighbour- 

Joining data by performing 1,000 resamplings (Felsenstein, 1985). 

The 16S rRNA gene sequences have been deposited in the 

GenBank database under accession numbers GQ357927- 

GQ357995. 

Data analysis 

Nematicidal activity (NA) was calculated using the formula: NA = 

IN/SN×100%, where IN represents the number of immobile 

nematodes and SN represents the sum of all nematodes counted 

(SN 100). Data were analyzed using the analysis of variance 

(ANOVA), and means were compared by the test of least significant 

difference (LSD) at P=0.05 using SPSS 11.0 for Windows (SPSS 

Inc., Chicago, USA). 

RESULTS 

Nematicidal activity of actinobacteria 

From each soil sample, 20 strains of actinobacteria were 

selected for nematicidal bioassay and these strains were 

selected mainly based on the differences of colony 

features, or randomly picked if colony features were 

similar, which resulting in a total of 4000 strains from 200 

samples for virulence test. Of the 4000 isolates tested, 

533 (13.2% of total) exhibited nematicidal activities with 

NA values more than 30% against the free-living 

nematode P. redivivus. These isolates showed

Isolate sum 

Isolate sum 

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Figure 2. Statistics of nematicidal actinobacteria from different sampling sites with different nematicidal 

activity (NA) to nematodes P. redivivus (A) and B. xylophilus (B). 

significant differences on their NAs against P. redivivus, 

which consisted of 55 isolates with NAs of 90-100%, 100 

with NAs of 80-90%, 127 with NAs of 60-80%, and 251 

with NAs less than 60% but more than 30% (Figure 2). 

Bioassay of the 4000 actinobacteria to plant-parasitic 

nematode B. xylophilus also was performed and 488 

isolates (12.2% of total) showed nematicidal virulence 

with NAs of 30-100%. The numbers of active isolates with 

NAs of 90-100%, 80-90%, 60-80% and 30-60% were 37, 

85, 111 and 255, respectively (Figure 2). 

Among the 4000 actinobacteria evaluated, the most 

active isolates showed the highest host selectivity to P. 

redivivus or B. xylophilus. Only 101 isolates (2.5% of 

total) were toxic to both targets with NA>30%. Among the 

101 strains, 4 (SDYM18, SDYM7, NMYC10 and NMCG3) 

showed the most potency to produce nematicidal 

metabolites with high NAs of more than 80% against both 

nematodes. Of the 155 actinobacteria strains with NAs > 

80% to P. redivivus, only 18 displayed similar NAs to both 

targets without statistically significant differences (P 

>0.05), while 54 isolates had no nematicidal activity 

against B. xylophilus. Similarly, of the 125 strains with 

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NAs>80% to B. xylophilus, the NAs of 11 isolates to the 

two tested nematodes were not significantly different 

(P>0.05) while 41 isolates had no nematicidal activity 

against P. redivivus. 

The four potential strains (SDYM18, SDYM7, NMYC10 

and NMCG3) causing high mortality against both hosts 

were assayed further after diluting the culture suspension 

by 2, 5 and 10 folds. Our results indicated that the 

original culture broths of the four actinobacteria exhibited 

powerful virulence (85.2% NA 100%) to both P. redivivus 

and B. xylophilus, but the former was more susceptible 

than the latter (P


Table 1. Nematicidal activity (NA) of four potential actinobacteria. 

Strain Nematode 

SDYM18 

SDYM7 

NMYC10 

NMCG3 

Mean NA (%)±SD under different diluent 

Origin broth 2 fold 5 fold 10 fold 

P. redivius 100±0 a 93.2±1.22 80.8±0.83 64.2±0.85 

B. xylophilus 95.7±0.72 b 89.3±0.52 78.2±1.04 50.6±0.91 

P. redivius 99.4±1.31 a 72.6±0.87 57.7±0.88 44.3±1.21 

B. xylophilus 88.3±0.89 c 71.7±1.11 51.6±0.62 37.2±0.82 

P. redivius 99.2±1.11 a 80.5±0.85 68.7±0.93 34.3±1.12 

B. xylophilus 83.2±1.14 d 79.8±1.21 50.8±1.08 34.8±0.94 

P. redivius 99.6±1.42 a 80.3±1.13 67.5±0.89 50.3±1.34 

B. xylophilus 85.2±0.26 c 75.6±0.92 56.8±1.22 41.3±0.82 

Means in the “Origin broth” column followed by the same letter do not differ significantly at p 0.05, according to multiple 

comparisons. 

amplified using primer pair of 27f and 1492r, and 

submitted for sequencing. The resulting sequences, 

about 1380 nucleotides, were compared with those 

available in GenBank using the BLAST network service 

(Table 2), and a phylogenetic tree was constructed to 

determine their approximate taxonomic affiliation (Figure 

3). Phylogenetic analysis placed the 69 strains in three 

families (Streptomycetaceae, Pseudonocardiaceae and 

Nocardiaceae) of the Actinobacteria with sequence 

similarity of 97-100% (Figure 3 and Table 2). 

The largest group was Streptomycetaceae and it consisted 

of 58 isolates (84.1% of the total) showing 97 to 

100% sequence identity to 27 species of the genera 

Streptomyces and 1 species of Kitasatospora. Among the 

Streptomyces strains, the isolates phylogenetically 

related to species of S. althioticus (8 isolates), S. 

flavotricini (7) and S. diastatochromogenes (6) 

dominated, with a total of 21 isolates (36.8% of the 

Streptomyces group). There were 13 isolates (accounting 

to 22.8% of Streptomyces members) respectively 

affiliated to S. xanthophaeus (4 isolates), S. lateritius (3), 

S. exfoliatus (3) and S. ciscaucasicus (3). Six isolates 

respectively identified as S. showdoensi (2 isolates), S. 

corchorusii (2) and S. viridochromogenes (2). The remaining 

17 isolates were respectively identified belonging 

to 17 different species of the genus Streptomyces. 

The Pseudonocardiaceae group contained 7 isolates 

(10.1% of the total) respectively showing 98.5-99.7% 

sequence identity to species of Amycolatopsis lurida (4 

isolates) and A. niigatensis (3). The last group, 

Nocardiaceae, only included 4 isolates (5.8% of the total) 

with 99-99.6% identity to a species N. fluminea. 

Of the four isolates showed strong nematicidal activity, 

NMYC10 and NMCG3 respectively showed 98.9 and 

99.3% sequence identity to S. diastatochromogenes and 

S. violaceochromogenes, while SDYM18 and SDYM7 

were assigned to the same species A. lurida with the 

homology more than 99%. 

DISCUSSION 

In this study, 4000 actinobacteria isolated from agricultural 

soils of a wide range of geographical regions were 

evaluated for their nematocidial activities. The proportions 

of strains capable of killing P. redivivus and B. xylophilus 

were 13.3 and 12.2% respectively when taking NAs 30% 

as a cutoff for virulence activity. These results indicated 

that there were a large number of nematocidial 

actinobacteria distributed in agricultural soils. Of the 69 

active actinobacteria submitted for phylogenetic analysis, 

57 (accounted to 82.6%) showed 97 to 100% homology 

to 27 species of the genus Streptomyces. Ruanpanun et 

al. (2010) isolated 83 actinobacteria from plant-parasitic 

nematode infested soils and found the predominant 

actinobacteria taxa was Streptomyces (97.6%). Similarly, 

Luo et al. (2006) reported that in the 20 actinobacteria 

isolated from eggs and females of root-knot nematodes, 

16 strains were members of Streptomyces. Most 

actinobacteria commonly isolated from soil belonged to 

the genus Streptomyces because they are more common 

in the environment and tend to have rapid growth rate 

and good sporulation compared to other actinobacteria 

(Williams and Vickers, 1988). This may explain why the 

nematicidal Streptomyces spp. had high occurrence 

frequencies. Beside the biocontrol application of S. 

avermitilis (Jayakumar, 2009; Wright et al., 1983), the 

producer of avermectins, many other species of 

Streptomyces also had the potential as biocontrol agents 

against parasitic nematodes. CR-43 T , a strain of S. 

costaricanus isolated from a nematode-suppressive soil, 

exhibited both antinematodal and antifungal activities in 

laboratory, greenhouse, and field trials (Dicklow et al., 

1993). An application for a patent for CR-43 T as a


Table 2. Nematicidal actinobacteria used for phylogenetic analysis in this study and their closest affiliation according to the partical 16S 

rRNA gene. 

Strain [Accession No.] Sample location The closest relative in the database [Accession No.] Similarity (%) 

SDYM9 [GQ357978] Shandong S. lateritius LMG 19372 T [AJ781326] 99.8 

BJH10 [GQ357963] Beijing S. lateritius LMG 19372 T [AJ781326] 99.9 

SCTS20 [GQ357973] Sichuan S. lateritius LMG 19372 T [AJ781326] 99.9 

HNHS7 [GQ357938] Henan S. exfoliatus NBRC 13191 T [AB184324] 99.8 

SXYC14 [GQ357967] Shanxi S. exfoliatus NBRC 13191 T [AB184324] 99.6 

HNYM7 [GQ357937] Henan S. exfoliatus NBRC 13191 T [AB184324] 99.9 

NXFQ8 [GQ357970] Ningxia S. griseoplanus AS 4.1868 T [AY999894] 99.9 

NMXM7 [GQ357930] Neimenggu S. kurssanovii NBRC 13192 T [AB184325] 99.9 

BJS2 [GQ357965] Beijing S. mauvecolor LMG 20100 T [AJ781358] 99.1 

GSPG8 [GQ357950] Gansu S. xanthophaeus NBRC 12829 T [AB184177] 99.7 

SCYT16 [GQ357972] Sichuan S. xanthophaeus NBRC 12829 T [AB184177] 99.7 

SCLZ7 [GQ357981] Sichuan S. xanthophaeus NBRC 12829 T [AB184177] 99.8 

SCL7 [GQ357982] Sichuan S. xanthophaeus NBRC 12829 T [AB184177] 99.9 

NXHG4 [GQ357980] Ningxia S. flavotricini NBRC 12770 T [AB184132] 99.9 

GSPT14 [GQ357949] Gansu S. flavotricini NBRC 12770 T [AB184132] 99.9 

HNDD1 [GQ357943] Henan S. flavotricini NBRC 12770 T [AB184132] 99.9 

NXHG3 [GQ357983] Ningxia S. flavotricini NBRC 12770 T [AB184132] 99.8 

HNGL19 [GQ357939] Henan S. flavotricini NBRC 12770 T [AB184132] 99.3 

NXLJ11 [GQ357979] Ningxia S. flavotricini NBRC 12770 T [AB184132] 99.9 

NXFQ2 [GQ357928] Ningxia S. flavotricini NBRC 12770 T [AB184132] 99.9 

GSSZ10 [GQ357948] Gansu S. showdoensis NBRC 13417 T [AB184389] 98.2 

GSSZ14 [GQ357953] Gansu S. showdoensis NBRC 13417 T [AB184389] 98.2 

BJL6 [GQ357961] Beijing S. ciscaucasicus NBRC 12872 T [AB184208] 99.8 

GZ22 [GQ357951] Guizhou S. ciscaucasicus NBRC 12872 T [AB184208] 99.7 

XZYM3 [GQ357971] Xizang S. ciscaucasicus NBRC 12872 T [AB184208] 99.9 

SHJ24 [GQ357969] Shanghai S. longwoodensis LMG 20096 T [AJ781356] 99.3 

HaNXJ7 [GQ357947] Hainan S. corchorusii NBRC 13032 T [AB184267] 97.9 

HaNYM16 [GQ357944] Hainan S. corchorusii NBRC 13032 T [AB184267] 97.4 

CZYS9 [GQ357960] Xizang S. mirabilis NBRC 13450 T [AB184412] 100 

SHDQ4 [GQ357977] Shanghai S. cinereoruber subsp. fructofermentans 

NBRC 15396 T [AB184647] 

BJH1 [GQ357966] Beijing S. phaeochromogenes NBRC 3180 T [AB184738] 99.9 

SHLJ2 [GQ357976] Shanghai S. diastatochromogenes ATCC 12309 T [D63867] 98.6 

BJS8 [GQ357964] Beijing S. diastatochromogenes ATCC 12309 T [D63867] 99.4 

SHLJ5 [GQ357975] Shanghai S. diastatochromogenes ATCC 12309 T [D63867] 99.1 

GXLY20 [GQ357952] Guangxi S. diastatochromogenes ATCC 12309 T [D63867] 98.7 

NMYC10 [GQ357929] Neimenggu S. diastatochromogenes ATCC 12309 T [D63867] 98.9 

SXTD7 [GQ357968] Shanxi S. diastatochromogenes ATCC 12309 T [D63867] 99.3 

NMCG3 [GQ357931] Neimenggu S. violaceochromogenes NBRC 13100 T [AB184312] 99.3 

JLLJ17 [GQ357927] Jilin S. flavoviridis NBRC 12772 T [AB184842] 99.9 

DLHG2 [GQ357958] Liaoning S. viridodiastaticus NBRC 13106 T [AB184317] 99.8 

DLDG2 [GQ357959] Liaoning S. longispororuber NBRC 13488 T [AB184440] 99.3 

GSL5 [GQ357956] Gansu S. coeruleofuscus NBRC 12757T [AB184840] 99.6 

BJL2 [GQ357962] Gansu S. alboflavus NBRC 3438T [AB184775] 99.9 

HaHD12 [GQ357942] Hainan S. coelicoflavus NBRC 15399T [AB184650] 99.9 

DLTS7 [GQ357957] Liaoning S. viridochromogenes NBRC 3113T [AB184728] 99.6 

JLIM5 [GQ357940] Jilin S. viridochromogenes NBRC 3113T [AB184728] 99.5 

HaNYM8 [GQ357945] Hainan S. malaysiensis ATB-11T [AF117304] 99.9 

98.8


Table 2. Contd. 

HaNXJ11 [GQ357946] Hainan S. glauciniger NBRC 100913 T [AB249964] 99.4 

JLGC14 [GQ357941] Jilin S. variabilis NBRC 12825 T [AB184884] 99.9 

JLYM14 [GQ357933] Jilin S. althioticus NRRL B-3981 T [AY999791] 98.3 

JLFQ8 [GQ357936] Jilin S. althioticus NRRL B-3981 T [AY999791] 97.5 

GSPG5 [GQ357954] Gansu S. althioticus NRRL B-3981 T [AY999791] 97.6 


JLFQ12 [GQ357935] Jilin S. althioticus NRRL B-3981 T [AY999791] 97.6 

GSPG4 [GQ357955] Gansu S. althioticus NRRL B-3981 T [AY999791] 97.6 


SHN26 [GQ357974] Shanghai S. althioticus NRRL B-3981 T [AY999791] 97.4 

BJY6 [GQ357985] Beijing N. fluminea S1 T [AF277204] 99 

SXYM10 [GQ357986] Shanxi N. fluminea S1 T [AF277204] 99.4 

SXS5 [GQ357987] Shanxi N. fluminea S1 T [AF277204] 99.6 

NMBC12 [GQ357988] Neimenggu N. fluminea S1 T [AF277204] 99.3 

DLYT2 [GQ357984] Liaoning K. arboriphila HKI 0189 T [AY442267] 99.7 

HaNHS4 [GQ357989] Hainan A. niigatensis LC11 T [AB248537] 98.5 



NXQZ2 [GQ357995] Ningxia A. lurida DSM 43134 T [AJ577997] 99.3 

SDYM18 [GQ357994] Shandong A. lurida DSM 43134 T [AJ577997] 99.4 

GSPG7 [GQ357993] Gansu A. lurida DSM 43134 T [AJ577997] 99.7 

SDYM7 [GQ357990] Shandong A. lurida DSM 43134 T [AJ577997] 99.7 

A: Amycolatopsis, K: Kitasatospora, N: Nocardia, S: Streptomyces. 

biocontrol agent for nematodes was filed by Research 

Corporation Technologies, Tucson, Ark, under exclusive 

license from the University of Massachusetts at Amherst 

(Esnard et al., 1995). Several strains of Streptomyces 

with antifungal activities also significantly reduced rootlesion 

nematode population densities in roots of both 

susceptible and resistant alfalfa varieties grown in field 

(Samac and Kinkel, 2004). Several species of 

Streptomyces, e.g. S. dicklowii (US Patent 5549889), S. 

cyaneogriseus (US Patent 5030650), have been patented 

as nematicidal biopesticides (http://www.patentstorm.us). 

Many other actinomycete genera also produced biologically 

active secondary metabolites of medical importance 

and showed promising biological activity including 

parasitism and antibiosis (Goodfellow and O’Donnell, 

1989). Rickards et al. (1998) reported that a strain belonged 

to the genera Amycolatopsis or Amycolata could 

produce the cyclic decapeptide antibiotic quinaldopeptin 

with nematicidal activity. A strain of Streptoverticillium 

albireticuli was found to show strong nematicidal activity 

against Caenorhabditis elegans and several fungal 

pathogens. This species exhibited hyphal growth on both 

external and internal surfaces of C. elegans and 

ultimately killed the nematode (Park et al., 2002). Of the 4 

isolates with strong nematicidal activity in this study, 

NMYC10 and NMCG3 showed 98.9 and 99.3% homology 

to S. diastatochromogenes and S. violaceochromogenes 

respectively, while the other two (SDYM18 and SDYM7) 

were assigned to A. lurida. To isolate a wider spectrum of 

actinobacteria, pretreating soils such as dry-heating 

(Nonomura and Ohara, 1969), using Streptomyces’ 

specific lytic actinophage (Kurtböke et al., 1992), and 

applying selective media supplemented with antibiotics 

(Williams et al., 1993) could all be employed. 

In this study, we used a free-living nematode and a 

plant parasitic nematode as target for bioassay. Among 

the 533 isolates and 488 isolates respectively showed 

nematicidal activity to P. redivivus and B. xylophilus, only 

101 isolates showed nematicidal properties to both target 

hosts. The host selectivity or sensitivity between 

actinobacteria and nematodes suggested that the 

nematicidal pathways or microbial actions were diverse. 

To determine the control targets of a nematicidal 

biopesticide, the host specificity should be taken into 

account. Additionally, the nematicidal actinobacteria in 

this study were screened based on the bioassay using 

culture broth. The metabolite composition of the four 

potential strains and their control effects in fields warrant 

further studies. 


This work was funded jointly by “National Key Sciences 

and Technology Program for Water Solutions” 

(2009ZX07102-004-04-01; 2012ZX07102-003), NSFC


Figure 3. Phylogenetic dendrogram obtained by neighbour-joining method of 16S rRNA gene sequences. Numbers at nodes 

are bootstrap values based on 1000 resamplings. Bar, 1% sequence divergence. Bifidobacterium psychraerophilum T16 T 

(AY174108) and Bifidobacterium mongoliense DSM 21395 T (AB433856) are used as outgroups. GenBank Accession 

Numbers for each strain are indicated in parentheses adjacent to each strain name.


(30970100), ZhengZhou Tobacco Research Institute 

(122009CZ0420), Yunnan Corporation of CNTC 

(2011YN03), National Development and Reform 

Commission, and Department of Science and Technology 

of Yunnan Province. 

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African Journal of Microbiology Research Vol. 5(16) pp. 2325-2328, 18 August 2011 


DOI: 10.5897/AJMR11.638 



A new recommended disinfectant for dental 

instruments 

Jamileh bigom Taheri 1 , Mahin Bakhshi 1 , Sedigheh Bakhtiari 1 *, Bahare Nazemi 2 , Fateme 

Fallah 3 , Sahand Rezaie 5 , Hamed Mortazavi 1 and Somayyeh Azimi 4 

1 Department of Oral Medicine, Faculty of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 

2 Department of Pediatric Dentistry, Faculty of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 

3 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 

4 Department of Oral Medicine, Faculty of Dentistry, Qazvin University of Medical Sciences, Qazvin, Iran. 

5 General Practitioner, Faculty of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 


Enduro hand sanitizer is a new disinfecting agent that has recently been introduced as an effective 

antibacterial, antifungal and antiviral agent. Considering the importance of infection control in dentistry, 

this study aimed to evaluate the antimicrobial effects of the foam and its ability to disinfect dental 

surfaces and instruments. An experimental study was designed to evaluate the microbial load of 50 

samples which were obtained randomly from dental surfaces and instruments before and after 

application of the disinfecting foam. All samples were cultured in blood agar and nutrient agar culture 

media and incubated at 37°C for 24 h. Colony count was subsequently performed after an additional 24 

h. Furthermore, to evaluate the efficacy of the foam on different bacterial species, a blank disc was 

coated with foam and approximated with the bacteria. The clear zone around each disc was measured 

and reported (mm) after 48 h. The significance of differences between the data retrieved before and 

after applying the foam was determined using the non parametric Chi-square test. None of the species 

were colonized after the application of the foam. It was also shown to significantly reduce the 

colonization of resistant bacterial strains and the standard microbial species (P


species/strains. Its antimicrobial effect remains for two 

hours after spraying the agent. Moreover, due to its nonalcoholic 

nature, the subcutaneous adipose tissue is 

preserved which makes Enduro hand sanitizer foam 

suitable for daily hand washing usage without causing 

excessive dryness and skin irritability. Longevity and 

biocompatibility of this potent antimicrobial agent, makes 

it superior to other disinfectants (Enduro hand sanitizer 

wwwoiltechnics.co.uk/Animalhygiene/Sanitizers/index). 

Hence, this study was performed to assess the efficacy of 

this product in disinfecting dental instruments. 


This study was a joint project conducted in the department of oral 

medicine and Microbiology, Department of Shahid Beheshti 

University of Medical Sciences, Tehran, Iran in 2009-2010. The disinfecting 

foam (Enduro Hand Sanitizer) was a product of Biotechnic 

Company in England. Fifty samples were obtained from moist 

dental instruments (surgical forceps, elevator and periodontal 

scaling instruments) using a swab before and after the application 

of the foam and were placed in BHI broth. The BHI was subsequently 

incubated at 37°C for 24 h. The samples were cultures on 

nutrient blood agar (simple agar) and the plates were re-incubated 

for an additional 24 h. Positive plates marked bacterial growth. In 

order to confirm the presence of bacteria and identify the bacterial 

strains in contaminated samples, lamellas were obtained from the 

positive plates and subjected to Gram staining after drying and 

fixing. For this purpose, each lamella was thoroughly covered with 

crystal violet dye for 1 min. and then rinsed out. Each specimen 

was then covered with lugol solution immediately for 1 min, washed 

out and immersed into alcohol for 20 to 30 s. Fuchsine was 

ultimately poured on the lamellas and washed after 1 min. The 

specimens were stored in a dry place and observed under ×100 

magnification to verify the presence and type of bacterial strains. 

In order to evaluate the effect of the foam on MRSA, Bacillus 

Subtilis and Entrobacteriacea, these bacteria were cultured on 

Muller-Hinton agar. A blank disk was coated with foam and three 

antibiogram discs comprising of Penicillin, Ciprofloxacin and 

Tetracycline were subsequently placed next to each other on the 

agar plate. The plates were incubated at 37°C for 24 h and the clear 

zone around the blank discs on the culture plates were measured 

and recorded. Clear zones with a diameter of 10 mm or more would 

indicate the effectiveness of the foam. Furthermore colony count 

was performed to determine CFU with standard experimental 

strains. The following bacteria with standard ATCC were used as 

control species to verify the growth of contaminating samples in 

agar plates: 

1. Bacillus ATCC 6633. 

2. Staph Aureus 2923. 

3. Entrococcus 13047. 

4. Staph Epidermis ATCC 12228. 

5. Candida ATCC 2091. 

The significance of differences between the data retrieved before 

and after applying the foam was determined using the non 

parametric Chi-square test (P

Table 1. Colony count of standard bacterial species before administration of 

Enduro hand sanitiser foam. 

Bacterial species Number of positive samples 

Stap.h epidermidis 36 

Pseudomonas 1 

Streptococcus 5 

Bacillus 15 

Entrococcus 5 

Lactobacillus 3 

Table 2. Colony count of standard bacterial species after administration of Enduro 

hand sanitiser foam. 

Bacterial species Number of positive samples 

Staph. epidermidis 5 

pseudomonas 0 

streptococcus 0 

Bacillus 1 

Entrococcus 0 

Lactobacillus 0 

anti microbial effects on different bacterial, viral and 

fungal species and effectively diminishes microbial contamination 

on dental surfaces (Enduro hand sanitizer). The 

results of the present study demonstrated that Enduro 

hand Sanitizer can effectively eliminate all pathogen 

microorganisms from dental instruments including surgical 

forceps and periodontal scalers. The importance of 

this data is that the tested pathogens are among the 

extremely contagious species which if disinfected effectively 

between every patient, would significantly prevent 

the spread of diseases and infection. Furthermore, the 

diameter of clear zone around the blank disk immersed in 

the foam was reported 15 mm for Bacillus, 12 mm for 

MRSA and 8 mm for Entroccocus. This indicates the 

sensitivity of common oral pathogens such as S. 

Epidermidis, Bacillus subtitles and candida species to the 

new product and further confirms its efficacy in 

eliminating the microbial load in dental environments. 

However, further evaluations are required to assess the 

efficiency of the foam in clinical settings and on different 

bacterial and viral species. 

The present study failed to evaluate the antiviral effect 

of this product however, according to the information 

provided by the manufacturers, the foam seems to render 

satisfactory results against Avian Flue H5N1 infection 

according to UK DEFRA standard. It has further been 

claimed that the foam can destroy spores of Aspergillus 

niger and Clostridum difficile (EN 1650 and EN 13704 

standards respectively). On the other hand, considering 

its strong anti bacterial effect against gram positive as 

well as gram negative resistant strains of bacteria, the 

antiviral effect of the foam may also be justified. 

Taheri et al. 2327 

Furthermore, due to the fact that HIV and HBV are highly 

sensitive microorganisms compared to vegetative 

bacterial pathogens, the veridical effect of this new agent 

may be conceived (Enduro hand sanitizer). Nonetheless, 

it appears that the antiviral and antifungal effect of the 

foam should be further studied. 

This study demonstrated strong bactericidal effects of 

the product however; the important point is the sensitivity 

of specific species such as Bacillus Subtilis to Enduro 

Hand Sanitizer. This bacterial strain is capable of 

producing spores and long lasting infections if not treated 

effectively. According to in vitro studies, the efficacy of 

disinfecting agents may be altered by the substances in 

culture media and thus need to be reported with cautious 

(Sen et al., 2009). The basic shortcoming of this study is 

therefore hidden within its design that is, investigating the 

efficacy of the new foam in controlled laboratory 

conditions on specific isolated microorganisms. Hence, it 

appears that future robust clinical trials should be 

designed to further study the antimicrobial effects of the 

Enduro hand sanitizer foam in accurate clinical settings. 

It is also important to note that despite the acceptable 

results retrieved from different disinfecting agents 

irrespective of the form (foam or solution), all of them are 

solely capable of reducing the microbial load on the outer 

surfaces of the instruments; the inner chamber of the air 

motor and the inner wall of the air and water sprays 

which are major sources of infection transmission, are not 

thoroughly cleaned. Therefore, researchers recommend 

the combination of disinfection, lubrication and heat 

sterilization of the air motors and hand pieces between 

every patient as an ideal measure to minimize the risk of


cross contamination. In other words, researchers believe 

that despite the satisfactory results retrieved from 

different disinfecting solutions and foams, these products 

are solely used to reduce the microbial load prior to heat 

sterilization. Furthermore with constant change in the 

bacterial flora and the emergence of new highly resistant 

species of bacterial pathogens in health care environments, 

there is always a great concern regarding the 

resistance of microorganisms to the available antibiotics 

and disinfectants. Therefore, there is still room for further 

studies on new products and benefitting from successful 

experiences of leading companies. 

Conclusion 

The present study provided an overall evaluation of the 

Enduro hand sanitizer foam. Considering the fact that the 

manufacturer has provided a list of sensitive microorganisms 

to this product, studies should be designed to 

assess bacterial and viral pathogens independently. 

Once the efficacy of the foam is firmly documented, it can 

be widely used in dental environments. 


This article was based on a undergraduate thesis by Dr 

Rezaie, which was successfully completed under the 

supervision of Dr Taheri with the close cooperation of the 

Oral Medicine department of dental school of Shahid 

Beheshti University of Medical School. 

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Bonten MJ (2002). Infection in the intensive care unit: Preventive 

stratrgies. Curr. Opin. Infect. Dis., 15: 401-405. 

CDC Guide line for infection in dental health care setting (2003). 

December, 14; 1-16. Available at http://www.cdc.gov/oral health 

/infection control /guidelines/index.htm 

Enduro hand sanitizer. Available at: 

http//wwwoiltechnics.co.uk/Animalhygiene/Sanitizers/index.html 

Oosthuysen J, Potgieter E, Blignaut E (2010). Compliance with infection 

control recommendations in South African dental practices: a review 

of studies published between 1990 and 2007. Int. Dent. J., 60: 181- 

189. 

Rauter S, Sigge A, Wiedeck H, Trantmann M (2002). Analysis of 

transmission pathway of pseudomonas aeroginos between patient 

and tap water outlets. Crit. Care Med., 30: 2222-2228. 

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acid on Candida albicans .Oral Surg. Oral Med. Oral 

Pathol. Oral Radiol. Endod., 90: 651-655. 

Silverman SJR (1987). Infection disease control and the dental office: 

Aids and other transmissible disease. Int. Dent. J., 37(2):108-113. 

Smith A, Creanor S, Hurrell D, Bagg J, McCowan M (2009). 

Management of infection control in dental practice. J. Hosp. Infect., 

71: 353-358. 

Yüzbasioglu E, Saraç D, CanbazS, Saraç S, Cengiz S (2009). A survey 

of cross-infection control procedures: knowledge and attitudes of 

Turkish dentists. J. Appl. Oral Sci., 17: 565-569. 

Zanetti F, Vannini S, Bergamaschi A, Baldi E, Stampi S (2004). 

Infection control in dental health care setting:results of a survey on 

current disinfection practices. Ig Sanita Publ., 60: 229-242.



DOI: 10.5897/AJMR11.642 



The functional roles of the residue tyrosine at position 

26 in staphylococcal enterotoxin C2 

Hongbo Wang 1,2 , Junyi Zhou 3 , Mingkai Xu 1 *, Huiwen Zhang 1 and Chenggang Zhang 1 

1 Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. 

2 Graduate School of Chinese Academy of Science, Beijing 100039, China. 

3 Shenyang Pharmaceutical University, Shenyang 110016, China. 


Bacterial superantigen Staphylococcus aureus enterotoxin C2 (SEC2) is a very potent activator of T 

cells. Previous crystal studies on SEC2 showed that residue Tyr26 which is located near the T cell 

receptor (TCR) binding sites could potentially participate in T cell activating. In SEC1, a superantigen 

highly homologous to SEC2, the residue Val26 played a critical role in stimulating certain V expansion. 

Moreover, residues 20, 22 and 26 in SEC1 determined its serologic cross-reaction. In order to 

investigate the potential roles of Tyr26 in SEC2, two SEC2 mutants SEC2(Y26V) and SEC2(Y26A) were 

constructed by site-directed mutagenesis. The activities and toxicities of the two mutants were 

determined both in vivo and in vitro. Results showed that both SEC2(Y26V) and SEC2(Y26A) remained 

potent immune stimulating activities compared with native SEC2. SEC2(Y26V) had a decreased 

pyrogenic effect on rabbit model. Both of the two mutants had significantly decreased binding affinities 

to anti-SEC2 IgG. Our result indicated that Tyr26 is a critical site for the serological characters of SEC2 

rather than for its superantigen activity. This may also provide an insight in constructing a novel mutant 

to substitute native SEC2 in cancer immunotherapy avoiding being neutralized by anti-SEC2 IgG in 

vivo. 

Key words: Staphylococcal enterotoxin C2, Site-directed mutagenesis, superantigen, serological, property. 

INTRODUCTION 

Microbial superantigens are a family of enterotoxins that 

share the ability to trigger excessive and aberrant 

activation of T cells (Llewelyn and Cohen, 2002). Unlike 

conventional antigens, SAgs bind to invariant regions of 

MHC class molecules outside the antigen-binding 

groove and are presented as unprocessed proteins to T 

lymphocytes expressing appropriate motifs on the 

variable domain of the chain(V ) of the T cell receptor 

(TCR) (Müller-Alouf et al., 2001). As a consequence, 

SAgs can activate an unusually high percentage of T 

lymphocytes and initiate massive releasing of cytokines 

which plays a pivotal role in T cell-mediated immune 

responses. The best characterized SAgs are 

Staphylococcal enterotoxins (SEs) and Streptococcal 

*Corresponding author. E-mail: mkxu@iae.ac.cn. Tel: +86 24 

8397 0380. Fax: +86 24 8397 0381. 

pyrogenic exotoxins (Spes) to date. Staphylococcal 

enterotoxins (SEs), a series of serological types of heat 

stable enterotoxins, produced by S. aureus, are the 

leading causes of gastroenteritis resulting from consumption 

of contaminated food (Balaban and Rasooly, 

2000). These toxins share common phylogenetic relationship, 

structure, function, and sequence homology. SECs 

are a group of highly conserved proteins with significant 

immunological cross-reactivity (Marr et al., 1993). 

According to minor epitope differences, SECs were 

further classified into three subtypes (C1-C3), which differ 

in only several amino acid residues near the N terminus 

(Hovde et al., 1990). 

Staphylococcal enterotoxin C2 (SEC2) can effectively 

activate the immune system and result in tremendous 

releasing of cytokines such as tumor necrosis factors- 

(TNF- ), interleukin-2 (IL-2), and interferon- (INF- ). 

This attribute makes SEC2 an effective ingredient for 

cancer immunotherapy in clinic in China (Chen, 2007).


But as an enterotoxin produced by S. aureus, SEC2 can 

cause toxic shock syndrome (TSS) at certain concentration, 

which limits its clinical application. Moreover, anti- 

SEC2 antibodies in patients’ serum could neutralize 

injected SEC2 and impair its effects. In Sweden, a mutant 

SEA has been fused to the Fab-fragment of tumortargeted 

monoclonal antibody for treatment of solid 

tumors in phase II trial. But toxicity and anti-SEA antibody 

were also the limiting factors for therapy (Shaw et al., 

2007). For construction of novel superantigen variants 

with decreased toxicity and low reactivity to human anti- 

SEC2 antibodies, it is necessary to give insight into the 

key residues in SEC2. 

In SEC1, valine in position 26 (Val26) was likely to 

interact with TCR directly and cause certain V expansion 

(Deringer et al., 1996). Previous researches on 

antigenic uniqueness indicated that residues 20, 22 and 

26 were critical for forming SECl- and SEC2-specific 

epitopes (Turner et al., 1992). While in SEC2, the 

crystallographical data showed that tyrosine in position 

26 (Tyr26) might locate in putative TCR binding site 

(Schad et al., 1997; Fields et al., 1996), and it was likely 

to expose on the molecular surface and influence 

molecular interaction between SEC2 and TCR (Turner et 

al., 1992; Papageorgiou et al., 1995). 

SEC2 and SEC1 are different in only seven residues 

near the N terminus (Bohach and Schlievert, 1989), while 

their superantigen activities exhibited some differences 

(Hovde et al., 1994; Wang et al., 2009a). Based on the 

high homology between SEC1 and SEC2, we 

hypothesize that the residue of Tyr26 might be important 

to the superantigen activities and serological characters 

of SEC2. 

In order to investigate the potential roles of Tyr26 in 

SEC2, in this study, two SEC2 mutants SEC2(Y26V) and 

SEC2(Y26A) have been constructed and their 

superantigen activities and toxicities were determined 

both in vivo and in vitro. Our results showed that the 

Tyr26 was not function-related with the immune stimulating 

activities of SEC2, but important for the serological 

characters of SEC2. The mutant SEC2(Y26V) with potent 

immune stimulating activities showed decreased toxicity 

and significantly reduced binding by SEC2-specific IgG, 

which suggested that the newly constructed mutant 

protein could be used as a potentially powerful candidate 

for cancer immunotherapy in the future. 


Plasmids, bacterial strains and cell line 

Expression vector pET28a(+) from Novagen (Darmstadt, Germany) 

was used to transform SEC2 mutant gene into the host. E. coli 

BL21(DE3) from Novagen was cultured in Luria–Bertani (LB) 

medium for expressing SEC2 mutant protein. Murine hepatoma 

Hepal-6 cell line used in this study was purchased from American 

Type Culture Collection (ATCC) and maintained in RPMI 1640 

medium with 10% (v/v) fetal bovine serum (Gibco, USA). 

Recombined plasmid pET-28a-SEC2 which contains the entire 

SEC2 cDNA (Accession number AY450554) was constructed and 

conserved in our lab (Xu and Zhang, 2006). 

Chemicals and enzymes 

All chemicals are analytical grade. Pfu DNA polymerase and 

restriction enzymes were purchased from Takara Biotechnology Co 

(Dalian, China); isopropyl-D-thiogalactopyranoside (IPTG) and 

methylthiazol tetrazolium (MTT) were from Sigma Chemical Co (St 

Louis, MO, USA); Ni-NTA His-Bind resin was from Qiagen 

(Germany); the DNA Gel Extract kit and Mini-preparation of plasmid 

kit were from BioDev-Tech Co (Beijing, China). 

Experimental animals 

Female BALB/c mice (6-8 weeks old, 20-25 g) and adult New 

Zealand white rabbits weighing 2.0-2.5 kg were purchased from the 

Experimental Animal Center, China Medical University (Shenyang, 

China). 

All experimental animals were maintained under specificpathogen-free 

conditions on a 12 h light/dark cycle. All experiments 

were carried out under the guiding principles for the care and use of 

laboratory animals approved by Animal Care Committee of China 

Medical University. 

Site-directed mutagenesis 

We constructed two mutant SEC2, SEC2(Y26V) and SEC2(Y26A), 

by converting the Tyr at position 26 to Val and Ala respectively. The 

conceptual ideas behind this substitution design were as follows: 

we introduce Val into position 26 of SEC2 because valine is the 

residue in position 26 in SEC1, furthermore we introduce Ala into 

position 26 of SEC2 to diminish the effect of Tyr in this position. 

The SEC2 mutant genes were constructed by sequence overlap 

extension. All the primers related with over-lap PCR were listed in 

Table 1. The PCR-generated fragments containing substituted 

oligonucleotides were digested by EcoR I and Xho I, and ligated 

into plasmid pET28a(+) digested with the same enzymes. The 

recombined expression plasmid was transformed into E. coli 

BL21(DE3) and positive clone was identified by DNA sequencing. 

Expression and purification of mutant protein 

For expressing SEC2 mutant protein, the positive clone of 

transformed E. coli BL21(DE3) was incubated in LB for 10 h. The 

culture was then inoculated at a ratio of 1:100 (v/v) into LB medium 

supplemented with 50 g kanamycin ml -1 and incubated at 37°C 

with vigorous shaking until the OD600 reached 0.7. Then 1.0mM 

IPTG was added into the culture and the incubation was continued 

for 4 h more with vigorous shaking at 30°C. The cells were 

harvested by centrifugation at 5,000 rpm and resuspended with icecold 

loading buffer (50 mM NaH2PO4, 500 mM NaCl, 10 mM 

imidazole, pH 7.9). The cells were disrupted by sonication on ice 

followed by centrifugation (10,000 rpm, 30 min). The supernatants 

were collected and loaded onto a Ni-saturated chelating Sepharose 

column pre-equilibrated with loading buffer. After removing nonspecifically 

binding protein with washing bufferl (50 mM NaH2PO4, 

500 mM NaCl, 40 mM imidazole, pH 7.9), SEC2 mutant protein was 

released from the column by washing buffer (50 mM NaH2PO4, 500 

mM NaCl, 250 mM imidazole, pH 7.9) and dialysed against PBS 

(137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 

pH 7.4) at 4°C for 48 h to remove the high concentration of

Table 1. Primers for amino acid substitution by over-lap PCR. 

Primers Amino acid substitution Primer sequence a 

P-1 5 -CGGAATTCGAGAGTCAACCAGA-3 

P-2 26Y V 5 -CATCATATAATACTTTCATATTACCCATCG-3 

P-3 5 -GGGTAATATGAAAGTATTATATGATGATC-3 

P-4 26Y A 5 -CATCATATAATGCTTTCGTATTACCCATCG-3 

P-5 5 -GGGTAATACGAAAGCATTATATGATGATC-3 

P-6 5 -TCGCTCGAGTTATCCATTCTTTGTTG-3 

a The primers were designed for special amino acid substitution. The sequences of mutant residues are stressed in 

boldface. The recognition sites of EcoR I and Xhol I are underlined. 

imidazole. The purity of SEC2 mutant protein was estimated by 

SDS-PAGE and the concentration of mutant protein was 

determined by Bradford assay using BSA as the standard. 

Stimulating murine T-cell proliferation assay 

To investigate the activity of stimulating T cell proliferation by 

mutant SEC2, splenocytes from 6- to 8-week-old BALB/c female 

mice were collected as experimental target. The mutant SEC2 and 

the native SEC2 dissolved in 1640 medium were filled into the wells 

of 96-well microtitre plates in four different concentrations (10, 100, 

1000, and 10,000 ng/ml). There were three repeated wells at each 

concentration. RPMI 1640 medium with 10% FBS was served as 

negative control. Murine T cells were seeded into the experimental 

and negative control wells at a density of 8×10 5 cells per well. 

The proliferation index (PI) was determined by MTT assay. The 

splenocytes were incubated at 37°C with 5% CO2 for 72 h. The 

incubation was continued for 4 h more after 25 l MTT (5 mg/ml, 

dissolved in PBS) had been added to each well. Then the T cells 

were collected by centrifugation at 1,000 rpm for 10 min. 120 l 

DMSO was added into each well to redissolve the pellet on the 

bottom. To fully dissolve the formazan produced by T cells, the 

plate was vigorously shaking for 20 min. The absorbance was 

measured with a microplate reader at 570 nm, using a reference 

wavelength of 630 nm. The final absorbance is the difference 

between these two readings. The proliferation index (PI) was 

calculated with the following equation: 

PI=Abs value of the experimental well / Abs value of the negative 

control well. 

Antitumor activity 

To further investigate the superantigen activity of mutant SEC2, the 

inhibitory effects on tumor cell was determined by MTT assay. 

Splenocytes from 6- to 8-week-old BALB/c female mice were 

served as effector cells and Hepal-6 cell as target. Native SEC2 

and the mutants diluted with RMPI 1640 containing 10% FBS were 

separately filled into the wells of 96-well microtitre plate at 10ng/ml, 

100 and 1,000 ng/ml in triplicate. In experimental wells, effector 

cells (5×10 5 cells/well) and target cells (2.5×10 4 cells/well) were 

mixed in the present of native SEC2 or the mutants in different 

concentrations. Wells contained protein and lymphocytes only were 

served as lymphocytes releasing control. Wells contained Hepal-6 

cells alone were served as unsettled tumor cell control. The blank 

wells were RMPI 1640 with 10% FBS only, and BSA was served as 

negative control. 

After incubation for 72 h at 37°C with 5% CO2, 25 l MTT was 

added into each well, and the cells were incubated for 4 h more. 

Wang et al. 2331 

The cells were collected by centrifugation and redissolve with 

DMSO. The absorbance was measured with a microplate reader at 

570 nm, using a reference wavelength of 630 nm. Tumor growth 

inhibition (%) was calculated with the equation: 100 - [(Abs value in 

protein-treated cells well - Abs value in lymphocytes-releasing well) 

/ (Abs value in unsettled tumor cells control wells - Abs value in 

blank control wells)] ×100. 

Rabbit model assay 

Rabbit model was used to compare the febrile response of SEC2 

and the mutants in vivo. Each of the experimental animals was 

fixed and the rectal temperature was measured for at least 90 min 

before injection. Qualified rabbits used in this assay must have 

stable body temperatures ranging from 38.6 to 39.5°C. Native 

SEC2 and the mutants dissolved in PBS were injected into rabbits 

at the does of 10 g/kg (body weight). Three animals were injected 

with each protein. PBS was served as negative. Rectal temperatures 

of rabbits were measured with indwelling rectal thermometers 

right after pyrogen administration continually for 3 h. The rectal 

temperature change ( T) of rabbit was accepted as an index of 

pyrogen effect in vivo, and it was calculated by subtracting the 

temperature immediately before injection from the temperature at 

each time point after the pyrogen injection. 

Proteolysis susceptibility assays 

To investigate whether the trypsin-resistance of SEC2 mutants 

were affected by amino acid substitution, purified mutanted proteins 

and native SEC2 (100 g/ml) were respectively incubated at 37°C 

with trypsin at a trypsin/protein ratio of 1:25 (w/w). After desired 

periods of time, the enzymolysis was terminated by boiling in SDS- 

PAGE sample buffer for 5 min and analyzed by SDS-PAGE. 

Detection of mutant SEC2 by ELISA 

ELISA assay was carried out in accordance with the instruction of 

the kit (National institute for the control of pharmaceutical and 

biological products, Beijing, China). Briefly, the polystyrene plates 

were coated with 100 l of 5 g/ml rabbit anti-SEC2 IgG diluted in 

carbonate buffer overnight. The excessive IgG which did not bind to 

the wells was washed off by PBS containing 0.05% Tween 20 

(PBS-T20), then unabsorbed sites were blocked with 1% gelatin in 

PBS for 30 min at 37°C. Mutant SEC2 and the native dissolved in 

PBS-T20 buffer were added into the wells separately. The plate 

was incubated for 2 h at 37°C. Then the wells were emptied and 

washed by PBS-T20 buffer. Rabbit anti-SEC2 IgG-HRP conjugate 

was added, and the plate was incubated for 1.5 h at 3 7°C. The


Figure 1. Results of murine T-cell proliferation by SEC2 mutants and native SEC2. 

Purified proteins of SEC2 mutants or the native were incubated with murine splenocytes 

for 72 h before the proliferation effects were determined by MTT assay. Value on the yaxis 

represents the average proliferation index (PI)± SD of triplicate values. Each 

purified protein was tested in at least three separate assays. 

uncombined enzyme-linked antibody was washed off by PBS-T20, 

and 100 l substrate (0.04% ortho-phenylene-diamine) was added 

into the wells and incubated for 30 min at 37°C. Reactions were 

terminated by addition of 50 l 2 mol/L H2SO4, and the absorbance 

was measured at 450 nm. 


Results are presented as the mean ± SD. Statistical analysis was 

performed using Student’s t test. A P-value < 0.05 was considered 

statistically significant. 

RESULTS 

Construction and expression of the mutant SEC2 

To construct SEC2 mutant protein, over-lap PCR was 

employed. The mutant SEC2 genes were successfully 

constructed and cloned into the expression vector pET- 

28a (+). The recombinant expression vectors were 

transformed into E. coli BL21 (DE3) respectively for 

expressing mutant proteins. The positive clones were 

identified by DNA sequencing. 

For expressing SEC2 mutant proteins, the positive 

clones with the right mutagenesis introduced were 

respectively cultured in LB medium containing 50 mg 

kanamycin ml -1 till the OD 600 reached 0.7. The 

expressions of mutant proteins were performed by IPTG 

inducing at 30°C for 4 h. After purification with Ni-NTA 

His Bind Resin, both of the two mutant proteins shown as 

a single band on SDS-PAGE were of purity of more than 

95%. 

Murine T-cell proliferation activity 

The activities of stimulating T cell proliferation by SEC2 

and the mutants were investigated. Series of ten-fold 

dilutions of native SEC2 and the mutants dissolved in 

PBS were filled into the wells of a 96-well microtitre plate 

with murine T cells. After 72 h incubation, the proliferation 

index of murine T cell was determined by MTT assay. As 

the experimental data reflected, both native SEC2 and 

the mutants could induce murine T cells proliferation in a 

dose-dependent manner. The ability to induce T cell 

proliferation of SEC2 mutants were similar with that of the 

native, without significant differences at any concentration 

(P>0.05; Figure 1). This result indicated that the 

amino acid substitution at position 26 with Ala or Val had 

not significantly affected the stimulating activity of SEC2. 

Examination the antitumor activity 

To further investigate the superantigen activity of mutant 

SEC2, MTT assay was employed to compare the

Figure 2. MTT assay was employed to compare the tumor cell growth inhibition effects between 

SEC2 and the mutants. Tumor cells and the splenocytes were mixed and seeded into the wells at 

the density of 2.5×10 4 cells/well and 5×10 5 cells/well respectively. Different concentrations of SEC2 

or the mutants were incubated with the cells at 37°C for 72 h before the absorbance was 

measured. BSA was used as negative control. Data shown are representative of at least three 

separate experiments, and the values represent the mean ± standard error of the mean. 

anti-tumor activity of SEC2 and the mutants. Hepal-6 

cells (target cells) and splenocytes (effector cells) were 

incubated in the present of proteins in different concentrations 

for 72 h before the absorbance was measured. 

All the proteins tested in this study exhibited predominant 

anti-tumor abilities compared with negative control 

(P0.05). This indicated that the amino 

acid substitution in position 26 had little influence on the 

superantigen activity of SEC2. 

Toxicity 

The Rabbit model assay was carried out to investigate 

the pyrogenic effect of mutant SEC2. The rectal 

temperature of each experimental animal was continually 

measured for 3 h after pyrogen injection. The results 

showed that both SEC2 and the mutants could induce the 

enhancement of temperature in experimental animal 

significantly compared with negative control (P0.05). Whereas SEC2(Y26V) exhibited a 

decreased ability to induce fever at 2h, 2.5h, and 3h 

compared with SEC2. Furthermore, the enhancement of 

temperature in experimental animal injected with 

SEC2(Y26V) reached its top at 1.5 h after pyrogen 

injection. But it is noteworthy that the body temperature of 

animal injected with SEC2 or SEC2(Y26A) rose 

continually although the experiment. 

Susceptibility to trypsin 

In order to determine whether the minor decreased 

pyrogenic effect of SEC2(Y26V) in the later stage of 

pyrogen experiment was resulted from its degradation, 

trypsin digestion assay was performed to evaluate the 

stability of the two mutants. The result was determined by 

15% SDS-PAGE and showed that there was no significant 

difference in the trypsin susceptibility between native 

SEC2 and the mutants (Figure 4). This result suggested 

that the amino acid substitution at position 26 did not 

affect the instability of SEC2, and the decreased 

pyrogenic activity of SEC2(Y26V) was not resulted from 

its degradation.


Figure 3. The pyrogenicities of SEC2 and the mutants in rabbit model. The body 

temperature of experimental rabbit was continually measured for 3h right after 

protein injection. PBS solution was served as negative control. The value of y-axis 

represents the rise of temperature. 

Figure 4. Susceptibilities of SEC2(Y26V), SEC2(Y26A), and 

native SEC2 to trypsin were compared. Each protein was 

mixed with trypsin at a trypsin/protein ratio of 1:25 (w/w). The 

digestive reaction was performed at 37°C for time gradient 

and terminated by boiling for 5 min at 100°C. The result was 

analyzed by 15% SDS-PAGE.

Figure 5. Comparison the anti-SEC2 IgG binding affinity of native SEC2 and the mutants 

through ELISA. The absorbance was measured and calculated as the mean readings of 

triplicate samples ± SD. 

Evaluating epitope change of mutant SEC2 by ELISA 

ELISA assay was employed to investigate whether the 

Tyr at position 26 determines the epitope of SEC2. Both 

of the two mutants showed significant decreased binding 

affinities to SEC2-specific IgG compared with native 

SEC2 (P


side chain of Try26 was completely removed by Ala 

substitution in SEC2(Y26A). A reasonable explanation for 

this phenomenon is that Try26 was not the key residue 

for the immune stimulating activity of SEC2. 

Previous crystal structure study on SEC2 conveyed 

that residues 20, 22 and 26 potentially interacted with 

TCR (Papageorgiou et al., 1995). Although there was no 

direct evidence that Tyr26 in SEC2 participated in the 

interaction of SEC2 with TCR, some researchers 

reported that Val26 in SEC1 recognized the specificity of 

TCR V of SEC1. Mutant study in SEC1 showed that 

SEC1(V26 Y)with a single amino acid substitution at 

position 26 could significantly enhance the expansion of 

V 13.1 but decreased the expansion of V 3 (Deringer et 

al., 1996). 

So, on the basis of the high homology between SEC1 

and SEC2, we constructed SEC2(Y26V) and 

SEC2(Y26A) to respectively change and diminish the 

TCR V recognization ability of SEC2. It was widely 

accepted that the affinity of SAgs for TCR can 

significantly affect their mitogenic potency (Andersen et 

al., 2001). The weak affinity of the TCR-Sag interaction 

results in poor T cell response. But in this study, both of 

the two mutants exhibited comparable stimulating ability 

with SEC2, which suggested that substitution in position 

26 could not alter the TCR affinity of SEC2. 

In rabbit model assay, SEC2(Y26V) exhibited a 

decreased ability to induce fever at 2h, 2.5h, and 3h 

compared with SEC2. This promoted us to investigate 

whether the decreased toxicity in vivo was due to its 

instability resulted from amino acid substitution. Our 

result from proteolysis susceptibility assays has exhibited 

that both of the two mutants maintained comparable 

resistance to trypsin with SEC2, which indicates that the 

amino acid substitution in position 26 did not result in the 

exposure of additional tryptic cleavage sites. So it is 

suggested that the decreased toxicity of SEC2(Y26V) 

does not due to its degradation. SEC2(Y26V) has potent 

immune stimulating activities compared with SEC2 in all 

concentration, which is inconsistent with the previous 

theory that the febrile activity of SE is a direct 

consequence of T cell stimulation. On the other hand, 

macrophages and monocytes stimulated by superantigen 

could secrete some endogenous pyretogenic cytokines 

such as TNF- and IL-1, which was the major cause of 

immune-mediated diseases such as fever (Roggiani et 

al., 1997). Further investigation was needed to interpret 

it. 

Previous researches on antigenic uniqueness indicated 

that residues 20, 22 and 26 were critical for forming 

SECl- and SEC2-specific epitopes. SEC1 mutants with 

substitutions at all three positions reacted only with an 

SEC2-specific antibody (Turner et al., 1992). On the 

basis of the homology between SEC1 and SEC2, we 

hypothesized that Tyr26 was important for the serological 

characters in SEC2. The result from ELISA confirmed this 

hypothesis that SEC2(Y26V) and SEC2(Y26A) showed 

significantly decreased binding affinities to SEC2-specific 

IgG. The residue of Val26 in SEC2(Y26V) was homologous 

to Val26 in native SEC1, so Val26 could mediate 

the cross-reactivity of SEC2(Y26V) with rabbit anti-SEC2 

polyclonal IgG used in this study. It could be a possible 

explanation for that SEC2(Y26V) showed higher binding 

affinity to SEC2-specific IgG than SEC2(Y26A) did. For 

the lack of SEC1-specific IgG, we could not determine 

whether SEC2(Y26V) and SEC2(Y26A) were able to 

induce cross-reactivity with SEC1-specific IgG. However 

it was quite sure that the mutation introduced into position 

26 influenced the recognition of SEC2 by SEC2-specific 

antibodies, which suggested that Tyr 26 was critical for 

the antigenic uniqueness of SEC2. 

Concluded from our result, the roles of residue at 

position 26 in SEC2 are different from those of in SEC1, 

which may result from continuous evolution. This slight 

difference can display different serological specificity to 

avoid the cross-reactivity with antibodies from the host. 

In summary, we have investigated the potential roles of 

Tyr26 in SEC2 by site-directed mutagenesis. Our results 

indicated that Tyr26 is not important for the immune 

stimulating activities, but a critical site for the serological 

characters of SEC2. A low toxic mutant SEC2(Y26V) 

constructed in this study with potent superantigen 

activities and significantly decreased binding affinities to 

anti-SEC2 IgG has the potentiality to be a novel mutant to 

substitute native SEC2 in cancer immunotherapy. 


This work was supported by a grant from the National 

Science and Technology Major Specific Projects of China 

for "Significant Creation of New Drugs" (2009ZX09103- 

692). 

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DOI: 10.5897/AJMR11.136 



Prevalence study of cytomegalovirus (CMV) infection 

among foreign manpower in Jeddah Saudi Arabia 

N. A. Redwan 1 , M. M. M. Ahmed 1,2 * and M. S. H. AL Awfi 1 

1 Department of Biological Sciences, Faculty of Science, P. O. Box 80203, King Abdulaziz University, Jeddah, 21589, 

Saudi Arabia. 

2 Nucleic Acids Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), 

Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt. 


Human cytomegalovirus (HCMV) is a species-specific DNA virus of the Herpetoviridae family. 

Cytomegalovirus (CMV) is more widespread in developing countries and in areas of low socioeconomic 

conditions. It causes high morbidity and mortality. After primary infection CMV is not 

eradicated but establishes life-long infection in its host. CMV dispersed and become dormant or latent 

in multiple end organs, and can later be reactivated by a number of different stimuli, including 

immunosuppresion and inflammation. To determine CMV prevalence in a sample of the foreign 

manpower population in Jeddah region, Saudi Arabia, we tested serum samples for CMV-specific 

immunoglobulin G from participants aged 20 to 60 years (n = 514) by enzyme linked immunosorbent 

assay (ELISA). The prevalence of CMV infection was 80.7% in studied population. CMV prevalence 

differed significantly by sex (p

through the epithelium of the upper alimentary, 

respiratory, or genitourinary tracts. 

However, the initial infection of epithelial cells is not 

essential, as demonstrated by infections from blood 

transfusions and organ transplantation. CMV is spread 

through the body with the aid of leukocytes and vascular 

endothelial cells (Halpern et al., 2004). Dissemination of 

CMV through the blood is typically followed by infection of 

the ductal epithelial cells. CMV spreads through the body 

quickly into many sites. Historical in-vitro studies 

characterize CMV as a slowly replicating virus, but in a 

recent study based on changes in viral load in humans, 

CMV has been shown to replicate quickly with a doubling 

time of approximately one day during active infection 

(Gerna et al., 2004). CMV infects a wide range of tissues 

and cell types: it has been found in salivary glands, lung, 

liver, pancreas, kidney, ear, eye, placenta, alimentary 

tract, heart, ovaries, pituitary, brain, skin, thyroid, 

esophagus, prostate, testes, and adrenals (Reddehase et 

al., 2002). 

In the vast majority of healthy individuals the immune 

system quickly reaches homeostasis with CMV. While the 

immune system of healthy individuals is usually able to 

prevent CMV from producing clinical manifestations, the 

immune system very rarely completely eliminates CMV 

from the body - the viral genome remains in a latent 

stage waiting for reactivation. While the latent stage of 

CMV infection has been researched extensively, the 

identity of cell types that harbor latent CMV, the ranges of 

genes expressed during latency, and the mechanisms of 

reactivation continue to be controversial (Jarvis and 

Nelson, 2002). 

A crucial part of the immune system defense against 

CMV is the development of CMV specific antibodies. 

Antibodies bind to CMV, thereby inhibiting its ability to 

infect new cells and marking it for removal from the body. 

The first type of antibody to develop in response to CMV 

is Immunoglobulin M (IgM), which develops within a few 

days following primary infection. While CMV IgM remains 

detectable for six to nine months, medium to high levels 

of CMV IgM can be detected during the first three months 

of a primary infection. IgM can also be detected during 

some secondary infections, both reactivation and 

reinfection, and is therefore not a valid marker of primary 

infection. The second antibody type to respond to CMV is 

IgG. This antibody develops within 1 to 2 weeks after 

infection and, once developed, can be detected throughout 

life. Consequently, IgG is commonly used and widely 

accepted measure of previous CMV infection (Drew, 

1988). 

CMV seropositivity rates are higher in females, older 

people, those of lower socioeconomic status (SES) and 

residents of developing countries. A recent study also 

shows a relationship between ethnicity and seroprevalence 

that is independent of factors such as SES. 

Worldwide, seroprevalence in adults in the general 

population varies from 40 to 90% (Dowd et al., 2009). 

Redwan et al. 2339 

Tests for antibodies in an individual's serum are called 

serologic tests. If antibodies are not detected in the 

serum, the individual is seronegative. For 

Immunoglobulin G (IgG), this is commonly interpreted to 

mean that the individual's immune system has never 

encountered the virus and they have never been infected 

with CMV. Individuals are called seropositive if antibody 

to the virus is detectable in their blood. This means that 

the individuals have been previously infected with CMV. If 

upon an individual's first CMV IgG test they are seronegative 

and on a later test are seropositive, the individual 

is said to have undergone seroconversion, indicating the 

occurrence of a primary infection. 

The aim of this study was a prevalence of infected 

CMV in worker population in Jeddah region, Saudi 

Arabia. In addition, find out the relationship of our result 

with age, gender and ethnicity. 


Serum samples collection 

A total of 514 serum samples were tested for CMV seroprevalence 

using enzyme linked immunosorbent assay (ELISA). Samples were 

collected from 4 diagnostic laboratories throughout Jeddah, 

including: the Medical Laboratory of Ghulail Dispensary, Medical 

Laboratory of Badr Adeen Dispensary, Medical laboratory of AL- 

Quds dispensary and Medical laboratory of University street 

hospital. These laboratories supplied remnant sera from samples 

that had been submitted for serological testing and would otherwise 

have been discarded. Sera from subjects who were known to be 

infected with human immunodeficiency virus, hepatitis B virus and 

hepatitis C virus were excluded. Sera were identified at the referring 

laboratory by the sex of the subject, age or date of birth, date of 

collection, and nationality. The samples were coded by date of 

collection, nationality/territory of origin, sample number and 

referring laboratory. All serum samples were stored at -20°C until 

use. 

Study population 

Serum samples were collected from foreign workers between 20 

and 60 years of age and stratified into the following age groups: 20 

to 24, 25 to 29, 30 to 34, 35 to 39, 40 to 44, 45 to 49, 50 to 54 and. 

55 to 60, Serum samples were not available for less than 20 year of 

age. Approximately equal numbers of males and females were 

tested. Also, approximately equal numbers of seven nationalities 

including: Egyptian, Yemeni, Sudan, Pakistani, Indian, Bangladesh 

and Ethiopian were tested. Prevalence was calculated separately 

for each age group, for each nationality, for male and female 

separately, and for foreign workers as a whole. The CMV prevalence 

for the group aged 61 years and above was assumed to be 

the same as for the group aged 55 to 60 years. Sample sizes were 

calculated to achieve a 95% confidence interval (CI) of 

approximately ±5% for each age group. 

Serological testing 

Serum samples were tested for HCMV-specific immunoglobulin G 

(IgG) using a HCMV IgG enzyme-linked immunosorbent assay 

(ELISA) technique using DRG kit (DRG International, Inc., USA).


Principle of the test 

The antigen composed of partially purified and inactivated 

cytomegalovirus is bound to the solid phase (8-well strips). The 

specific immunoglobulin is bound to the antigen through incubation 

with diluted human serum. After washing to eliminate the proteins 

which have not reacted, incubation is performed with the conjugate, 

composed of human IgG monoclonal antibodies conjugated to 

horse radish peroxidase. The unbound conjugate is eliminated, and 

the peroxidase substrate added. The blue color which develops is 

proportional to the concentration of specific antibodies present in 

the serum sample. When the enzymatic reaction is interrupted by 

the addition of sulphuric acid solution, the yellow color, which 

develops, can be easily read by using a microplate reader (Wisdom, 

1976). 

Reagent preparation 

All reagents should be allowed to reach room temperature (18 to 

25°C) before use. Dilute 1 volume of wash buffer with 19 volume of 

distilled water. For example, dilute 50 ml of wash buffer into 950 ml 

distilled water to prepare 1000 ml of wash buffer which is stable for 

1 month at 2 to 8°C mixed well before use. 

Sample and controls dilution 

Prepare 1:40 dilution of test samples, negative control, positive 

control and calibrator by adding 5 l of the samples and controls to 

195 l of sample diluents and mixed well. Procedure is as in assay 

(ELISA) technique using DRG kit. 

RESULTS 

Calculation of the result 

Qualitative results 

The CMV IgG Index of each determination was 

calculated by dividing the absorbance value of each 

sample by absorbance value of cut-off. Samples with 

CMV index less than 0.90 was sero-negative for IgG 

antibody to CMV, those with CMV index between 0.91 

and 0.99 is equivocal and the samples should be 

repeated. 

Quantitative results 

For quantitative determination of anti-CMV IgG levels of 

positive specimens in IU/ml, the O.D of cut-off and 

positive calibrators are plotted on the Y-axis of a group 

against their corresponding anti-CMV IgG concentration 

of 0, 1, 2, 6, 18 IU/ml on the X-axis. The estimates of 

levels in patient sera are read off the graph using their 

individual O.D. values. 

Interpretation of the results 

Qualitative results 

The results were interpreted according to the 

manufacturer’s instructions. Samples with CMV index 

less than 0.90 was seronegative for IgG antibody to 

CMV, those with CMV index between 0.91 and 0.99 is 

equivocal and the samples should be repeated. Samples 

with CMV index of 1.00 or greater was seropositive for 

IgG antibody to CMV. 

Quantitative result 

The results were interpreted according to the 

manufacturer’s instructions. when the anti-CMV IgG 

concentration in the sample is less than 0.8 IU/ml, the 

sample was no immune, those with anti-CMV IgG 

concentration is more than 1.2 IU/ml, the sample was 

immune. If the result is between the two values, in this 

case it is advisable to repeat the test in duplicate. 


The percentages of individuals with positive, negative, 

and equivocal results were determined for each age 

group and sex. SPSS program version 15 was used for 

the analysis and comparison of sero-statuses among age 

groups, male and female and each nationality. Ninetyfive-percent 

confidence intervals were calculated where 

appropriate, and P values of < 0.05 were considered 

statistically significant. 

Demographic characteristics of the studied 

population 

Serum samples according to sex divided to two groups' 

males (51.4%) and female (48.6%) with their ages varied 

from 20-60 years old. Serum samples were stratified 

according to age into the following groups: 20 to 24 

(12.6%), 25 to 29 (12.6%), 30 to 34 (12.8%), 35 to 39 

(12.8%), 40 to 44 (12.4%), 45 to 49 (12.3%), 50 to 54 

(12.3%) and 55 to 60 (12.1%) year. Serum samples 

were not available for less than 18 years of age. Serum 

samples according to nationality were divided to the 

following groups: Egyptian (14.6%), Yemen (14.2%), 

Sudan (14.4%), Pakistan (14.6%), Indian (14%), 

Bangladesh (14.2%) and Ethiopian (14%). According to 

race/ethnicity serum samples were divided to African 

(43%) and Asian (57%). Approximately equal numbers of 

males and females were tested. Also approximately 

equal numbers of each nationality and ages groups were 

tested. Prevalence of CMV- IgG in each group were 

tested using enzyme linked immunosorbent assay and

Table 1. Demographic characteristics of the studied population. 

Characteristic 

Age(years) 

No. of samples 

No. 

Positives 

No. 

P value 

20-24 12.6 65 53.8 35


Sex 

Figure 1. Prevalence of cytomegalovirus by sex (Males and females). 

Table 3. Prevalence of cytomegalovirus by age groups from 20 to 60 years old. 

Age group No. of samples 

Positive 

No. % 

Compared to age group 20 - 24 

2 

(p) (95%CI) 

20-24 65 35 50.8 Referent 41.0-61.8 

25-29 65 43 66.2 2.051(>0.05) 61.5-72.1 

30-34 66 46 69.7 3.486(>0.05) 62.2-77.8 

35-39 66 58 87.9 18.418(

Figure 2. Prevalence of cytomegalovirus by age groups from 20-60 years old. 

Table 4. Prevalence of cytomegalovirus in females by age groups from 20 to 60 years old. 


Positive 

No. % 


Compared to age group 20 - 24 

2 

(p) (95%CI) 

20-24 32 21 65.6 Referent 60.1-69.9 

25-29 33 26 78.8 1.406(>0.05) 74.8-83.6 

30-34 32 29 90.6 5.851(


Table 5. Prevalence of cytomegalovirus in males by age groups from 20 to 60 years old. 


Positive Compared to age group 20 - 24 

No. % 

2 

(p) (95%CI) 

20-24 33 14 42.4 Referent 34.3-50.1 

25-29 32 17 53.1 0.746(>0.05) 47.5-60.5 

30-34 34 17 50 0.387(


Table 6. Prevalence of cytomegalovirus by Nationality: (Egyptian; Yemeni; Sudan; Pakistan; Indian Bangladesh and Ethiopian). 

Nationality No. of samples 

Positive Compared to age group 20 - 24 

No. % 

2 

(p) (95%CI) 

Indian 72 48 66.7 Referent Referent 

Egyptian 75 59 78.7 2.671(>0.05) 72.9-85.1 

Yemen 73 56 76.7 1.804(>0.05) 73.5-79.5 

Sudan 74 65 87.8 9.350(


Table 7. Prevalence of cytomegalovirus by ethnicity (African and Asian). 

Ethnicity No. of sample 

No. 

Positive 2 (p) (95%CI) 

African 221 188 85.1 4.671(

toddlers. Infected infants and children, in particular those 

under 30 months old, actively excrete the virus in their 

saliva and urine. Thus, one hypothesis to explain the 

higher females CMV seroprevalence would be that 

women have more contact with children and have more 

opportunities for HCMV infection during pregnancy, 

delivery, and menstruation. 

In the study reported herein, the seroprevalence of 

CMV IgG among the studied population varied with 

nationality and/or ethnicity ranging from 66.7% in Indian, 

78.7% in Egyptian, 76.7% in Yemeni, 87.8% in Sudan, 

82.7% in Pakistan, 83.6% in Bangladesh, to 88.9% in 

Ethiopian. The prevalence varied significantly with 

nationality when all nationalities compared with Indian 

nationality as reference (p< 0.05) except for the Egyptian 

and Yemeni nationality (p>0.05). In general the seroprevalence 

of cytomegalovirus among the African population 

(85.1%) varied significantly from Asian population 

(77.5%). The differences in the prevalence rate in 

different nationalities perhaps could be related to socioeconomic 

factors, environmental, and climatic factors. 

The result of the present study is consistent to the 

results of other researches carried out in different parts of 

world. Badami et al. (2009) reported that the ethnicity 

appeared to be related to seroprevalence of cytomegalovirus 

(CMV) in New Zealand between 2003 and 2006 

and it ranging from 93.2% in Pacific Islanders, 54.8% in 

Caucasians, 80.4% in Maori, 77.6% in Asian, to 71.4% in 

Maori/Caucasian (Badami et al., 2009). The result of 

present study is also consistent to the result of another 

study carried out by Staras et al. (2006) between the year 

1988 and 1994 to determine CMV prevalence in the US 

population. CMV seroprevalence differed by race and/or 

ethnicity as follows: 51.2% in non-Hispanic white 

persons, 75.8% in non-Hispanic black persons, and 

81.7% in Mexican Americans. Racial and/or ethnic 

differences in CMV seroprevalence persisted when 

controlling for household income level, education, marital 

status, area of residence, census region, family size, 

country of birth, and type of medical insurance (Staras et 

al., 2006). Also similar results to our results shown in a 

study carried out by Gaytant et al. (2005) to investigate 

the incidence of congenital cytomegalovirus infections in 

the Netherlands. A significant difference (P


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OI: 10.5897/AJMR11.169 



Antibacterial and cytotoxic activity of Eremurus 

persicus (Jaub and Spach) Boiss 

Mojdeh Hakemi Vala 1 , Jinous Asgarpanah 2 *, Mohammad Hossein Hedayati 3 , Jeilan Shirali 2 

and Fatemeh Bagheri Bejestani 4 

1 Microbiology Department, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 

2 Pharmacognosy Department, Pharmaceutical Sciences Branch and Pharmaceutical Sciences Research Center, 

Islamic Azad University (IAU), Tehran, Iran. 

3 Pasteur Institute of Iran (Karaj branch), Iran. 

4 Microbiology Department, Pharmaceutical Sciences Branch, Islamic Azad University (IAU), Tehran, Iran. 


Traditional medicine has a key role in health care worldwide. Obtaining scientific information about the 

efficacy and safety of the plants is one of the researcher's goals. In this research, the flowering aerial 

parts of Eremurus persicus were collected from Golpayegan (Isfahan, Iran) in May 2010. The extract was 

tested for its antibacterial activity against 4 Gram-positive bacteria strains (Staphylococcus aureus, 

Staphylococcus epidermidis and Bacillus cereus, Streptococcus pyogenes) and 5 Gram-negative 

bacterial strains (Escherichia coli, Salmonella typhi, Shigella dysantriae, Klebsiella pneumoniae, 

Pseudomonas aeruginosa). Its cytotoxic activity was also investigated using MTT assay. Here we 

reported the antibacterial activity of E. persicus against S. aureus (MIC = 125 mg/ml), B. cereus (MIC = 

15.62 mg/ml), E. coli (MIC = 125 mg/ml), S. typhi (MIC = 31.25 mg/ml), S. dysantriae (MIC = 0.48 mg/ml) 

for the first time. Furthermore the cytotoxic potentials of E. persicus on two cell lines, HeLa and Caco-2, 

were studied. 

Key words: Eremurus persicus, antibacterial activity, cytotoxic activity. 

INTRODUCTION 

The genus Eremurus (Liliaceae) comprising nearly 50 

species, is mainly restricted to central and western Asia 

(Chong et al., 2000) and six species are known to exist in 

Iran. Eremurus persicus locally called "Serish” is widely 

distributed in south, east and west of Iran. The roots are 

used as natural glue. The leaves are traditionally used to 

relieve constipation and treatment of diabetes, liver and 

stomach disorders. Polysaccharides have been reported 

as chemical constituents of species in the genus 

Eremurus (Chong et al., 2000). Since the literature 

survey revealed that there is no any information on the 

biological activities of E. persicus, we prompted to investigate 

the antibacterial activity of methanolic extract of 

flowering aerial parts of this plant by using the cup plate 

*Corresponding author. E-mail: asgarpanah@iaups.ac.ir. Tel: 

+98 21 22640051. Fax: +98 21 22602059. 

method and determine its cytotoxic effect after infection 

by two cell lines (HeLa and Caco-2) and further by MTT 

assay. 


Plant material 

The flowering aerial part of E. persicus was collected from 

Golpayegan (Isfahan, Iran) in May 2010 and identified by Dr. Gh.R. 

Amin at the Pharmacognosy Department, Faculty of Pharmacy, 

Tehran University of Medical Sciences, Tehran, Iran. A voucher 

specimen (NO. 197) has been deposited in the herbarium of the 

Department of Pharmacognosy, Pharmaceutical Sciences Branch, 

Islamic Azad University (IAU), Tehran, Iran. 

Extraction and isolation 

The dried ground material was extracted by percolator apparatus


Table 1. IC50 of the methanolic extract of Eremurus persicus and DMSO in cell lines. 

Cell line 

HeLa 

Caco-2 

Eremurus persicus extract IC50 (mg/ml) 

4.74 

5 

using methanol (Merck). The extract was concentrated by rotary 

evaporator apparatus and the solvent removed to produce a dark 

brown gummy solid. The resulting extract was kept in a sterile vial 

in a dark and cool place for further tests. 

Bacterial strains 

Gram-positive bacteria including Staphylococcus aureus (PTCC 

1431), Staphylococcus epidermidis (PTCC 1435) and Bacillus 

cereus (PTCC 1247), Streptococcus pyogenes (PTCC 1447), and 

Gram-negative bacteria including Escherichia coli (PTCC1399), 

Salmonella typhi (PTCC 1639), Shigella dysantriae (PTCC 1188), 

Klebsiella pneumonia (PTCC 1053), Pseudomonas aeruginosa 

(PTCC 1430) were obtained from Persian Type Culture Collection, 

Iranian Research Organization for Science and Technology (PTCC, 

Iran). 

Antibacterial assay 

Antibacterial activity of the methanolic crude extract of E. persicus 

was investigated against 9 bacterial strains by the cup plate method 

(Fazyl Bazzaz et al., 2005). An overnight bacterial culture 

containing 1.5 x 10 8 CFU/ml was used to culture on surface of 

Muller-Hinton agar plates. The wells were made on agar plates. 

1000, 500 and 250 mg, respectively, of the extract were dissolved 

in 1 ml DMSO 10% and then filtered. 80 l of each test solution was 

added to each well. Following diffusion of solutions into agar, the 

plates were incubated at 37°C for 24 h. The diameter of inhibition 

zones around each well were determined in comparison with the 

well of ciprofloxacin (31.25 mg/ml) which was used as positive 

control. A well added by 80 l DMSO 10% instead of the extract 

solution, served as negative control too. The experiments carried 

out 3 times and the results were presented as mean ± SD. 

Minimum inhibitory concentration (MIC) 

After confirmation of the antibacterial activity in methanolic crude 

extract of E. persicus, MIC of the extract was determine by testing 

10 concentrations of the extract against sensitive Gram-positive 

and Gram-negative tested bacteria by the micro plate dilution 

method. The reconstituted extract was diluted to give concentrations 

of 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.95, 0.97 and 

0.48 mg/ml, respectively. The lowest concentration of the extract 

that could inhibit the bacterial growth was considered as MIC 

(Mehregan et al., 2008). The data was subject to statistical analysis 

of SPSS software. 

Cytotoxic activity 

Cell culture 

HeLa (Human cervix carcinoma) and Caco-2 (Human colon 

carcinoma) cell lines were obtained from Iranian Cell Bank of 

Institute Pasteur of Iran. HeLa cells was grown in a flask and RPMI 

DMSO 10% IC50 (v/v) 

4.45 % 

4.73 % 

1640 media (Gibco, Germany) which was supplemented with 10% 

fetal bovine serum; FBS (Gibco, Germany) and 20mg/ml gentamicin 

(Sigma) was added. In addition, Caco-2 cell line was grown in 

DMEM media (Gibco, Germany) which was supplemented with 20% 

FBS and 20 mg/ml gentamicin. All plated were incubated in a CO2 

incubator at 37°C. 100 µl of 3 x 10 5 cells/ml suspension of HeLa cell 

line and 100 µl of 6 x 10 5 cells/ml suspension of Caco-2 cell line 

transferred to a 96- well microplate and incubated at the same 

condition. 

MTT assay 

Based on determined Ic50 (Table 1), 17 mg methanolic extract of E. 

persicus dissolved in DMSO 1%, separately for HeLa and Caco-2 

assay and 100 µl of each concentration was transferred in a row of 

separate microplate to make serial dilution. After 72 h incubation in 

a CO2 incubator at 37ºC, 20 µl of MTT (5 mg/ml in PBS) was added 

and incubated at the same condition for 3 to 5 h for MTT assay. 

After Formazan crystallization, 50 µl of isobutyl alcohol was added 

and OD (Optical density) was read at 570 nm wave length (Figures 

1 to 2). 

RESULTS 

The inhibitory effects of methanolic extract of E. persicus 

against different test organisms are shown in Table 2 (the 

p-value mentioned in Table 2 is only for the 1000 mg/ml 

concentration of extract). This extract indicated significant 

antibacterial activity (growth inhibition zone diameters 

ranging from (12.5 to 32 mm) against some Grampositive 

bacteria including S. aureus, B. cereus, S. 

epidermidis and Gram-negative bacteria including E. coli, 

S. typhi and specially S. dysantriae. 

Also, the MIC of the extract was determined by using 

the micro plate dilution method for Gram-positive 

bacteria, S. aureus and B. cereus. In addition, the 

methanolic extract could inhibit the growth of other Gramnegative 

bacteria at 125 and 15.62 mg/ml, respectively; 

the extract could effectively inhibit other test bacteria, E. 

coli, S. dysantriae and S. typhi, even at concentrations as 

low as 125, 0.48 and 31.25 mg/ml, respectively. 

The cytotoxicity effect of methanolic extract of E. 

persicus on HeLa cell and Caco-2 cell culture was 

evaluated by MTT assay. The result shows that the more 

concentration of extract in the cell culture the less OD 

was detected. It means, after increasing the concentration 

of methanolic extract the viability of cells were 

decreased. Similarly, both cell lines showed decrease in 

cell viability after the concentration of extract was 

increased and decrease in viability begins when 2.125 

and 4.25 mg/ml of methanolic extract was added to

Eremurus persicus 

Figure 1. Absorbance value versus concentration of E. persicus extract and DMSO in Caco-2 cell line. 


Figure 2. Absorbance value versus concentration of E. persicus extract and DMSO in HeLa cell line. 

Table 2. Antibacterial activity of E. persicus methanolic extract against various bacteria, as obtained by cup plate method (n=6). 

S/No. 

1 

2 

3 

4 

5 

6 

Organism 

S. aureus 

S. epidermidis 

B. cereus 

E-coli 

S. typhi 

S. dysantriae 

Mean zone of inhibition in mm and standard deviation (SD) 

A 

B 

C 

D 

Mean ± SD Mean ± SD Mean ± SD Mean ± SD 

0.0±0.0 

0.0±0.0 

0.0±0.0 

0.0 ±0.0 

0.0±0.0 

28.0±0.0 

11.0±1.1 

0.0±0.0 

0.0±0.0 

0.0 ±0.0 

0.0 ±0.0 

31.0±0.0 

17.5±0.5 

15.0±0.0 

13.5±1.6 

12.5± 0.5 

14.0±1.1 

32.0±0.0 

60.0±0.0 

57.0±3.3 

46.0±0.0 

50.0±0.0 

48.0±2.2 

60.0±0.0 

P- value* 



Figure 3. Effects of E. persicus extract and DMSO on cell viability in Caco-2 cell line. 


Figure 4. Effects of E. persicus extract and DMSO on cell viability in HeLa cell line. 

shows more sensitivity than Caco-2 cell line against both 

E. persicus extract and DMSO concentrations as positive 

control. 

DISCUSSION 

Since the presence of anthraquinones has been indicated 

in the aerial parts of other species of this genus (Chong 

et al., 2000), we suggest the strong antibacterial effect 

due to anthraquinones because they are more likely to 

possess antibacterial activity (Chukwujecwu et al., 2006). 

The cytotoxic activity is also related to the anthraxquinones 

because it is shown that they are able to induce 

opoptosis due to the fact that they are suitable substrate 

for one-electron-reducing enzymes and effective redox 

cycler which lead to the production of oxygen derived free 

radicals that eventually induce opoptotic cell death 

(Kagedal et al., 1999). Based on the results of this study, 

further in-vivo and ex-vivo confirmatory tests are 

recommended. 


This work was supported by Pharmaceutical Sciences 

Branch, Islamic Azad University (IAU), Tehran, Iran and 

the authors are thankful for this support. We would like to 

thank Mr. M. Asoudeh for collecting the plant material. 

REFERENCES 

Chong L, Jian G, Peng Zhang Y, Zhong Z (2000). Constituents of 

Eremurus chinensis. J. Nat. Pro., 63: 653-656. 

Chukwujecwu JC, Coombes PH, Mulholland DA, Vanstaden J (2006). 

Emodin, an antibacterial anthraquinone from the roots of Cassia 

occidentalis. South Afr. J. Bot., 72(2): 295-297. 

Fazly Bazzaz BS, Khajehkaramadin M, Shokooheizadeh HR (2005). 

Antibacterial activity of Rheum ribes extract obtained from various 

plant parts against clinical isolates of Gram-negative pathogens. Iran. 

J. Pharm. Res., 2: 87-91. 

Kagedal K, Bironaite D, Ollinger K (1999). Anthraquinone cytotoxicity 

and opoptosis in primary cultures of rat hepatocytes. Free. Radic. 

Res., 31(5): 419-428. 

Mehregan H, Mojab F, Pakdaman SH, Poursaeed M (2008). 

Antibacterial activity of Thymus pubescence methanolic extract. Iran. 

J. Pharm. Res., 7(4): 291-295.



DOI: 10.5897/AJMR11.174 



Optimization of ultrasound-assisted extraction 

conditions using orthogonal matrix design to enhance 

the antimicrobial activity of extracts from Cichorium 

intybus root 

Quanzhen Wang 1 *, Haitao Liu 1 , Jinhong Du 1 , Jian Cui 2 , Guo Chen 1 and Yuyan Liu 1 

1 Department of Grassland Science, College of Animal Science and Technology, Northwest Agriculture and Forestry 

University, 712100 Yangling, Shanxi Province, P. R. China. 

2 Department of Plant Science, College of Life Science, Northwest Agriculture and Forestry University, 712100, 

Yangling, Shanxi Province, P. R. China. 


Plant-derived compounds used as alternatives to chemical preservatives have been extensively 

researched for use as natural medical ingredients or food preservatives. Conditions for the ultrasoundassisted 

extraction (UAE) of chicory (Cichorium intybus) root (including type of solvent, impregnation 

time, number of sonication steps and ultrasonic power) were optimized to determine the best extract 

antibacterial activity by using orthogonal matrix design [L16 (4 5 )]. The combination of 70% ethanol v/v, a 

36 h impregnation time, three sonication rounds and 300 W ultrasonic power input provided the best 

antimicrobial activity results. Our results demonstrate that solvent composition has the largest effect 

on antimicrobial activity. Several extracts demonstrated antibacterial activity against Escherichia coli, 

Staphylococcus aureus, Bacillus thuringiensis and Salmonella typhi, and all extracts exhibited weak 

activity against Bacillus subtilis. To our knowledge, these results represent the first example of 

ultrasound-assisted chicory extracts aimed at increasing its potential for use in food industry. 

Key words: Cichorium intybus, antibacterial, ultrasound-assistant extract, optimal conditions, orthogonal matrix 

design. 

INTRODUCTION 

There are a variety of useful compounds from higher 

plants that have not yet been identified. Medical and food 

industries tend to use natural antimicrobials, such as 

plant-derived compounds, as an alternative to chemical 

preservatives (Bachir and Mohamed, 2010). These 

preferences have led to the search for plant products that 

have natural antimicrobial and antioxidant effects. Such 

products have been extensively researched and used as 

both natural medical ingredients and food additive 

agents (Issa-Zacharia et al., 2010; Kumaravel et al., 

2010; Liu et al., 2010; Mini et al., 2010; Somchit et al., 

2010). 

*Corresponding author. E-mail: wangquanzhen191@163.com. 

Tel: 0086-29-87091953 or 0086-13759942845. Fax: 0086-29- 

87092164. 

Chicory (Cichorium intybus) is a perennial plant of the 

Asteraceae family, which is a native of to the 

Mediterranean region and widely grown in Europe, 

Western Asia, Egypt and North America. In traditional 

Indian medicine, chicory has been used to treat fever, 

diarrhea, spleen and liver enlargement, jaundice, gout 

and rheumatism (Mulabagal et al., 2009). In Belgium, 

France and the United States, chicory root has been 

used as a coffee additive for its bitter taste, which is 

caused by sesquiterpene lactones (Peters and 

Amerongen, 1996; Poli et al., 2002). 

A number of studies report that chicory extracts 

possess antimicrobial activities. It was reported that 

water, ethanol and ethyl acetate extracts from chicory 

have antibacterial properties, and that root extracts have 

more intensive antibacterial activity than extracts from 

whole plants (Petrovic et al., 2004). Moreover, it was 

found that the sesquiterpene lactones extracted from


Table 1. Assignment of levels and condition factors using orthogonal matrix L16 (4 5 ). 

Levels 

Factors 

A (Solvent) B [Impregnation time (h)] C (Sonication repetitions) D [Ultrasonic input power (W)] 

I petroleum ether 12 1 200 

ii ethyl ether 24 2 300 

iii ethyl acetate 36 3 400 

iv 70% ethanol v/v 48 4 500 

chicory root can inhibit the growth of zoophilic and 

anthropophilic dermatophytes (Mares et al., 2005). Many 

gastrointestinal foodborne infections in humans, particularly 

those in developing countries, are often caused by 

enterobacteria (Ajayi and Akintola, 2010). Increasing 

concern over pathogenic and spoilage microorganisms in 

food is due to the growing prevalence of food borne 

disease outbreaks (Rahman and Kang, 2009). Considering 

the questionable safety of synthetic food 

preservatives, consumers are demanding more natural 

and fresh foods with fewer synthetic additives. However, 

to increase safety and maintain a long shelf life, food 

manufacturers are compelled to use natural or mild preservation 

techniques. Therefore, alternative sources of 

safe and effective natural preservatives must be explored 

(Abbasi et al., 2010; Negi et al., 2008; Hussain et al., 

2009). 

Orthogonal matrix design has been used to evaluate 

the interaction of different production parameters and 

how they affect product recovery. Furthermore, this 

method has been used to optimize multiple production 

parameters to establish optimum conditions (Hedayat et 

al., 1999). At present, ultrasound-assisted extraction 

(UAE) is considered a desirable method for organic 

compound extraction from different matrices (Khan et al., 

2010). This method can shorten extraction time because 

the increased pressure favors penetration and transport 

while the temperature decreased may improve solubility 

and diffusivity (Pena et al., 2006). 

In order to optimize conditions to achieve the best 

antibacterial activity for chicory extract use as a natural 

food preservative, the solvent, impregnation time, sonication 

repetitions and ultrasonic input power were evaluated 

by orthogonal matrix design. The L16 (4 5 ) experiment was 

adopted and sixteen different extraction groups were 

analyzed. The antibacterial activities of extracts derived 

using different extraction protocols were investigated. 


Plant materials 

Five-year-old Puna chicory roots were freshly harvested in 

September 2009 in an experimental field at the Grassland Science 

Department at Northwest Agriculture and Forestry University 

(Shaanxi Province, China). The plant was identified by associate 

professor Quanzhen Wang (Northwest Agriculture and Forestry 

University, China). A voucher specimen was deposited in the 

Herbarium of the Laboratory of Grassland Science at the Faculty of 

Animal Science and Technology (Northwest Agriculture and 

Forestry University, China). 

Preparation of plant extracts 

Four levels for each of four different extraction conditions resulting 

in sixteen extraction method combinations (Table 1) were studied 

by an L16 (4 5 ) orthogonal matrix design (Hedayat et al., 1999). For 

each sample, 100 g dried ground powder (40 meshes) was soaked 

in 800 ml of various solvents for different times at room temperature 

until extraction was exhausted. The extraction temperatures were 

30°C (ethyl ether), 50°C (petroleum ether and ethyl acetate) and 

65°C (70% ethanol v/v), respectively. An ultrasonic apparatus (KQ- 

500DE, Kunshan Ultrasound Instrument Co., Ltd., China) was used 

for accelerated extraction. A beaker was partially submerged in an 

isothermal water bath to maintain the extraction temperature for 30 

min. After extraction, the resulting mixture was passed through filter 

paper (Whatman No. 1.) by vacuum. The filtrate was concentrated 

on a rotary evaporator (SHB-III, Zhengzhou Science and Industrial 

Foreign Trade Co., Ltd., China) at 45°C and then stored at 4°C for 

further use. All samples were redissolved in dimethylsulfoxide 

(DMSO) at a concentration of 10 mg/mL and stored at 4°C. 

Bacteria 

The antibacterial activity of chicory extracts was determined using 

the following food-related bacteria obtained from the Department of 

Life Science, Northwest Agriculture and Forestry University: E. coli, 

Staphylococcus aureus, Bacillus thuringiensis, Bacillus subtilis, 

Salmonella typh. Cultures of each strain were maintained on beef 

cream-peptone culture media at 4°C (Monadi et al., 2010; Moussa 

and Hessan, 2010). 

Antibacterial assays of disc diffusion 

Antibacterial activity tests were conducted based on the disc 

diffusion method (Yan et al., 2009). A suspension of bacteria (2×10 8 

CFU/ml) was spread on solid media plates. Sterile paper discs (6 

mm diameter) were individually impregnated with solvent at 10 

mg/ml. Discs with the solvent used for dissolution were used as 

negative controls and the standard reference antibiotic streptomycin 

(10 g/disc) was used as a positive control for the bacteria in 

question. The plates were incubated at 37°C for 24 h after which 

the inhibition zone was measured in millimeters. Each assay in this 

experiment was performed in triplicate. 


All experimental results are expressed the mean of four repeats.

Table 2. The L16 (4 5 ) matrix associated with analytical results. 

Group�� 

Factors and levels 

A B C D 

Staphylococcus 

aureus b 

Bacillus 

subtilis b 

Zone of inhibition in mm a 

Bacillus 

thuringiensis b 


Salmonella 

typhi c 

Escherichia 

coli c 

1 1 1 4 3 10.2 7.2 9.7 8.6 9.7 

2 2 1 1 1 11.6 6.5 9.4 8.2 8.1 

3 3 1 3 4 8.5 8.5 13.1 12.3 11.1 

4 4 1 2 2 12.2 8.9 10.8 7.5 9.5 

5 1 2 3 2 12.1 7.1 11.8 10.2 9.2 

6 2 2 2 4 11.7 8.1 8.9 10.1 11.5 

7 3 2 4 1 9.2 9.6 9.6 11.6 12.0 

8 4 2 1 3 9.8 8.2 12.7 8.6 11.7 

9 1 3 1 4 11.0 7.9 8.6 10.4 7.6 

10 2 3 4 2 9.4 9.8 10.7 14.2 11.2 

11 3 3 2 3 8.6 7.5 10.2 10.9 10.9 

12 4 3 3 1 11.1 6.7 10.3 12.2 13.6 

13 1 4 2 1 8.3 8.7 10.2 7.4 10.2 

14 2 4 3 3 8.4 7.1 9.7 9.7 6.9 

15 3 4 1 2 9.1 7.5 9.4 11.4 8.1 

16 4 4 4 4 11.5 9.1 11.5 7.1 10.1 

a Values are means (mm) of four separate treatments. b Gram positive bacteria. c Gram negative bacteria. 

Analysis of variance was performed by ANOVA. SAS software 

(version 8.2, USA) was used for statistical analysis. Results were 

considered statistically significant at p ultrasonic input power (D) > impregnation 

time (B) > sonication repetition number (C) (Table 3). The 

optimal extraction parameters were defined as the 

following: treatment with 70% ethanol v/v (A4), 300 W 

ultrasonic input power (D2), 12 h impregnation time (B1) 

and two rounds of sonication (C2). The impact of 

variables on the inhibition zone against Bacillus subtilis 

was measured in the following order: C > D > A > B. The 

optimal combination of parameters for this bacterium was 

to use four sonication rounds (C4), 300 W ultrasonic input 

power (D2), ethyl ether (A2) and a 36 h impregnation time 

(B3). The impact of variables on the inhibition zone of B. 

thuringiensis was in the following order: A > C > B > D. 

The optimal combination was to use ethyl acetate (A3), 

three sonication rounds (C3), a 12 h impregnation time 

(B1) and 500 W ultrasonic input power (D4). The impact of 

variables on the inhibition zone of Salmonella typh was in 

the following order: B > A > C > D. The optimal parameter 

combination was to use a 36 h impregnation time (B3), 

ethyl ether (A2), four sonication rounds (C4), and 300 W 

ultrasonic input power (D2). The impact of variables on 

the inhibition zone of against E. coli was in the following 

order: B > A > C > D. The optimal parameter combination 

was found using a 36 h impregnation time (B3), 70% 

ethanol v/v (A4), three sonication rounds (C3), and 200 W 

ultrasonic input power (D1). According to the results for 

antibacterial activities against all five types of bacteria, 

we found that extraction using 70% ethanol v/v, a 24 h 

impregnation time, three sonication rounds and 300 W 

ultrasonic input power is the optimal synthetic 

combination. 

Variance analyses of the models of all bacteria but B. 

thuringiensis were found to be significant (Table 4. Pr < 

0.001). Factors A, B and D were significantly effective for 

inhibiting S. aureus whereas factors C and D were significant 

against Bacillus subtilis (Table 4). Factors A, B and 

C consistently inhibited the Gram-negative bacteria S. 

typhi and E. coli; however, factor D (ultrasonic input 

power) was not significant (Table 4). Solvent type was 

found to be the most influential factor; increasing solvent 

polarity resulted in better antibacterial activities primarily 

because substances such as carbohydrates, proteins,


Table 3. Averages of levels with factors and ranges. 

Factors A B C D A B C D 

Staphylococcus aureus Bacillus subtilis 

Level i 10.4 10.63 10.38 10.05 7.73 7.78 7.53 7.88 

Level ii 10.35 10.70 10.20 10.70 7.88 8.25 8.30 8.33 

Level iii 8.85 10.03 10.03 9.25 8.28 7.98 7.35 7.50 

Level iv 11.15 9.33 10.08 10.68 8.23 8.1 8.93 8.40 

Range a 2.3 1.37 0.35 1.45 0.55 0.47 1.58 0.90 

Order 1 3 4 2 3 4 1 2 

Bacillus thuringiensis Salmonella typhi 

Level i 10.08 10.75 10.03 9.88 9.15 9.15 9.65 9.85 

Level ii 9.68 10.75 10.03 10.67 10.55 10.13 8.98 10.83 

Level iii 10.58 9.95 11.23 10.58 11.55 11.93 11.10 9.45 

Level iv 11.33 10.2 10.38 10.53 8.85 8.90 10.38 9.98 

Range a 1.65 0.8 1.2 0.79 2.7 3.03 2.12 1.38 

Order 1 3 2 4 2 1 3 4 

Escherichia coli 

Level i 9.18 9.6 8.87 10.98 

Level ii 9.43 11.1 10.53 9.5 

Level iii 10.53 10.83 10.2 9.8 

Level iv 11.23 8.83 10.75 10.08 

Range a 2.05 2.27 1.88 0.58 

Order b 2 1 3 4 

a R means the average range for four average responses in each level for the inhibition zone against Staphylococcus aureus, Bacillus 

subtilis, Bacillus thuringiensis, Salmonella typhi, Escherichia coli, respectively. 

b The ordinal numeral for the range sequence of the eight factors in decreasing order. 

Table 4. Variance analysis for the model and experimental factors. 

Factor 

Model 

A 

B 

C 

D 

Staphylococcus 

aureus b 

Bacillus subtilis b 

Bacillus 

thuringiensis b 

Salmonella 

typhi c 

Escherichia coli c 

F Value 3.85 3.72 1.87 4.93 4.33 

Pr > F 0.0003 0.0005 0.0608 F 0.0003 0.3348 0.0248 0.0007 0.0040 

F Value 3.49 0.88 1.14 8.11 6.19 

Pr > F 0.0221 0.4567 0.3415 0.0002 0.0011 

F Value 0.22 9.74 2.15 3.58 3.89 

Pr > F 0.8807 F 0.0113 0.0350 0.4933 0.2560 0.0956 

b Gram positive bacteria. c Gram negative bacteria. 

alkaloids, tannins, glycosides and amines increase in 

abundance with increased solvent polarity. Impregnation 

time, sonication repetition number and ultrasonic input 

power were found to be subordinate factors. Most

ioactive compounds such as polyphenolics (e.g., tannins 

and flavonoids) exist in higher polarity solvents. 

Additional compounds found in the extracts may either 

enhance or attenuate the effect of phenolic compounds. 

The 70% ethanol and acetic ether extracts exhibited 

significant antibacterial activity against many of the 

bacteria in this study. This antibacterial activity may be 

ascribed to polyphenols, tannins and coumarins found in 

crude extracts (Kilani et al., 2008). Phenolic compounds 

can attack cell walls and membranes by affecting their 

permeability, enabling the release of intracellular constituents 

and interfering with membrane functionality (Bajpai 

et al., 2009). There were differences in chicory extract 

antibacterial activity against Gram-positive and Gramnegative 

bacteria. Gram-positive bacteria were found to 

be more susceptible to chicory extracts than Gramnegative 

bacteria, possibly because the hydrophilic cell 

walls of Gram-negative bacteria are composed of 

lipopolysaccharide (LPS), which inhibits the accumulation 

of phenolic compounds in a target cell membrane (Bezic 

et al., 2003). 

Chicory contains several sesquiterpene lactones, 

particularly in the roots (Beek et al., 1990; Peters and 

Amerongen 1996; Poli et al., 2002). Poli et al. has 

reported the isolation of two guaianolides with different 

chemical structures, including 8-deoxylactucin and 11 , 

13-dihydrolactucin, from the root extracts of C. intybus 

var. ‘Rosso di Chioggia’ (Picman, 1986). Two pure 

isolated lactones possess similar biological activity to 

chicory extracts against several phytopathogens, which 

cause morphological anomalies that have been observed 

using scanning electron microscopy (Mares et al., 2005). 

Conclusions 

The antibacterial activity of chicory extracts was found to 

be affected by the type of solvent used for extraction. The 

optimal UAE conditions were obtained to produce the 

best chicory root extract antimicrobial activity. It was 

concluded that combining 70% ethanol v/v, a 36 h 

impregnation time, three sonication rounds and 300 W 

ultrasonic input power could produce the highest 

antibacterial activity in the extracts. Extracts from chicory 

roots can be used as a natural food preservative as well 

as an antibacterial agent. To our knowledge, these 

results represent the first example of a practical application 

for UAE aimed at increasing the potential use of 

chicory extracts in food industry. Chicory root 

antibacterial activities against additional bacterial strains 

should also be studied. 


This work was supported by The Ministry of Science and 

Technology of the Peoples Republic of China, 

International Cooperation Research Project 

(2008DFA31650) and Shaanxi Province International 


Cooperation Research Project (2008KW-29). We are 

grateful to Prof. Hu Tianming and postgraduate students 

Sun Luanzhi and Wu Chunhui. 

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DOI: 10.5897/AJMR11.273 



Optimization of the production of exopolysaccharides 

by Bacillus thuringiensis 27 in sand biological soil 

crusts and its bioflocculant activity 

Z. R. Wang 1.2 , J. P. Sheng 1 , X. L. Tian 1 , T. T. Wu 1 , W. Z. Liu 1 and L. Shen 1 * 

1 College of Food Science and Nutritional Engineering, China Agricultural University, Beijing100083, China. 

2 School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China. 


To improve the yield of exopolysaccharides (EPS) by Bacillus thuringiensis 27 from sand biological soil 

crusts in Gurban Tonggut Desert, Xinjiang, China and to analyze its bioflocculant activity, orthogonal 

matrix method was used and this method enabled us to obtain maximum EPS production. By studying 

the optimal medium condition of beef extract 3 g/l, peptone 10 g/l, maltose 40 g/l, and NaCl 4g/l we 

observed that the optimal medium condition was pH 6.0, incolum size 8%, liquid volume 40 ml in 200 ml 

flak and temperature 28°C. The maximum EPS production was 20.19 g/l which is about five times more 

than that at the basal condition. Furthermore, results obtained indicated that the flocculation activity of 

the extracellular polymer can be achieved at over 80.4% in kaolin suspension and this occurred at a 

concentration of 0.4 mg/l. This paper describes the optimum condition of exopolysaccharides 

production by Bacillus thuringiensis 27 and showed that exopolysaccharides had high bioflocculant 

activity. This work provides a scientific foundation to explore new exopolysaccharides and 

bioflocculation in sand biological soil crusts. Maximum production of exopolysacchaides under the 

optimal medium and condition can be achieved and exopolysaccharides have high bioflocculating 

activity in kaolin system. 

Key words: Biological soil crusts, exopolysaccharides, flocculation, optimization, Bacillus. 

INTRODUCTION 

Extreme environments, once thought to be too hostile to 

permit survival of living organisms, are the natural habitat 

of certain microorganisms. It is now recognized that the 

microorganisms living in extreme environments have 

different genetic background and metabolic pathway 

when compare to general microbiology and their seconddary 

metabolites have special function (Kennedy et al., 

2001). Among the secondary metabolites from extreme 

environments, polysaccharide for biotechnological applications 

has been reported in Literature (Nicolaus et al., 

2004). Therefore a wide search for bacteria that are able 

*Corresponding author. E-mail: pingshen@cau.edu.cn. Tel: 

8610-62738456. Fax: 8610-62737604. 

Abbreviation: EPS, Exopolysaccharide. 

to produce new polysaccharides with potentially useful 

properties has been undertaken. Sand biological soil 

crusts (BSCs) as extreme environment are a unique mininature 

landscape in desert district as well as the obvious 

sign of fixing mobile dune. They are composed of living 

microorganisms, their product of metabolism (mainly 

extracellular polysaccharides) and sand granule 

(Gundlapally and Garcia-Pichel, 2006). 

Exopolysaccharides (EPS) produced by microorganisms 

in BSCs are barely reported in the recent research. 

Medium condition and other bacteria growth conditions 

are important factors for EPS production. There are large 

numbers of reports on optimization of EPS production by 

statistical optimization techniques (Xu et al., 2003; 2010; 

Kaditzky and Vogel, 2008; Hao et al., 2010) Orthogonal 

design as one of the important statistical methods has 

been successfully applied to improvement of the


Table 1. Orthogonal matrix table of the L9 (3 4 ). 

Factor 1 2 3 

Beef extract (g/100ml) 0.2 0.3 0.4 

Peptone (g/100ml) 0.5 1.0 1.5 

Maltose (g/100ml) 2 3 4 

NaCl (g/100ml) 0.4 0.5 0.6 

production of primary and secondary metabolites in 

cultivation process (Xu et al., 2003). 

This work is an attempt to isolated strain producing 

EPS from sand BSCs, analysize suitable media and 

culture condition for the production of EPS from B. 

thuringiensis 27 and examines the bioflocculaton activity 

of EPS. To the best of our knowledge, the nutritional 

requirements and culture condition of EPS production 

form B. thuringiensis 27 isolated from sand BSCs and its 

bioflocculation activity has not been demonstrated. 


Bacterial strain 

Bacillus thuringiensis 27 was originally isolated from BSCs collected 

in the Gurban Tonggut Desert. It was maintained on agar slants 

containing (g/l): beef extract 3; peptone 10; NaCl 5 and agar 20 (pH 

7.0-7.4). The slants were incubated at 35°C for 24 h and the fully 

grown slants were stored at 4°C. 

Culture medium 

Basal medium, beef extract 3 g, peptone 10 g, NaCl 5 g, water 

1000 g, pH 7.0-7.4. Fermentation medium, beef extract 3 g, 

peptone 10 g, NaCl 5 g, water 1000 g, pH 7.0-7.4, temperature 

35°C. 

Single factor experiments of culture requirement 

To find the optimal culture requirements, the following factors were 

investigated using the one-factor-at-a-time method, including 

carbon sources nitrogen sources inorganic ions, initial pH value, 

cultivation temperature, inoculum size, liquid volume triangle flask. 

All experiments were performed in triplicates (n = 3). 

Orthogonal matrix method 

The orthogonal L9 (3 4 ) was used to obtain the optimal medium after 

the test by the one-factor-at-a-time method. This enables us to 

determine which process variables affect the response. A logical 

next step is to determine the point in the important factors that 

leads to the best possible response (Di et al., 2003; Li et al., 2001). 

The levels of components of the culture medium are listed in Table 

1. All experiments were performed in triplicate (n = 3). The software 

SPSS was used for experimental design, data analysis and model 

building. The optimal fermentation conditions for enhanced yield of 

EPS and cell growth were obtained by solving the regression 

equation using the software Origin 8.0. 

Determination of flocculating activity 

A kaolin suspension was used to measure the flocculating activity 

of the EPS crude extracts. One milliliter of 90 mmol/l CaCl2 and one 

milliliter were added into 50 ml 4.0 g/l kaolin suspension. The 

mixture was vigorously stirred for 0.5 min, and stand for 4 min. The 

optical density (OD) of the clarifying solution was measured with a 

spectrophotometer at 550 nm. A control experiment was conducted 

in the same manner by replacing distilled water. The flocculating 

activity was calculated according to the following equation (Kurane 

et al., 1986): 

η= (A-B) ×100/A 

where; A is the optical density of the control experiment at 550 nm 

and B is the optical density of the sample experiment at 550 nm. All 

experiments were performed in triplicates (n = 3). 

Analytical methods 

Samples collected at various culture conditions from shake flasks 

were centrifuged at 7000 g for 15 min. The 2 ml resulting 

supernatant was precipitated with threefold times 95% ethanol, 

stirred vigorously, and left overnight at 4°C. The precipitated 

polysaccharides were collected by centrifugation at 7000 g for 15 

min, discarding the supernatants, and dissolved with distilled water, 

dialyzing for 24 h, adding distilled water to 20 ml. Phenol-Sulfuric 

Acid Method was used for determining EPS yield (Pazur et al., 

1994), using glucose solution as a standard reference. The EPS 

yield was expressed as gram per liter. 

RESULTS 

Effect of carbon and nitrogen source on EPS yield 

To find out the optimal carbon and nitrogen source for the 

EPS production of B. thuringiensis 27 five 2% carbon 

sources (glucose, maltose, sucrose, lactose and glycerol) 

were separately provided in the basal medium and five 

1% nitrogen sources (ammonium chloride, ammonium 

sulfate, peptone, beef extract and yeast extract) were 

separately instead of peptone employed in the basal 

medium. A high level of EPS was obtained when maltose 

and glycerol were used as the carbon source. Among the 

carbon sources tested, maximum EPS (10.45 g/l) was 

obtained in the maltose medium. Based on this study 

maltose is a good candidate. The maximum EPS

EPS prduction (g/l) 

12 

10 

8 

6 

4 

2 

0 

Basal medium 

Glucose 

Frucose 

Maltose 

Carbon sources 

Lactose 

Glycole 

Figure 1. Effect of carbon and nitrogen sources on EPS production. 

production (10.53 g/l) was obtained in the peptone 

(Figure 1). So we selected maltose peptone as our 

optimal nitrogen source. 

Effect of concentration of carbon and nitrogen 

source on EPS yield 

To find out the suitable concentration of maltose for the 

EPS yield B. thuringiensis 27 five different concentration 

(1, 2, 3, 4 and 5%) maltose were separately instead of 

2% maltose above. The maximum EPS production (10.25 

g/l) was obtained when the concentration is 3% (Figure 

2). To find out the suitable concentration of nitrogen 

source for the EPS yield five different concentration (0.1, 

0.2, 0.5, 1.0 and 1.5%) peptone were separately instead 

of 1% peptone above. The maximum EPS production 

(10.74 g/l) was obtained when the concentration is 1% 

(Figure 2). 

Effect of inorganic ions and its concentration on EPS 

yield 

To find out the suitable mineral elements for the EPS 

yield of B. thuringiensis 27 six different 0.5% mineral 

elements (sodium chloride, magnesium chloride, copper 

12 

10 

8 

6 

4 

2 

0 

NaNO3 

NH4Cl 

NH4SO4 

Nitrogen sources 

peptone 


Yeast extracts 

sulfate, zinc chloride, ferric chloride and manganese 

chloride) were separately in basal medium. The maximum 

EPS production (15.36 g/l) was obtained in sodium 

chloride (Figure 3). Besides magnesium chloride, others 

depress the EPS production. Based on the result we 

selected sodium chloride as our selection. 

Further, to find out the suitable concentration of NaCl 

for the EPS yield five different concentration (0.3, 0.4, 

0.5, 0.6 and 0.7%) sodium chloride were separately 

instead of 0.5% sodium chloride above. 

The maximum EPS production (15.36 g/l) was obtained 

when the concentration is 0.5% (Figure 3). The difference 

in EPS yield between the peptone concentrations of 0.5% 

and 0.6% was not significant because the cell could not 

absorb more mineral element. But considering the 

economic factor, we use the 0.5% NaCl as our optimal 

option. 

Effect of initial pH and temperature on EPS yield 

To find out the optimal temperature for and EPS 

production, B. thuringiensis 27 was cultivated at various 

temperatures ranging from 23 to 45°C. The EPS (12.12 

g/l) were observed at 28°C (Figure 4). B. thuringiensis 27 

was grown at initial pH 7.0 for polysaccharide production 

and optimum EPS production is 14.96 g/l) (Figure 4).


EPS Production (g/l) 


Figure 2. Effect of maltose and peptone concentration on EPS production. 

16 

14 

12 

10 

8 

6 

4 

2 

(g/l) 

14 

12 

10 

8 

6 

4 

FeCl 3 

10 20 30 40 50 

ZnCl 2 

Maltose Concentration (g/l) 

MnCl 2 

MgCl 2 

Inorganic ions 

CuSO 4 

Figure 3. Effect of inorganic ions and NaCl concentration on EPS production. 

Effect of inoculum size and liquid volume on EPS 

yield 

To examine the effect of inoculum size, B. thuringiensis 

27 was varying the inoculum size (2, 5, 8 and 11%) in 

basal medium. The results indicated 8% inoculums size 

NaCl 

14 

12 

10 

8 

6 

4 

16 

14 

12 

10 

8 

6 

4 

2 

1 2 

5 

10 

Pepton Concentration (g/l) 

15 

3 4 5 6 7 

NaCl (g/l) 

was fit for EPS production (Figure 5). 

To find out the optimal oxygen rate four different culture 

medium volumes (25, 40, 55 and 70 ml) added in 200 ml 

flask were separately instead of 60 ml basal medium. The 

maximum EPS production (14.38 g/l) was obtained when 

the culture medium volume is 25 ml in 200 ml flask

(Figure 5). 


EPS Yield (g/L) 

Figure 4. Effect of initial pH value and temperature on EPS production. 

16 

14 

12 

10 

8 

6 

16 

14 

12 

(g/L) 

10 

8 

6 

4 

5 6 7 8 9 

Initial pH value 

25 40 55 70 

Liquid Volume (ml/200ml flask) 

Figure 5. Effect of liquid volume and inoculum size on EPS production. 

Optimization by orthogonal matrix method 

According to the orthogonal method, the effect of those 

medium on EPS production was analyzed and the results 

are shown in Table 2. Based on the magnitude order of R 

value (maximum difference), the order of effects of all 

factors on EPS production was maltose > peptone > 

NaCl > beef extract. In terms of the maximum K-value of 

16 

14 

12 

10 

8 

6 

4 

16 

14 

12 

10 

8 

6 

4 

23°C 28°C 33°C 38°C 43°C 

Temperature 

2 5 8 11 

Inoculum Size (%) 


each column in Table 3, optimal level of each medium 

ingredient for EPS yield was 3 g/l beef extract glucose, 

10 g/l peptone, 40 g/l maltose, 4 g/l NaCl. 

Based on the magnitude order of R value (maximum 

difference), the order of effects of all factors on EPS yield 

was maltose >peptone >NaCl >beef extracts. According 

to ANOVA (Table 4), the factors, peptone and maltose 

had significant effect on EPS yield (P


Table 2. Results of L9 (34) orthogonal test of exopolysaccharide production of Bacillus strain 27 strain in shake flask culture. 

Run 1 2 3 4 

Factor Beef extract (g/L) Peptone (g/L) Maltose (g/L) NaCl (g/L) Results (g/L) 

1 2 5 20 4 5.63±0.2 

2 2 10 30 5 15.98±1.07 

3 2 15 40 6 13.24±0.73 

4 3 5 30 6 5.83±0.31 

5 3 10 40 4 18.08±2.26 

6 3 15 20 5 5.59±0.52 

7 4 05 40 5 10.54±2.21 

8 4 10 20 6 8.57±0.22 

9 4 15 30 4 13.48±0.2 

Values are means ± SD of triple determinations. 

Table 3. Analysis of media on exopolysaccharide production of Bacillus strain. 27 in shake flask culture with 

L9 (34) orthogonal test. 

Beef extract (g/l) Peptone (g/l) Maltose (g/l) NaCl (g/l) 

k1 11.61 7.33 6.59 12.39 

k2 9.83 14.21 11.76 10.70 

k3 10.86 10.77 13.953 9.21 

R 1.78 6.87 7.356 3.18 

Ki =Σ exopolysaccharide in thrice experiment at Xi. Values are means ±SD of triple determinations 

R = maximum Kix -minimum Kix. Values are means ± SD of triple determinations. 

Table 4. Variance analysis of L9 (34) orthogonal experiment on EPS yield. 

Sum of squares Degree of freedom Mean square Significance 

Beef extract 4.809 2 1.000 

Peptone 70.933 2 14.750 * 

Maltose 85.611 2 17.802 * 

NaCl 15.221 2 3.165 

Error 4.81 2 

inoculum size 8%, liquid volume 40 ml in 200 ml flask) 

was carried on. Consequently, the maximal EPS 

production was 20.17 g/l. The EPS yield on optimal 

condition was about five times than that at the basal 

culture condition. 

Bio-flocculating activity 

The flocculating activity of the EPS was measure by 

using a kaolin suspension. In order to neutralize the 

charge, one milliliter of 90 mmol/L CaCl2 were added into 

50 ml 4.0 g/l kaolin suspension. The optical density (OD) 

of the clarifying solution was measured with a 

spectrophotometer at 550 nm. After calculation the 

flocculating activity of B. thuringiensis 27 is 80.4%. 

DISCUSSION 

Carbon source plays a very important role in growth 

especially in polysaccharide production. A substantial 

change in the polysaccharide production was observed 

with different carbon sources. Maltose supported 

maximum EPS production which was difference with 

other bacillus (Lee et al., 1997a). The difference in EPS 

production among the maltose concentration of 3, 4 and 

5% was not significant because the cell maybe not 

absorb more maltose. It also is report that see that the 

EPS production increased as the initial carbon resource 

concentration increased from 20 to 100 g/l (Lee et al., 

1997b). Nitrogen sources in the form of proteins and 

nucleic acid play an important role in the cell mass. 

Peptone as organic nitrogen source is the best nitrogen

source in our work. Additionally, it was found in the study 

that organic nitrogen source are more suitable for the 

EPS production. It was reported that organic nitrogen 

sources were absorbed by the cells easier than the 

inorganic ones (Hwang et al., 2003; Gandhi et al., 1998). 

Inorganic ions affected EPS production by combined with 

enzyme. The result showed NaCl could provide the 

maximum EPS production, which did not match those of 

other researcher (Lung and Huang, 2010). 

Optimum pH for polysaccharide production for bacteria 

ranges from 6.0 to 7.5 (Kumar et al., 2007), which exactly 

accorded with those of B. thuringiensis 27. Optimum EPS 

production (14.96 g/l) was obtained in the neutral pH 

range. The maximum EPS production by B. thuringiensis 

27 were observed at 28°C, which is comparable to many 

kinds of bacillus that have relatively low temperature 

optimal (example 20 to 25°C) in their submerged 

cultures(Cerning et al., 1992). Among several bacteria 

physiological properties inoculum size may play an 

important role in biological development (Gancel and 

Novel, 1994). Inoculum size and liquid volume may play 

an important role in cell reproduction and EPS production 

(Chen et al., 2008). Result showed 8% inoculum size and 

25 ml in 200 ml flasks was fit for EPS production. 

From the single factors experiments the three critical 

factors affect the EPS yield were identified. Considered 

beef extract as an important nitrogen source it is selected 

to further optimize. To investigate the relationship among 

various factors the orthogonal experimental design 

technique as a mathematical method were used the 

orthogonal matrix method was obviously a serviceable 

experimental design to simultaneously investigate the 

relationship between the effect of medium components 

and their optimal concentrations (Gundlapally and 

Garcia-Pichel, 2006). In addition, the orthogonal matrix 

can comprehensively investigate central composite 

design, and thus facilitates economical benefit, experimental 

convenience. In our work by orthogonal method 

the EPS production arrived to 20.17 g/l, which is not only 

about five times than those of the basal medium but more 

than those of the maximum result of orthogonal design. 

That is means the orthogonal matrix method can be used 

for optimizing the EPS production in a submerged 

fermentation process. Furthermore it is applied to 

optimization of culture media for the production of primary 

and secondary metabolites in fermentation processes 

(Chen et al., 2008). 

Flocculation technology as a kind of effective and quick 

method is used for wastewater treatment. And bioflocculation 

is a dynamic process resulting from the synthesis 

of extracellular polymers by living cells. In recent years, 

the use of microbial flocculants has been promoted as a 

solution to environmental problems because their 

intermediates are harmless and biodegradable. Thus 

searching and usage bioflocculation arouse the 

interesting of researcher in the world. The flocculating 

activity of 27 is 80.4% which is below the bioflocculant p- 

KG03 (Yim et al., 2007). To obtain new and better 


bioflocculant, we need screen more strain to obtain 

functional EPS in BSCs. Otherwise new usage for 

microbiology in the desert and desert biological soil 

crusts is proved a scientific basis. 

Conclusion 

The traditional separation, screening and purification 

methods were used, 27 strains of producing EPS were 

isolated. Under optimal condition the strain could produce 

a large amount of EPS. And the EPS possess a certain 

degree of flocculation, the flocculation rate was 80.4%, 

which is lower than the previously reported strain isolated 

from the soil (flocculation rate can reach 97%). Further 

screening and optimization to increase the flocculation 

rate still carry on. It is well known that microbial 

flocculants as a kind of sewage treatment agent are nontoxic, 

harmless, no secondary pollution. In this subject, 

the discovery of new sources of microbial flocculant has 

brought new interest in dealing with the pollution of water 

resource. And new usage for the desert biological soil 

crusts is provided a scientific basis. 


This work was supported by the Commonweal Industry 

Scientific Researcher Project from the Ministry of 

Agriculture (No. 200803033). 

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DOI: 10.5897/AJMR11.893 



A simple and efficient method that uses low 

concentration fetal bovine serum to culture and purify 

Schwann cells 

Han Feng, Qu Wei*, Jiang Huajun, Fu Chongyang, Lu Ming, Zhang Weiguo and Lv Decheng 

Department of Orthopaedic Surgery, First Affiliated Hospital of Dalian Medical University, Dalian, China. 

Accepted 18 April, 2011 

To develop a simple and efficient method for the isolation of highly purified Schwann cells (SCs) from 

the sciatic nerves of newborn rats, cells were initially cultured in 10% FBS for 6 days, and then cultured 

in 2% FBS for 6 days. And we used the immunocytochemical characterization, flow cytometry, RT-PCR, 

to observe the condition of SCs. After the 12-day treatment protocol, we obtained a high yield of SCs 

with an average purity of 96.9%. Furthermore, the condition of SCs had no change. In our purification 

protocol, we had shown that our procedures did not alter the cell cycle and neurokines’ secretion. Our 

simple and efficient method yielded pure and healthy SCs and did not require treatment with antimitotic 

agents or any special equipment. 

Key words: Schwann cells, cell culture, low fetal bovine serum concentration, tissue engineering, fibroblast. 

INTRODUCTION 

Tissue engineering, the use of cells and engineering 

methods to modify biological functions, has already been 

applied to diverse clinical fields. In neurology, tissue 

engineering appears to have certain advantages over 

nerve cell transplantation. There are three critical 

meterials of tissue engineering: (i) biodegradable 

materials; (ii) trophic factors; and (iii) cells, typically 

autologous cells that do not cause antigenic reactions 

(Mackinnon and Dellon, 1990; Vacanti et al., 2001). In 

peripheral nerve tissue engineering, it is critical to rapidly 

obtain large numbers of pure Schwann cells (SCs) 

(Fansa and Keilhoff, 2004). SCs are the principal 

supporting cells of the peripheral nervous system, and 

are crucially involved in the functional recovery of injured 

peripheral nerves (Bunge, 1993). SCs play an important 

role in nerve regeneration and are at the core of 

peripheral nerve tissue engineering, a procedure that 

requires a large number of cells. Diverse methods have 

been proposed for the purification of SCs: antimitotic 

agent and antibody-mediated cytolysis methods (Brockes 

*Corresponding author. E-mail: fhtg001@sina.com. 

et al., 1979); repeated explantation methods (Oda et al., 

1989); the cold jet technique (Jirsová et al., 1997); 

immunoselective methods (Calderon-Martinez et al., 

2002); in vivo or in vitro predegeneration methods 

(Komiyama et al., 2003; Mauritz et al., 2004); differential 

adhesion methods (Pannuzio et al., 2005); differential 

detachment methods (Jin et al., 2008); and a combination 

of the in vitro predegeneration and cold jet technique 

(Haastert et al., 2007; 2009). These methods can yield 

highly purified SCs with various efficiencies, but all 

require special equipments, complicated procedures 

and/or high costs. 

Preparation of SCs for tissue engineering requires a 

safe, rapid, easy and high yield methodology for 

purification. We aimed to develop an economical method 

besides those requirements from 7 days old Sprague 

Dawley (SD) rats by use of low concentration FBS. 


Animals 

Postnatal SD rats (7 days old, 11.3±0.5 g) were obtained from the 

Dalian Medical University Animal Center (Dalian, China). All animal


protocols were approved by the Animal Experiment and Care 

Committee of Dalian Medical University. 

Primary culture 

Rats were sacrificed by decapitation, and sciatic nerve segments 

(10 to 15 mm in length) were harvested aseptically under a 

dissecting microscope and were temporarily maintained in 60 mm 

culture plates (Corning, USA). Then, 2 ml of ice-cold D-Hanks 

solution (Sigma USA) was added, and the sciatic nerve segments 

were removed from the epineurium and cut into pieces (1 mm 3 ) 

under a dissecting microscope. Finally, nerve fragments were 

placed in 15 mL conical centrifuge tubes (Corning, USA) and 

centrifuged (140 g × 5 mins). The supernatant was discarded. 1 mL 

each of 0.25% trypsogen (Gibco, USA) and collagen 0.2% type- II 

(Gibco, USA) were added, then incubated in a cell incubator 

(Thermo, USA) with 5% carbon dioxide at 37°C for 30 min and heat 

shocked once every 5 mins. Addition of 2 mL of Dulbecco’s 

Minimum Eagle’s Medium (DMEM) (Hyclone, USA) that contained 

10% FBS (Gibco, USA). The mixture was centrifuged again (140 g 

× 5 min), and the supernatant was again discarded. Removed 

nerve fragments to 60 mm culture plates. Then, 1.5 mL 

DME/10%FBS containing 50 U/ml penicillin and 50 µg/ml 

streptomycin (Gibco, USA) was added, and all plates were 

maintained in a cell incubator within a humidified atmosphere with 

5% CO2 at 37°C. The growth medium was changed every 48 h. 

Purification of SCs 

Cells isolated from nerve segments after the first 6 days were 

divided into three groups: Group A (control) were fed every 2 days 

using DMEM with 10% FBS; Group B were fed every 2 days using 

DMEM with 2% FBS and cells were collected on day 6; Group C 

were fed every 2 days using serum-free DMEM, and cells were 

collected on day 6. 

Immunocytochemical characterization of SCs and SC purity 

Immunohistochemical staining was accomplished in the culture dish 

after 12 days. Cells were marked with the Strept Avidin-Biotin 

Complex (SABC) kit and rabbit anti-S100 protein antibody (Boster, 

China) according to the manufacturer’s instructions. Then, cells 

were washed two times with PBS (pH 7.2) (Gibco, USA), fixed for 

90 mins in 4% paraformaldehyde, and then immersed in a mixture 

of 30% H2O2 and pure methanol (1:50) for 30 mins. After that, 5% 

BSA (confining liquid) was added drop-wise at room temperature in 

20 mins. BSA was shaken out of the dish. Then anti-S100 protein 

antibody was added drop-wise at 37°C for 1 h, washed 3 times with 

PBS, incubated with secondary antibodies at 37°C for 20 mins, 

washed 2 times with PBS. Then, SABC was added drop-wise at 

37°C for 20 mins, washed 4 times with PBS. Finally, cells were 

stained by DBA (Hsu and Ree, 1980; Klosen, 1989) using a kit 

(Boster, China), at room temperature for 8 min, then post-stained 

with hematoxylin and examined under a microscope. Cells with a 

bipolar or tripolar shapes were identified as SCs, whereas flat or 

polygonal cells were identified as fibroblasts (Pannuzio et al., 

2005). 

SC purity was expressed as the percentage of SCs (SC purity = 

[number of SCs]/[number of SCs + number of fibroblasts]) from 

samples of ten visual fields at 200 × magnification. 

Flow cytometry 

Cells from Groups A and B were digested with 0.25% trypsogen and 

0.02% EDTA, treated into the single cell suspension, centrifuged 

(314 g × 5 min) and washed with PBS. Then, centrifuged again, and 

the precipitate were resuspended and fixed in 2 mL of cold 80% 

reagent grade ethanol and left overnight at 4°C. Then, centrifuged 

and used 0.01% RNAase (Sigma, USA) to resuspend the 

precipitate and heat shocked at 37°C for 10 min. The cells were 

centrifuged and0.5%PI (propidium iodide) (Sigma, USA) was added 

to the precipitate and cell concentration was adjusted to 1 × 10 6 per 

mL. 

Finally, the cells were stained in darkness for 30 min and 

detected by flow cytometry. 

Reverse transcriptase polymerase chain reaction (RT-PCR) 

Total RNA was extracted separately from Groups A and B after 12 

days and cDNA was synthesized from the total RNA using a RNA 

PCR Kit (AMV) Ver.3.0 (Takara, China) according to the 

instructions. The sequences of the primers for NGFb (Yan et al., 

2007), BDNF (Yan et al., 2007), β-actin are depicted in Table 1. The 

PCR conditions were 94°C for 30s (denaturation), 56°C for 30 s 

(annealing) and 72°C for 1 min (extension) for a total of 30 cycles 

(NGFb, BDNF), or 55°C for 30 s (annealing) for β-actin. And a 

DL2000 DNA-ladder (TaKaRa, Japan) was run in parallel to the 

samples. Amplification products were separated by 2.0% agarose 

gel electrophoresis. After the gels were scanned, the relative 

intensity of NGFb and BDNF bands was determined by using Gelpro 

Analyzer software (Media Cybernetics, USA). 

Statistics 

SC purity and cell yields are presented as means ± SDs. Values 

were subjected to a Student’s t-test, with a P value less than 0.05 

considered to be statistically significant. 

RESULTS 

Primary culture and purification of SCs 

During the first 48 h of the primary culture, two distinct 

types of cells split off from the nerve fragments (Figure 

1a). Phase-contrast microscopy (× 200) indicated that 

most of the cells attached to the bottom of the culture 

dishes could be classified as SCs (spindle-shaped, 

bipolar, and sometimes tripolar, with a small cytoplasmto-nucleus 

ratio) or as fibroblasts (flat polygonal shaped, 

with a prominent ovoid nucleus and abundant cytoplasm). 

Most of the fibroblasts appeared to be scattered among 

the SCs, but some were beneath the SCs. During the first 

48 h, SCs proliferated faster than fibroblasts. However, 

96 h later, fibroblasts gradually became the dominant cell 

type, and SCs stopped growing and migrated onto 

fibroblasts. SCs and fibroblasts were both growing rapidly 

after 4 days. 

After 6 days of primary culture of Group A cells (DMEM 

with 10% FBS), most of the fibroblasts growing among 

the SCs were easily detached from the culture plates, 

whereas those growing under or interacting with SCs 

could not be removed (Figure 1b). For group B cells 

(DMEM with 2% FBS), the fibroblasts started to die after 

48 h, quite a number of cell were gone after 96 h of

Table 1. The sequences of the primers for NGFb, BDNF, β-actin. 

Feng et al. 2369 

Forward primer Reverse primer Ta (°C) Amplicon length (bp) 

NGFb 5'-GGCCACTCTGAGGTGCATAG-3' 5'-CATGGGCCTGGAAGTCTAAA-3' 56 349 

BDNF 5'-AAACCATAAGGACGCGGACT-3’ 5'-GATTGGGTAGTTCGGCATTG-3' 56 393 

β-actin 5’-TCTACGAGGGCTATGCTCTCC-3’ 5-GGATGCCACAGGATTCCATAC-3’ 55 320 

Figure 1. Observe the results of the growing cells with inverted microscope. A as SC, B as fibroblast, C as nerve 

segment and D as dead cells. During the 2nd day of the primary culture, two distinct types of cells emerged from 

the nerve fragments (a). And, at the 6th day of the primary culture, SCs and fibroblasts were growing fast (b). 

However, used DMEM with 2% FBS, numerous fibroblasts died within 96 h; (c) and almost all of the fibroblasts 

were cleared away by the 6th day and SCs morphology was not changed with 2% FBS (d). But after 6 days 

barely any cells survived at the culture plate in Group C. scale bar: 100 um). 

culturing (Figure 1c), and almost all fibroblasts were gone 

after 6 days of culturing. The morphology of SCs did not 

change during this time (Figure 1d). For group C cells 

(serum-free DMEM), a few cells died after 48 h, but after 

96 h, both types of cells had died in significant numbers. 

The death rate continued to increase, and after 6 days 

of culturing, almost all cells had died (Figure 1e). 

Immunocytochemical characterization of SC and SC 

purity 

S-100 is a well-established sensitive and specific marker 

for SCs (Raff et al., 1979). We observed S-100 staining in 

the SCs body and along the processes (Figure 2). Our 

purified SCs had typical bi- or tri-polar morphology and 

oval nuclei. In contrast, the contaminating fibroblasts 

were negative for S-100 staining. Because our 

immunological staining was performed in culture dishes, 

we had acquired very high yield and high-quality cells. 

Since so many of the Group C cells died, we could not 

collect enough cells for immunocytochemical staining. 

Table 2 shows the percentage of cells positive for S-100. 

SC purity in Group B was significantly better than in 

Group A (P < 0.01). However, the total numbers of SCs 

were too little to discriminate Group A from B (p > 0.05). 

Flow cytometry 

To further provide qualitative evidences of the characters 

of our SCs, we compare the SC cycle of Groups A and B 

cells (Figure 3). As shown in Table 3, there was no 

significant difference in the cells cycles between Groups


A and B cells (p > 0.05). 

Figure 2. Immunocytochemical characterization of SCs with 

S100 showed that the purified SCs had typical bi- or tri-polar 

morphology and oval nuclei. (a) In contrast, the contaminating 

fibroblasts were negative for S-100 staining (b). Used DMEM with 

10% FBS, numerous fibroblasts are shown at (A). Used DMEM 

with 2% FBS (B), by contrast, cells were very purified (scale bar: 

100 um). 

Table 2. Summary of results in the present method for SCs preparation. 

View Number of S100 protein positive cells/total number of counted cells 

Group A Group B 

1 646*/753 (0.857902)** 779*/824 (0.945388)** 

2 988*/1173 (0.842285)** 1045*/1094(0.955210)** 

3 1111*(/12340.900324)** 1368*/1415(0.966784)** 

4 845*/978 (0.864008)** 722*/746 (0.967828)** 

5 750*/849 (0.883392)** 901*/936 (0.962607)** 

6 731*/862 (0.848028)** 1711*/1752(0.976598)** 

7 1130*/1270(0.889764)** 749*/762 (0.982940)** 

8 1083*/1210(0.895041)** 939*/956 (0.982218)** 

9 1092*/1206(0.905473)** 839*/860 (0.975581)** 

10 592*/738 (0.802168)** 836*/856 (0.976636)** 

* There was an insignificant difference between SCs total at Groups A and B (p>0.05); **There was a 

significant difference between SC purity at Groups A and B (p

DISCUSSION 

Main findings 


Figure 3. The cell cycle of SCs detected by flow cytometer showed that used DMEM with 10%FBS(A). DIP 

G2:0.53%, S:47.37%, and used DMEM with 2%FBS(B) DIP G2:0.10%, S:44.92%. 

Table 3. Summary of results in the present method for SCs cycle. 

Cell cycle 

Group A 

Group B 

G2 a (%) S b (%) G2 a (%) S b (%) 

0.53 47.37 0.10 44.92 

0.37 45.44 0.68 48.23 

0.13 44.80 0.42 46.04 

a There was a insignificant difference between G2 percentage at Groups A and B (p>0.05); b There was a 

insignificant difference between S percentage at Groups A and B (p>0.05). 

Our method of culturing the sciatic nerves of newborn SD 

rats in DMEM with 10% FBS allowed SCs and fibroblasts 

to split off from intact nerve fragments. Once SCs have 

migrated in high densities, they proliferate actively due to 

the production of autocrine growth factors, IGF-2, NT-3 

and PDGF-BB (Jessen and Mirsky, 1999), regardless of 

being cultured in a medium with low FBS concentration. 

However, cells in serum-free DMEM (Group C) did not 

have adequate nutrition for growth and proliferation. 

Thus, although there were few fibroblasts in Group C, the 

growth of SCs was also greatly affected. There was no 

significant difference in the number of SCs in Groups A 

and B, but the SC purity was significantly greater in 

Group B. Our results indicate that culturing of nerve cells 

in DMEM with 2% FBS can gradually reduce the 

percentage of fibroblasts, while having no effect on SC 

differentiation, morphology or character. 

The immuno staining results indicated that staining was 

evident in both SC cell body and processes during the 

culture period. The flow cytometry results indicated that 

the number of cells in S period and G2 period were similar 

in Groups A and B. NGFb and BDNF are the main growth 

factors secreted by SCs, which are known to have 

neurotrophic effects on nerve regeneration (Schicho et 

al., 1999; Serpe et al., 2005). 

Semi-quantitative analysis of NGF mRNA and BDNF 

mRNA by RT–PCR showed that SCs of Group B had 

similar productive function of secretion the normal 

cultured SCs. Thus, we conclude that our culturing 

protocol is a simple and efficient method to purify SCs 

that also preserves the function and morphology of SCs. 

Comparison with the existing literature 

Gross dissection of nerve cells may lead to contamination 

of the desired cells (Jessen and Mirsky, 1999) and 

peripheral nerve regeneration with tissue-engineered 

cells requires highly purified SCs for the construction of 

artificial nerve grafts. Many previous studies have


500bp 

250bp 

500bp 

250bp 

M 

5 

6 

M 1 2 

3 4 

Figure 4. Total RNA was analyzed for the expression of NGFb and BDNF by RT–PCR at 12 days.1: BDNF 

(Group A); 2 BDNF (Group B); 3: NGFb (Group A); 4::NGFb (Group B); 5: β-actin(Group A);6: β-actin(Group B); 

M: Marker. 

attempted to reduce the fibroblast contamination and 

increase SC purity by use of: (i) antimitotic treatment 

(Schicho et al., 1999); (ii) a combination of antimitotic 

treatment and antibody-mediated cytolysis with complements 

(Brockes et al., 1979); (iii) repeated explantation 

methods 6 (Serpe et al., 2005; Smith and Greenfield, 

2003); (iv) differential adhesion methods (Pannuzio et al., 

2005; Wood, 1976); and (v) immunoselective methods 

(Manent et al., 2003; Askanas et al., 1980; Wrathall et 

al., 1981). Those previously proposed methods have 

been shown to enrich SCs with various efficiencies, but 

all have certain limitations. In particular, antimitotic agents 

are harmful to SC function (Serpe et al., 2005; Assouline 

et al., 1983) and can reduce SC yield because of their 

non-specific antimitotic effects. Antibodies and 

complements are expensive for large-scale SC 

preparations, so do not provide an economical approach 

to SC purification. Repeated explantation and 

differential adhesion can be comparatively complicated 

and time-consuming procedures, and may lead to a loss 

of SCs and delay of therapy. Immunoselection is a good 

method for achieving high purity, but requires expensive 

antibodies and special facilities. 

In a previous study, Needham et al. (1987), described 

the use of serum-free medium (S4) that allowed for 

optimal SC proliferation (up to 90% purity after 10 days), 

and suppressed fibroblast overgrowth in primary cultures 

of neonatal rat sciatic nerves (Assouline et al., 1983). But 

this serum-free media, though suitable for selective SC 

migration, was not as conducive for SC proliferation. 

However, use of a growth medium with 10% FBS 

supported not SC proliferation but an explosive fibroblast 

overgrowth. Komiyama et al. (2003) described the use of 

low FBS concentration for the purification of SCs, and 

obtained an average purity of 98% after 8 days by using 

DMEM for the first 6 days and changing to DMEM with 

2.5% FBS on day 6. However, this method yielded low 

cell counts, and the resulting SC purity was only 88.0%.

Another method described by Komiyama et al. (2003) 

cultured cells in DMEM with 10% FBS for the first 3 days, 

then in serum-free DMEM for 3 days, then, on day 6 in 

DMEM with 2.5% FBS. The SC purity was 93.6% at 14 

days, but was reduced to 89.5% at 21 days. Thus, 

regardless of method, the total cell counts were 

increasing, but the percentage of SCs decreased over 

time. 

This phenomenon was also noted in our study. 

IMPLICATIONS FOR FUTURE RESEARCH 

The protocol described here is a simple, economical, 

rapid and efficient method for the purification of SCs. Our 

method has a high yields and results in highly pure cells. 

We suggest that our method might be useful for the 

development of tissue-engineered nerves and for other 

studies related to peripheral nerve injuries. 


This research was supported by National Natural Science 

Foundation of China (30973066). 

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DOI: 10.5897/AJMR09.455 



First report on Enterobacter sakazakii from Sudanese 

patients 

Humodi A. Saeed¹* and Rania M. Musallam² 

Department of Microbiology, College of Medical laboratory Science, Sudan University of Science and Technology, 

Arbab El Agaid Street, P. O. Box 407 Khartoum, Sudan. 


Enterobacter sakazakii (E. sakazakii) has been identified as emerging opportunistic pathogens that can 

cause enterocolitis, bacteraemia, meningitis, brain abscess, and urinary tract infection. They have been 

particularly associated with meningitis in neonates where infant milk formulae have been 

epidemiologically linked to the disease. This study was carried out during the period of November 2008 

to March 2009; to determine the occurrence of E. sakazakii in clinical specimens and its resistance to 

traditionally used antimicrobial agents in the Sudan. A total of 389 (311 urine specimens, 11 wound 

specimens, and 67 stool specimens) were collected from outpatients of three leading hospitals in 

Khartoum State. The urine specimens and wound swabs were cultured on blood and MacConkey's 

agars for primary isolation of pathogen, while stool specimens were cultured on selenite F broth and 

incubated overnight then subcultured on xylose lysine deoxycholate agar. Identification of the E. 

sakazakii was done by colonial morphology, Grams stain and biochemical tests using API 20E. Modified 

Kirby-Bauer disc diffusion method was adopted to determine the resistance rate of E. sakazakii to 

fifteen antimicrobial agents. Minimum inhibitory concentration (MIC) of antimicrobial agents was 

determined by E-test. The result showed that out of 389 specimens examined, 6 (1.5%) E. sakazakii were 

recovered, 4 (1.03%) from urine, 1(0.3%) from wound and 1(0.3%) from stool. The results more over 

revealed that the antimicrobial resistance of E. sakazakii was as follows; ceftazidime, amoxicillin, 

amoxyclav (100% each), co-trimoxazole, ticarcyline (83.3% each), chloramphenicol, tetracycline, 

ceftriaxone, nitrofuratoin, cephotaxime, tobramycin (66.7% each), ciprofloxacin, amikacin and nalidixic 

acid (16.7% each). None of the isolates were found to be resistant to gentamicin. In addition to that, the 

result indicated that the MIC, MIC50 and MIC90 of different antimicrobial agents range from 0.001 to > 240, 

0.1 to > 240 and 0.5 to > 240 µg/ml respectively. The results indicated for the first time the presence of E. 

sakazakii in the examined clinical specimens in Sudan. The occurrence was high and the antimicrobial 

resistance of the isolated E. sakazakii was also high. 

Key words: Enterobacter sakazakii, API 20E, E-Test, Khartoum, Sudan. 

INTRODUCTION 

Enterobacter sakazakii (E. sakazakii) is a Gram-negative 

rod belonging to the family Enterobacteriaceae (Fiore et 

al., 2008). The organisms have been identified as 

emerging opportunistic pathogens that can cause 

enterocolitis, bacteraemia, meningitis, brain abscess and 

urinary tract infection. They have been particularly

associated with meningitis in neonates (Ongrádi, 2002; 

MacLean et al., 2009). Neonatal pathologies also include 

wound exudates, appendicitis, and conjunctivitis (Conte 

and Passantino, 2008). The organism can be found in 

broad range of foods including powdered infant formula, 

cheese, meat, vegetables, grain, herbs, spices, tomato, 

and in water, in a variety of areas, including hospitals and 

houses (Fiore et al., 2008; Kim et al., 2008; Beuchat et 

al., 2009). 

Although most documented cases involve infant 

reports, infections in adult have been also described 

(Bhat et al., 2009). Adults with E. sakazakii infection 

usually have serious underlying disease or malignancies. 

The wound infection with E. sakazakii wound infection in 

an adult patient has been reported. The organism was 

resistant to multiple antibiotics and required prolonged 

treatment with broad spectrum antibiotics (Hamilton et al., 

2003). It is thus necessary to introduce various control 

measures to reduce the risk of contamination at various 

levels (Fiore et al., 2008). 

Antimicrobial agents from natural sources (muscadine 

seed extracts, rich sources of phenolic compounds and 

organic acids) demonstrated a strong antimicrobial effect 

against E. sakazakii (Kim et al., 2009). Control can be 

done using accurate antimicrobial agents. The later have 

been recovered from natural sources like, muscadine 

seed extracts, rich sources of phenolic compounds and 

organic acids to demonstrate a strong antimicrobial effect 

against E. sakazakii (Kim et al., 2009). 

Cited publications have demonstrated that E. sakazakii 

can be isolated in hospitals from clinical specimens taken 

from patients. Revising literature, we found that this 

organism has never been isolated in the Sudan. This 

study was designed to determine possible occurrence 

and antimicrobial resistance of E. sakazakii in clinical 

specimens. 


Study design 

This work is a descriptive and cross-sectional study that included 

389 patients, complaining from symptoms of UTI, enterocolitis, and 

wound infection in Khartoum state. Urine, stool, and wound swab 

*Corresponding author. E-mail: biotechsust@hotmail.com. Tel: 

+249 83773074. Fax: +249 83 771512. 

Humodi and Rania 2375 

collection and patient’s data were done in Kartoum Teaching 

Hospital (KTH), Gaffer Iben Auff Specialized Hospital for Children 

(GIASH) and Omdurman Teaching Hospital. Laboratory investigation 

was done in Research Laboratory, College of Medical Laboratories 

Science, Sudan University of Science and Technology. Data were 

collected in accordance with structured interview questionnaire. 

Collection of specimens 

Urine 

Mid-stream urine (MSU) was collected. Patients were asked to 

clean the pre-urethra area with soap and water. Adult females were 

asked to make swabbing of the urethra with sterile swab avoiding 

any antiseptic then were asked to pass the first drops of urine and 

collect 10-20 ml of mid stream urine in sterile, wide mouth 

containers. 

Wound swabs 

Sterile cotton wool swab was used to collect specimens of 

discharge from the infected wound. Where there was no discharge, 

swab was moistened with sterile normal saline, prior to collection of 

specimen and subsequently inserted in sterile tube. In case of 

deeply ulcerated wound, a syringe was employed to aspirate the 

sample from the side wall of the ulcer. Specimens were delivered to 

the laboratory as soon as possible. 

Stool 

Freshly passed stool specimens were collected in sterile wide 

mouth containers and cultures were performed immediately after 

collection. In case of delay, specimens were stored at 4°C. 

Cultivation of specimens 

Under aseptic conditions, urine and wound specimens were 

streaked on MacConkey's agar and blood agar (Oxoid Ltd., UK). 

Stool specimens were cultured in selenite F broth (Oxoid Ltd., UK), 

incubated overnight at 37°C then subcultured on xylose lysine 

deoxycholate agar (XLD), (Oxoid Ltd., UK) and incubated 

aerobically overnight at 37°C. 

Examination of growth 

Abundant growth was examined for lactose fermentation on 

MacConkey's agar, XLD agar and haemolysis on blood agar. The 

various morphological characters of the targeted organism were 

also observed and recorded. The isolates were then streaked on 

nutrient agar and incubated overnight at 37°C. The growth was 

checked for purity and stored in Bijou Bottles for further 

investigations.


Criteria of isolation of E. sakazakii 

Gram-negative rods were considered significant (Guptta et al., 

2003) and were investigated. 

Identification of E. sakazakii 

Bacterial colonies were examined for round, irregular, crenated or 

branching appearance, transparency or opacity, smoothness or 

roughness, dullness or shiny appearance. The color of the colonies 

was also examined (Murray et al., 2007). 

Gram's stain 

The method was carried out according to Murray et al. (2007). 

Oxidase test 

The test was carried out according to Barrow and Filtham (2003). 

Briefly, oxidase reagent discs were placed on sterile Petri dish and 

rubbed with colony of the test organism removed by using a 

wooden stick. Formation of a red-purple color within 20 s indicated 

positive oxidase test. 

Analytical profile index (API 20E) 

The API 20E (20 biochemical tests) was performed according to 

manufacturer instructions. These were ONPG, ADH, LDC, ODC, 

CIT, H2S, URE, TDA, IND, VP, GEL, GLU, MAN, INO, SOR, RHA, 

SAC, MEL, AMY and ARA. Identification was obtained with the 

numerical profile and performed using the database with the 

analytical profile index by looking up to the numerical profile in the 

list of profile. 

Antimicrobial sensitivity test 

Modified Kirby-Bauer disc diffusion method was performed (WHO, 

1996; NCCLS. 1997; Murray et al., 2007). Briefly, plates of Mueller- 

Hinton agar (Oxoid Ltd, UK) were prepared according to the 

manufacturer instructions; the sterilized molten medium was cooled 

to 45 to 50°C and poured in sterile, dry Petri plates on a leveled 

surface, to a depth of 4 mm. The inoculum was prepared and 

turbidity was adjusted to 0.5 McFarland standard. A sterile non-toxic 

cotton swab on a wooden applicator was dipped into the 

standardized inoculum and the soaked swab was rotated firmly 

against the upper inside wall of the tube to express excess fluid. 

The entire agar surface of the plate was streaked with the swab 

three times with turning the plate at an angle of 60 degrees 

between each streaking; the inoculum was allowed to dry for 5-15 

min with lid in place. Using sterile forceps the antimicrobial discs 

were placed and evenly distributed on the inoculated plate. The 

plate was then inverted and incubated aerobically at 35-37°C for 

18-24 h. After overnight incubation, the control and test plates were 

examined to ensure the growth is confluent or near confluent. Using 

a ruler on the under side of the plate, the diameter of each inhibition 

zone was measured in (mm). 

Interpretation of the zone size 

Using the interpretive chart, the inhibition zone around each 

antibiotic disc was measured and reported as sensitive, resistant or 

intermediate according to WHO (1996), NCCLS (1997) and Murray 

et al. (2007). 

Minimum inhibitory concentration (MIC) test 

The MIC test was performed to each isolate by the E-test technique 

as recommended by the manufacturer (HiMedia Laboratories Pvt. 

Limited). Briefly, each isolate was tested against the fifteen 

antimicrobial agents. A 200 mm Petri plate containing 90 ml of 

Mueller-Hinton agar (M173) was used. The inoculum was prepared 

as suspension in 0.85% NaCl. The suspension was adjusted to a 

0.5 McFarland standard. Plates were seeded with a cotton swab as 

described by manufacturer. The E-test strips were applied after the 

excess moisture had been absorbed into the agar. The plates were 

then incubated at 35-37�C and examined after 18-24 h. 

RESULTS 

During the course of this study (November 2008 to March 

2009) a total of 389 patients were recruited. 311 of these, 

presented with UTI, 11 with wound infection and 67 with 

enterocolitis. Out of the total number of urine samples 

collected, 163 (52.4%) showed significant bacterial 

growth (127 lactose fermeters (LF) and 36 non-lactose 

fermeters (NLF)). 10 (90.9%) of wound swabs and 61 

(91%) of stool specimens revealed significant growth, 4 

LF; 6 NLF and 61 LF 6 NLF respectively. 

Identification of E. sakazakii 

Colonial morphology 

Colonial morphology was observed on blood and 

MacConkey’s agars after overnight incubation for urine 

and wound specimens, and on XLD for stool. Suggestive 

E. sakazakii isolates produced large (3-4 mm), dull-gray, 

and dry or mucoid colonies on blood agar, large and pink 

or colorless, mucoid colonies on MacConkey ’ s agar and

Table 1. Criteria for identification E. sakazakii on API 20 E. 

Biochemical tests Reaction Result 

ONPG Yellow-pale yellow Positive 

ADH Red / orange Positive 

LDC Yellow color Negative 

ODC Red / orange Positive 

CIT Blue-green blue Positive 

H2S Colorless Negative 

URE Yellow color Negative 

TDA Yellow color Negative 

IND Colorless Negative 

VP Pink/ red Positive 

GEL Diffusion of black pigment Positive 

GLU Yellow color Positive 

MAN Yellow color Positive 

INO Blue/ blue-green Negative 

SOR Blue/ blue-green Negative 

RHA Yellow color Positive 

SAC Yellow color Positive 

MEL Yellow color Positive 

AMY Blue/ blue-green Negative 

ARA Yellow color Positive 

Table 2. Antimicrobial resistance of E. 

sakazakii isolates (n=6). 

Antibiotic Resistance (%) 

Chloramphenicol 66.7 

Ciprofloxacin 16.7 

Tetracycline 66.7 

Gentamicin 00.0 

Ceftazidime 100.0 

Amikacin 16.7 

Ceftriaxone 66.7 

Ticarcyline 83.3 

Nitrofuratoin 66.7 

Co-trimoxazole 83.3 

Amoxicillin 100.0 

Amoxyclav 100.0 

Nalidixic acid 16.7 

Cefotaxime 66.7 

Tobramycin 66.7 

pink color on XLD. 

API 20 E 


The results of API 20E revealed that ONPG, ADH, ODC 

and CIT were positive, H2S, LDC, URE, TDA and IND 

were negative. VP and GEL were also positive and all 

sugars GLU, MAN, RHA, SAC MEL, AMY and ARA were 

positive except INO, SOR, AMY which were negative 

(Table 1). 

The total number of E. sakazakii isolates were 6 (1.5%) 

including 4 (1.03%) from urine, 1 (0.3%) from wound and 

1(0.3%) from stool. The results revealed that the 

resistance rate of E. sakazakii was (100%) to amoxicillin 

and amoxyclav, ceftazidime, (0%) to gentamicin, (83.3 %) 

to co-trimoxazole and ticarcyline, (16.7%) to 

ciprofloxacin, amikacin, and nalidixic acid, (66.7%) to 

chloramphenicol, tetracycline, nitrofuratoin, cefotaxime, 

ceftriaxone, and tobramycin (Table 2). The Minimum 

Inhibitory Concentration (MIC), MIC50 and MIC90 values 

respectively were (3->240, 60 and >240 µg/ml) for 

nitrofurantoin, (0.5->240, 4 and 4 µg/ml ) for nalidixic 

acid, (0.1->240, >240 and >240 µg/ml) for cotrimoxazole, 

(0.01->240, 4 and 4 µg/ml) for 

chloramphenicol, (0.1-64, 0.1 and 0.5 µg/ml) for 

amikacin, (0.1-30, 5 and 30 µg/ml) for gentamicin, (1-16, 

4 and 8 µg/ml) for tobramicin, (0.001-0.5, 0.25 and 0.5 

µg/ml) for ciprofloxacin, (0.1-60, 30 and 30 µg/ml) to 

ceftriaxone, (0.1->240, 15 and 15 µg/ml) for ceftazidime, 

(0.01-3, 2 and 3 µg/ml) for tetracycline, (10->240, >240 

and >240 µg/ml) for ticarcycline, (0.1->240, >240 and 

>240 µg/ml) for amoxicillin, (0.1->240, 60 and 60 µg/ml ) 

for cefotaxime, (2->240, >240 and >240 µg/ml) for 

amoxyclav (Table 3). 

DISCUSSION 

This study was conducted to determine the frequency 

and antimicrobial resistance of E. Sakazakii. Three 

hundred and eleven urine specimens, eleven wound 

specimens, and sixty seven stool specimens were 

investigated. Of these, 163 (52.4 %) from urine 

specimens, 10 (90.9%) from wound specimens, and 

61(91%) from stool specimens showed significant various 

bacterial growth. 

E. sakazakii was isolated from 6(1.5%) out of 389


Table 3. MIC range, MIC50 and MIC90 of antimicrobial agents to E. sakazakii. 

Antibiotics MIC µg/ml No tested 

MIC range 

MIC µg/ml 

MIC50 MIC90 

Nitrofuratoin 6 3 to >240 60 >240 

Nalidixic Acid 6 0.5 to> 240 4 4 

Co-trimoxazole 6 0.1 to > 240 >240 >240 

Chloramphenicol 6 0.01 to > 240 4 4 

Amikacin 6 0.1 to 64 0.1 0.5 

Gentamicin 6 0.1 to 30 5 30 

Tobramicin 6 1 to >16 4 8 

Ciprofloxacin 6 0.001 to >0.5 0.25 0. 5 

Ceftriaxone 6 0.1 to > 60 30 30 

Ceftazidime 6 0.1 to > 240 15 15 

Tetracycline 6 0.01 to 3 2 3 

Ticarcyline 6 10 to > 240 >240 >240 

Amoxycillin 6 0.1 to > 240 >240 >240 

Cefotazime 6 0.1 to > 240 60 60 

Amoxyclav 6 2 to > 240 >240 >240 

MIC; Minimum inhibitory concentration. 

patients (urine, wound and stool). The isolates showed 

large, dull-gray, and dry or mucoid colonies on blood agar 

and large, pink, mucoid colonies on MacConkey's agar. 

These results were comparable to those described by 

Erickson and Kornacki (2002) and Engelkirk and Duben- 

Engelkirk (2007). The frequency 6 (1.5%) of E. sakazakii 

in three types of infections (UTI, Enterocolitis, Wound 

infection) is similar to the results obtained by Paterso et 

al. (2005), who reported that E. sakazakii was responsible 

for (0.4%) of the three types of infection in five 

international regions including Asia, Europe, Latin 

America, North America, and Middle East. 

In vitro activities of antimicrobial agents indicated that 

the resistance rate of E. sakazakii to amoxicillin and 

ceftazidime were (100%). This result is consistent with 

Farajnia et al. (2009) who found that the resistance rate 

of E. sakazakii to amoxicillin was (100%) in Iran but 

different from those of Sader et al. (2005) who found the 

resistance rate to ceftazidime was (17.5%) in United 

States. The resistance rate of E. sakazakii to nitrofuratoin 

(66.7%), however is widely different from those of 

Farajnia et al. (2009) who reported a 37.5% resistance 

rate to nitrofuratoin in Iran. 

The efficacy of cefotaxime, tobramycin, gentamicin and 

ciprofloxacin were evaluated and the result showed the 

resistance rates of E. Sakazakii to these antibiotics was 

(66.7%), (66.7%), (0%) and (16.7%) respectively. This 

differs from results obtained by Pfaller and Jones (2002), 

who reported that the resistance rate to cefotaxime, 

tobramycin and gentamicin is (23%), (16%) and (11%) 

respectively but similar to ciprofloxacin (16%) in 

European countries. However, the results indicated that 

resistance to nalidixic acid (16.7%) was different 

compared to that of Farajnia et al. (2009) who reported a 

resistance rate of 0.0 % to this antibiotic. Similarly the 

resistance rate to amikacin in this investigation is quite 

different from (0.0%) resistance rate reported by Farajnia 

et al. (2009). 

On the other hand, this study showed that the MIC50 

and MIC90 of amikacin, ciprofloxacin, tobramycin, and 

gentamicin were in agreement with Pfaller and Jones 

(2002). 

It is concluded that resistance rate of E. sakazakii is 

high to commonly used antimicrobial agents. It is also 

concluded that gentamicin is the first choice for treatment 

of E. sakazakii infections. Ciprofloxacin, amikacin, and 

nalidixic acid are the second choice of antimicrobial 

therapy with resistance rate of (16.7%). Nitrofurantoin,

tetracycline, ceftriaxone, cephotaxime, chloramphenicol, 

and tobramycin on the other hand are less effective 

antimicrobial agents due to their high resistance rate 

(66.7 %). Amoxicillin, amoxyclav, and ceftazidime are not 

effective, having a resistance rate of 100 %. However; 

further future studies including larger sample sizes are 

recommended, and to our knowledge this report 

represents the first record of E. sakazakii in the Sudan. 


The authors are grateful to Prof. A. Rahim M. El Husien 

for manuscript review. 

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DOI: 10.5897/AJMR09.462 



In search of enteroviruses in water media in Marrakech 

Amina Hssaine 1 , Jawhar Gharbi 2 , Rafik Harrath 2 , Rajae Harrak 3 , Abderrahman Chait 4 , 

Mahjoub Aouni 2 and Jamal Hafid 1 * 

1 Equipe Immuno-Parasitologie et Physiologie, Laboratoire Aliments, Environnement et Santé, 

Faculté des Sciences et Techniques, Gueliz, Marrakech, Morocco. 

2 Laboratoire des maladies transmissibles et substances biologiquement actives (MDT-01), Faculté de Pharmacie, 

5000 Monastir, Tunisie. 

3 Laboratoire régional du diagnostic épidémiologique et de l’hygiène du Milieu, Marrakech Morocco. 

4 Laboratoire d’Ecophysiologie, Faculté des Sciences Semlalia, Marrakech, Morocco. 

Accepted 7 April, 2010 

Enteroviruses are among the most common viruses infecting the human intestine; they are very 

widespread in nature and resistant to external agents. They are eliminated in the faeces and 

contaminate water and food. These viruses cause various clinical syndromes and constitute a big 

public health problem. The aim of our study was to search for enteroviruses in the water samples. For 

this, a total of 225 L of tap water coming from the national network office for drinking water (ONEP) and 

18 samples of waste water originating from Marrakech city were studied. These samples were 

concentrated, treated with polyethylene glycol 6000 and then analyzed by RT-PCR. Only two samples 

were found to be positive for enteroviruses by RT-PCR among the 18 waste water samples analyzed, 

which gave a rate of 11.11%. On the other hand, no positive samples were found in the tap water. This 

study made it possible on the one hand to apply for the first time RT-PCR for the detection of 

enteroviruses in water samples originating from Marrakech city, and on the other hand to show that tap 

water of this city does not present any risk of contamination by this type of germ. 

Key words: Drinking water, waste water, enterovirus, RT-PCR, Marrakech. 

INTRODUCTION 

Water is essential to life, its availability in quantity and 

quality sufficient to meet basic needs is essential both for 

improving health and for sustainable development. 

Combating and reducing diseases linked to water is one 

of the important and urgent tasks which can be achieved 

only through enhanced cooperation at all levels. 

Enteric viruses can be present naturally in aquatic 

environments. These viruses can be transported into the 

environment through groundwater, estuaries, sea water, 

rivers, aerosols, sewage, inadequately treated water, 

drinking water, wells etc. (Fong and Lipp, 2005). The risk 

of infection by enteroviruses in water is 10 to 10,000 

times greater than bacterial infections (Bosch, 1998). 

Enteroviruses were isolated from environmental 

samples for more than 60 years (Paul and Trask, 1941) 

and have subsequently been widely studied and qualified 

*Corresponding author. E-mail: hfjamal@yahoo.fr. 

as a real threat to the quality of drinking water. The 

contamination rate allowed was set by the World Health 

Organization to be 3.2 x 10 -5 particles per litre (WHO, 

2006). 

Viruses excreted in faeces are found in sewage, the 

viral concentration of waste water is variable depending 

on many factors, geographic, socio-economic, seasonal 

and especially health. Thus, the higher the proportion of 

children in a community, the lower the level of hygiene 

and the higher the quantity of virus found in waste water. 

Enteroviruses are transmitted by the faecal-oral route, 

their effects may exceed gastroenteritis, because they 

pass from the intestinal tract to other organs. The enteric 

virus infections are primarily associated with diarrhoea in 

humans (Kocwa-Haluch, 2001). Thus, in the United 

States, cases of acute gastroenteritis recorded exceeded 

50% of all waterborne diseases from 1946 until 1980 

(Lippy and Waltrip, 1984) and, viruses were identified as 

the cause of 12% of the cases registered during this 

period.

In Morocco, few studies have been devoted to sources of 

contamination of people by enteroviruses, which is why in 

the present work we focused on assessing the virological 

quality of water intended for public consumption as well 

as surface waste in the region of Marrakech. The aim of 

this study was to search for enteroviruses, by RT-PCR, in 

225 L of tap water coming from the national network 

office for drinking water (ONEP) and in 18 samples of 

waste water originating from Marrakech city. 


Samples 

Water faucet 

Samples were taken directly from the tap (Régie autonome de 

distribution d'eau et d'électricité de Marrakech: RADEEMA). 15 

samples were taken during the months of July, September and 

August 2007 and October 2008 making a total volume of 225 L. 

Wastewater 

Samples were taken from the waste water treatment plant of 

Marrakech. Eighteen samples of 250 ml were taken, in June, July 

and October 2008, in clean glass containers. Eleven were taken 

from the raw water at the entrance to the station and seven from 

the treated waste water as it exited. These samples were taken to 

the Environment, Food and Health Laboratory of Marrakech for 

extraction and concentration of the virus. The last pellets after 

concentration were kept at -20°C for analysis by RT-PCR in the 

Diseases and Biologically Active Substances Laboratory of 

Monastir, Tunisia. 

Water wells 

During the months of June and July 2007, eight samples of one litre 

each were collected in eight wells located within a 2 km radius 

around the Oued Tensift located 6.3 km north of the city of 

Marrakech at 410 m altitude. The wells were located in clusters of 

habitats near Oued Tensift. This water is used by the people both 

for drinking and for the preparation of food without any treatment. 

These villages are not served by the national drinking water 

system. 

Sample processing 

Concentration of enteroviruses 

The concentration of virus in samples of tap water and wells has 

been done by filtration through membranes of sterile cellulose 

nitrate with porosity of 0.22 microns. The filtration device was 

connected to a vacuum pump. Each filter is then placed in a moist 

chamber and stored at 4°C (Wallis et al., 1972). 

Elution 

Each filter was cut into very fine pieces separately and put in 20 ml 

of a solution of meat extract pH 9.5 and centrifuged at 10, 000 

rounds/min for 35 min. The float was recovered and its pH adjusted 

to 7.2 with 5M HCl. One milliliter of the mixture of antibiotics and 

Hssaine et al. 2381 

antifungal (penicillin, streptomycin and fungizone) was added and 

the final mixture was placed at 4°C overnight and stored at -20°C. 

Extraction and concentration from waste water 

We adapted the protocol described by Gerba et al. (1979), based 

on the adsorption of virus to acidic pH and elution at basic pH. 

Thus, 100 ml of sample were homogenized by magnetic stirring. A 

solution of AlCl3 was added until a final concentration of 0.0005 M 

and the pH was adjusted to 3.5 with 5N HCl. The mixture was 

stirred vigorously for 30 min then centrifuged at 2500 g for 15 min at 

4°C and the pellet was taken in 100 ml of 10% beef extract of at pH 

9. The mixture was stirred for 30 min and centrifuged at 10 000 g 

for 30 min at 4°C and the supernatant was collected in a sterile 

container. PEG 6000 was added until a final concentration of 10% 

and the mixture was homogenized and incubated overnight at 4°C. 

The next day, after a centrifugation step at 10 000 g for 45 min at 

4°C, the pellet was resuspended in 10 ml phosphate buffer pH 7.2 

and kept at -20°C. 

Extraction of viral RNA 

A volume of 120 µl of each sample was put in 500 l of Tri-reagent 

(Sigma) and vortexed for 15 s and left in contact 5 min at room 

temperature. Then, 200 l of chloroform were added to the mixture 

to separate the nucleic acids of proteins to which they are 

associated. The mixture was vortexed, left to react at room 

temperature for 15 min and then centrifuged at 13 500 rpm for 15 

min at 4°C. The aqueous phase was transferred to a new tube, and 

mixed with 500 l of isopropanol. The mixture was allowed to react 

for 15 min at -20°C and then centrifuged at 13500 rounds/min for 10 

min at 4°C to remove the cell debris. The pellet underwent two 

successive washes with 75% ethanol followed by centrifugation at 

7500 rounds/min at 4°C for 15 min for the first wash and 5 min for 

the second. The pellet was left to dry at room temperature for 15 

min. To standardize the RNA in the solution, the pellet was put in 

30 l of Diethylpyrocarbonate treated water. 

Detection of the viral genome 

We used RT-PCR, a method considered sensitive in the detection 

of enteroviruses in both environmental and clinical samples (Zoll et 

al., 1992; Rutjeset al., 2005; Soule et al., 2000) using reverse 

transcriptase (Moloney-Murine Leukemia Virus) and two primers 

from the conserved non-coding region of the viral genome. The 

product of amplification gives a 154 bp band. 

Choice of primers: In the case of enteroviruses, a sequence which 

is stable and common to all serotypes must be identified. Thus, 

several sequences have been described by Zoll et al. (1992). The 

primers used in this work are positioned at 445 - 464 and 599 - 580 

on the genome of Coxackievirus B3 and their nucleotide sequences 

are 5 'TCCTCCGGCCCCTGAATGCG 3' and 3 

'ATTGTCACCATAAGCAGCCA 5' (Invitrogen), respectively. 

Preparation of complementary DNA (cDNA): For the preparation 

of cDNA, we mixed 2.5 l of of reverse transcriptase buffer 

(Promega), 1 l of dithiothreitol (Promega), 0.5 l of NTPS 

(Boehring Mannheim), 0.5 l of antisense primer (25 pmole) and 

0.5 l of the enzyme reverse transcriptase (Promega). To this 

mixture, we added 5 l of extracted RNA and incubated for 30 min 

at 42°C and then amplified.


Table 1. Presence of enteroviruses in waste water 

samples of Marrakech. 

Samples Date Results 

RW1 

- 

02/06/2008 

TW1 - 

RW 2 

- 

18/06/2008 

TW2 - 

RW 3 

- 

20/06/2008 

TW3 - 

RW 4 

- 

25/06/2008 

TW4 - 

RW 5 

- 

27/06/2008 

TW5 - 

RW 6 

01/07/2008 

RW 7 

- 

02/07/2008 

TW7 - 

RW 8 

07/07/2008 

RW 9 

+ 

08/07/2008 

TW9 - 

RW 10 

RW 11 

25/10/2008 

26/10/2008 

RW: Raw water; TW: Treated water. 

Amplification: The amplification mixture consisted of 2.5 l Taq 

polymerase buffer, 0.5 l of NTPs: dATP, dCTP, dGTP and dTTP, 

0.5 l antisense primer (25pmole), 0.5 l of sense primer 25 pmole: 

oligo (dt) 12 - 18 (pharmacia Biotech), 0.15 l of Taq polymerase 

1.25 l (Invitrogen), 0.75 l of MgCl2 and 18 l of sterile doubledistilled 

water. 

The positive control (RNA Coxackievirus Nancy B) and negative 

control (sterile distilled water) were treated in the same conditions 

as the samples studied. The amplification was performed in an 

Eppendorf thermal cycler with a denaturing RNA-cDNA for 5 min at 

94°C and a series of thirty cycles of the following: 30 s at 94°C, 

hybridization of primers for a minute at 42°C, elongation 2 min at 

72°C and an extension of 10 min at 72°C, the final stretch. 

Revelation: After amplification, electrophoretic migration of the 

PCR product was performed on 2% agarose gel. Ten milliliters of 

each sample of PCR product mixed with 5 l of migration blue were 

deposited on the gel. Migration was done in TBE buffer (1x) at 90 V 

and lasted for approximately 45 min. The gel was then soaked in 

a solution of ethidium bromide (Sigma) at 10 mg/ml. Visualization of 

the specific band was conducted by transillumination under 

ultraviolet light (UV) against a size marker of 100 bp (Pharmacia 

Biotech). 

- 

+ 

- 

- 


During the study, a total of 41 samples were collected 

including 18 samples of waste water during the months of 

May, June, July and October 2008, 15 samples of tap 

water during the months of July, September and August 

2007 and October 2008 and 8 samples of water wells 

during the months of June and July 2007. 

Using RT-PCR enabled the detection of enterovirus 

RNA in 2 samples of waste water; this gives a rate of 

11.1% (2/18). These two positive samples were collected 

in July, known as the driest month of the year 2008 in the 

city of Marrakech. However, no positive result was 

obtained in the treated waste water, tap water or water 

wells (Table 1 and Figure 1). 

We have little data on the detection of enteroviruses in 

the environment in Morocco because there are few 

studies that have been done. This allows us to emphasize 

that our present work is original. Our results are 

compared to those recorded by researchers in other 

countries. 

Several studies have shown that the increase in 

enteroviruses is seasonal Summer-Fall (Troudi et al., 

1997), while other studies claim that the rainy season is 

the perfect time when the frequency of enterovirus 

reaches its peak (Bini et al., 2006). A more prolonged 

follow-up of our study throughout the year can confirm 

either study. We also noted that no positive sample of 

treated waste water coming out of the treatment plant has 

been detected. This can be explained by the fact that 

much of the suspended matter is removed during the 

treatment; where the viruses are adsorbed on this 

material under the influence of physical surface 

properties. Some authors argue that the setting of virus 

particles suspended in the water environment is linked 

not only to the species and serotype but also to the strain 

itself (Nestor and Brisou, 1986). For more sensitivity, it 

will be interesting to use nested-PCR for low levels of 

enteroviruses (Lee and Lee, 2008; Zhang et al., 2008). 

Indeed, RT-PCR is a sensitive technique but is influenced 

by the presence of inhibitors which are rich environmental 

samples (Sdiri et al., 2006), a factor that limits its 

effectiveness. However, many precautions must be taken 

both in the implementation of the RT-PCR (including 

choosing the method of virus concentration and 

extraction technique of RNA). Thus the large amount of 

genetic material from environmental samples makes 

possible a non-specific amplification of sequences unrelated 

to the virus searched. 

Regarding the tap water, 225 L of water were analyzed 

without any positive result. This could be explained bythe 

efficiency of the water decontaminating treatment 

process by the treatment plant. Our results are consistent 

with those found in Korea (Kyung and Jeong, 2004) where 

no enterovirus was detected in treated water for 

consumption. However they are different from the study 

of Coin (1966) which was the first to have isolated viruses 

in water intended for consumption in Paris, and those of

(-) PM 1 2 3 4 (+) PM 

Hssaine et al. 2383 

154bp 

Figure 1. Results of electrophoresis of amplification products by RT-PCR of gene 5'NC 

enteroviruses from samples of waste water. 

(-): negative control; (+): positive control (Coxackievirus B Nancy); 1, 4: positive samples; 2, 

3: negative samples; PM: Size Marker 100 bp. 

other studies that were conducted in Romagne (France) 

(Nestor and Costin, 1976) and Russia (Rabshko, 1974). 

The authors had explained the presence of the virus by 

inadequate treatment which did not allow effective 

disinfection. In addition, other studies have shown high 

contamination of drinking water by enteroviruses in Korea 

(Lee and Kim, 2002), South Africa (Ehlers et al., 2005) 

and Egypt (Ali et al., 2004) with positivity rates of 47.8, 

18.7% and 17.5 PFU/L respectively. These high rates 

have been explained by the insufficiency of the treatment 

used and the possible contamination of water pipes. 

As the methods used in environmental virology are 

limited by the low concentration of virus in drinking water, 

the techniques of adsorption/elution using electropositive 

filters or electronegative methods are most recommended 

by APHA (1985) and Karim et al. (2009). Nevertheless, 

our study has demonstrated the effectiveness of 

the treatment technologies for water consumption, the 

technique used and the volume of samples analyzed to 

meet international quality standards (EPA, 1999b). 

Regarding groundwater, we have made samples in 8 

wells and none was found to be positive. These wells 

which are not deep (between 6 and 10 m) are located 

near agricultural fields irrigated with waste water. Study 

should be conducted using wider areas and larger 

volumes of water. In other studies, conducted between 

1971 and 1982, groundwater accounted for 51% of 

waterborne diseases (Craun, 1986). Hepatitis viruses and 

rotaviruses are considered the main causes of these 

diseases (Gerba et al., 1985). These infections are 

mainly due to infiltration of waste water through the pits 

as well as due to the spread of waste water and sewage 

sludge, domestic discharge, oxidation ponds etc 

(Borchardt et al., 2007). With their small size, viruses are 

able to travel hundreds of meters through soil to reach 

groundwater (Gerba and Bitton, 1984) this movement 

depends mainly on the type of virus, the type of soil and 

climate. 

The present work allowed us to detect for the first time, 

enteroviruses in waste water of the region of Marrakech. 

This encourages us to continue the search for these 

viruses in a larger number of water and human biological 

samples. 

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viruses, Giardia and Cryptosporidium in two different types of drinking 

water treatment facilities. Water Res., 38: 3931-3939. 

American Public Health Association (APHA) (1985). Standard Methods 

for the examination of water and wastewater, 16 th ed. Washington, 

D.C. 

Bini JC, Ekaza E, Gnagne T, Borget AMY, Veh KA, Akran AV, Coulibaly 

ND, Faye-Kette H, Sess ED, Dosso M (2006). Apport de la RT-PCR 

pour la détection des entérovirus dans les eaux usées à Abidjan 

(Côte d’ivoire). Cah. Santé Publique, 5: 55-65. 

Borchardt MA, Bradbury KR, Gotkowitz MB, Cherry JA, Parker BL 

(2007). Human enteric viruses in groundwater from a confined 

bedrock aquifer. Environ. Sci. Technol., 15: 6606-6612. 

Bosch A (1998). Human enteric viruses in the water environment: a 

minireview. Microbiol., 1: 191-196. 

Coin L (1966). Modern microbioligical and virological aspects of water 

pollution. In Jaag, O (eds) Advances in water Pollution Research, 

Pergamon Pres, London, pp. 1-10. 

Craun GF (1986). ed. Waterborne diseases in the United States. Boca 

Raton: CRC Press, p.13. 

Ehlers MM, Grabow WOK, Pavlov DN (2005). Detection of 

enteroviruses in untreated and treated drinking water supplies in 

South Africa. Water Res., 39: 2253-2258. 

EPA (1999b). Drinking water criteria document for enteroviruses and 

hepatitis a: an addendum, p. 173. 

Fong TT, Lipp EK (2005). Enteric Viruses of Humans and Animals in 

Aquatic Environments: Health Risks, Detection, and Potential Water 

Quality Assessment Tools. Microbiol. Mol. Biol. R, 69: 357-371. 

Gerba CP, Bitton G (1984). Microbial pollutants: their survival and


transport pattern to groundwater. In Bitton G, Gerba CP (eds) 

Groundwater pollution microbiology. Wiley, New York, pp. 39-54. 

Gerba CP, Goyal SM, Labelle RL, Cech I, Bodgan GF (1979). Failure of 

indicatorbacteria to reflect the occurrence of enteroviruses in marine 

water. A. J. Pub. Health, 69: 1116-1119. 

Gerba CP, Sing SN, Rose JB (1985). Waterborne viral gastroenteritis 

and hepatitis. CRC Crit. Rev. Environ. Contr., 15: 213-216. 

Karim M, Rhodes E, Brinkman N, Wymer L, Fout GA (2009). New 

Electropositive Filter for Concentrating Enterovirus and Norovirus 

from Large Volumes of Water. Appl. Environ. Microbiol., 75: 2393- 

2399. 

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Human. Pol. J. Environ. Stud., 10: 485-487. 

Kyung Lee H, Jeong YS (2004). Comparison of Total Culturable Virus 

Assay and Multiplex Integrated Cell Culture-PCR for Reliability of 

Waterborne Virus Detection. Appl. Environ. Microbiol., 70: 3632- 

3636. 

Lee G, Lee C (2008). Molecular detection and characterization of 

human enteroviruses in Korean surface water. J. Microbiol., 46: 319- 

324. 

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Lippy EC, Waltrip SC (1984). Waterborne disease outbreaks-1946- 

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sédiments marins et fluviaux. Rev. Epidemiol. Sante Publique, 34: 

181-190. 

Paul JR, Trask JD (1941). The virus of poliomyelitis in stools and 

sewage. J. Am. Med. Ass., 116: 493-498. 

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the environemental objects. Gig. Sanit. 39: 105-106. 

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Husman AM (2005). Isolation and Detection of Enterovirus RNA from 

Large-Volume Water Samples by Using the NucliSens miniMAG 

System and Real-Time Nucleic Acid Sequence-Based Amplification. 

Appl. Environ. Microbiol., 71: 3734-3740. 

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culture cellulaire et de la RT–PCR pour la détection des entérovirus 

dans les eaux usées et les coquillages en Tunisie. Pathol. Biol., 

54: 280-284. 

Soule H, Genoulaz O, Gratacap-Cavallier B, Chevallier P, Liu J, 

Seigneurin J (2000). Ultrafiltration and reverse transcriptionpolymerase 

chain reaction: an efficient process for poliovirus, 

rotavirus and hepatitis A virus detection in water. Water. Res., 34: 

1063-1067. 

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circulation des entérovirus dans la région du Sahel. Tunis Med., 75: 

23-26. 

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bacterial and viral contamination of urban waters: a case study in 

Xi'an, China. Water Sci. Technol., 58: 653-560. 

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Galama JM (1992). General primer-mediated polymerase chain 

reaction for detection of enteroviruses: application for diagnostic 

routine and persistant infections. J. Clin. Microbiol., 30: 160-165.



DOI: 10.5897/AJMR09.389 


Short Communication 

Laboratory analysis of a fatal meningococcal case due 

to serogroup B Neisseria meningitidis belonging to ST- 

4821 complex 

Zengguo Wang*, Tiejun Hou, Zhijun Chen, Jinsong Li, Shouzhi Wu, Xiaoguang Wei, 

Yahui Sun and Quanli Du 

Xi’an Center for Disease Control and Prevention, 599 Xi Ying Road, Xi’an, 710054, PR China. 

Accepted 12 November, 2009 

We describle a suspicious Meningococcal death case, confirmating by the laboratory PCR methods. 

The infection developed rapidly within only 24 h until the patient died. Analyzing the close contact strain 

and the DNA template extracted from the serum of this patient, we characterized a serogroup B 

Neisseria meningitidis as the pathogen of this case, which, in terms of sequence typing, belonged to 

ST-4821 complex which first characterized in serogroup C Neisseria meningitidis as a new unique 

hypervirulent meningococcal lineages. 

Key words: Neisseria meningitidis, multilocus sequence typing, serogroup. 

INTRODUCTION 

As a major bacterial meningititis pathogen, Neisseria 

meningitidis could cause a rapidly progressive and often 

fatal illness mainly in children and young adults all over 

the world. 13 serogroups were known of this organism 

based on the chemical and serological properties of the 

capsular polysaccharide. Serogroup A meningococci 

were the main cause of meningitis and responsibled for 

several nationwide epidemics during the last century in 

China (Hu, 1991). After the mass vaccination campaign 

with mainly the MenA vaccine initiated in 1982, the 

incidence of meningococcal disease rapidly declined and 

only some sporadic case reported. However, several 

outbreaks caused by serogroup C, ST -4821 meningococci 

occurred in Anhui province in 2003 and then spread 

to other provinces (Shao et al., 2006). To control this 

disease, a mass vaccination using polysaccharide 

vaccine for serogroup A plus C were undertaken in 

several provinces and the A plus C vaccine was used 

more prevalent than before in China. 

An etiologic diagnosis of N. meningitidis is confirmed by 

the isolation of strain from cerebrospinal fluid, blood or 

other body fluids. However, this diagnosis was hindered 

*Corresponding author. E-mail: upwilly@gmail.com. Tel: 86-10- 

8551-2367. 

by the failure to isolate bacteria because of the early 

treatment of the patient (Cartwright et al., 1992). So the 

PCR-based methods have been extensively used for the 

diagnosis and also the serotying based on several genes 

special for Neisseria meningitidis or the serotype. In this 

study, we confirmed a suspicious death case of meningococci 

infection with laboratory methods such as PCR 

(multiplex-PCR) (Jordens et al., 2002), MLST (Maiden et 

al., 1998). We also found the etiologic serogroup B 

meningococci belonging to the ST-4821 complex which 

was a newly identified lineage that most prevalent in 

serogroup C meningococci in China (Zhang et al., 2007). 

CASE REPORT 

A 16 years old female student was characterized by a 

high fever and headache without other symptoms like 

cough, diarrhea, vomit, which began on March 11, 2009. 

The body temperature was 39.4°C. The symptom was 

exacerbated when the patient was checked in LanTian 

Hosiptal next day at 7:30 am. Several petechiate rashes 

emerged on the whole body with the blood pressure was 

118/68 mmHg and pulse rate of 132 per minute. The 

numbers of white cell in the blood were 1.6 × 1010/L. The 

patient receiving azithromycin, and within drug administration, 

the patient had nausea several times. After the


drug administration, the body temperature was normal 

but also with the headache. Then the patient was 

transferred to Tang Du Hospital at 10:50 am, the body 

temperature was 36°C and the blood pressure was 84/65 

mm Hg with pulse rate of 116 per minute. Neck stiffness 

and purpura fulminans were noted during clinical examination. 

The blood and CSF samples were obtained and 

submitted for laboratory examination. Laboratory investigations 

revealed thrombocytopenia, the TP, ALB and 

GLO were all decreased, the blood culture was negative 

for any bacterial. The patient was initially sta-bilized with 

volume resuscitation, dopamine infusion, until 13:30, the 

patient was shortness of breath and dysphoria again, and 

died of acidemia at 14:35. In accordance with the 

observed clinical symptoms, this case was diagnosed as 

suspicious meningococcal meningitis. The serum of the 

patient was transferred to Xi’an CDC laboratory for 

detection via PCR and MLST. 

The close contact in this case was defined as 5 

roomates with the patient. Within the 5 roommates’ 

oropharyngeal swabs, the ctrA gene was positive in two 

samples and 1 strain was isolated. The ctrA gene was 

also positive in the patient’s serum. To check the serotype 

we use the molecular serogroup methods to predict the 

possible serotype described elsewhere (Taha, 2000), 

(Bennett et al., 2004). A 450 bp fragment predicted 

serogroup B Neisseria meningitidis was positive in the 

two ctrA gene positive oropharyngeal swabs sample and 

also the patient’s serum. MLST analyses were verified 

here with the close contact strain. The sequence type of 

this close contact strain was ST -7623 which was a new 

ST type found in our study belonging to ST-4821 complex. 

MLST was also conducted using the DNA template 

extracted from the serum of this patient. Only three 

fragments (adk, aroE and gdh) were obtained and 

sequenced. To search the type in the PubMLST net, the 

allelic profile was identical to 12 sequence types that all 

belonging to ST-4821 complexs. Also, the sequence of 

these three fragments identical to the close contact strain. 

According to the above-listed, we conclude that the 

pathogen of this fatal case was serogroup B N. 

meningitidis which belonging to ST-4821 complexs. 

DISCCUSSION 

Currently, rapid laboratory diagnosis of meningococcal 

disease has been widely used as routine methods 

(Bryant et al., 2004; Hoang et al., 2005). Especially, when 

the bacterial could not been isolated, PCR based 

methods will be very important in confirming the 

meningococci infection. With the only sample like serum 

of patient, MLST could be used in epidemiology research 

(Zhang et al., 2006). In this case, PCR and MLST method 

was used in laboratory confirmation and epidemiology 

research as only the serum sample was obtained. To our 

knowledge, this is the first death case causing by 

serogroup B meningococci belonging to ST- 4821 

complexs in China. 

Serogroup C meningococci have been associated with 

the outbreaks in 2003. Then a unique ST-4821 clone has 

been characterized (Shao et al., 2006). ST-4821 complex 

isolates were observed as early as 1980 in China and 

existed in several serogroups like C, B, H, I and K (Yang 

et al., 2007; 2008; Zhang et al., 2007). In addition to ST- 

4821 complex has been seen in serogrop B 

meningococcal isolated from healthy carriers on the 

Chinese mainland, it also have been seen from invasive 

cases in Taiwan (Chiou et al., 2006). Here we also found 

the serogroup B meningococcal belonging to ST- 4821 

complex in a fatal case. Because the pathogen strain of 

the patient was not able to isolate and we can only get 3 

gene fragments from the patient’s serum. Moreover we 

isolated two N. meningitidis strains from the oropharyngeal 

swabs of patient’s other 10 classmates. This two 

strains also belongs to B serogroup and the sequence 

type was ST-4821 and ST-5586 each. The 3 gene 

fragments from the patient’s serum also indentical to the 

ST-4821 strain. We also got ctrA gene positive result from 

2 of 5 close contacts, one close contact strain with the 

exact sequence type is unsatisfactory. So we presumed 

there was not sufficient evidence to confirm the exact 

sequence type of this etiological strain. After all it seems 

belonging to ST-4821 complex. 

Multivalence vaccines A plus C have used widely in 

China after the serogroup C meningococci outbreaks. 

Thus, the B serogroup has been observed in healthy 

carrier is relative higher than before and even maybe the 

capsule switch between B and C (Beddek et al., 2009). 

The case presented here indicate the need for better 

surveillance of the epidemiology of meningococcal meningitis 

in China, especially the new unique hypervirulent 

meningococcal lineage ST- 4821 complexs. 


We thank all the researchers in hospitals and Wang Qian 

in the Baqiao district CDC. We also thank Dr Julia 

Bennett at the University of Oxford for the registration of 

the close contact strain as new ST in the MLST database. 

REFERENCES 

Beddek AJ, Li MS, Kroll JS, Jordan TW, Martin DR (2009). Evidence for 

capsule switching between carried and disease-causing Neisseria 

meningitidis strains. Infect. Immun., 77: 2989-2994. 

Bennett DE, Mulhall RM, Cafferkey MT (2004). PCR-based assay for 

detection of Neisseria meningitidis capsular serogroups 29E, X, and 

Z. J. Clin. Microbiol., 42: 1764-1765. 

Bryant PA, Li HY, Zaia A, Griffith J, Hogg G, Curtis N, Carapetis JR 

(2004). Prospective study of a real-time PCR that is highly sensitive, 

specific, and clinically useful for diagnosis of meningococcal disease 

in children. J. Clin. Microbiol., 42: 2919-2925. 

Cartwright K, Reilly S, White D, Stuart J (1992). Early treatment with 

parenteral penicillin in meningococcal disease. Bmj, 305: 143-147. 

Chiou CS, Liao JC, Liao TL, Li CC, Chou CY, Chang HL, Yao SM, Lee 

YS (2006). Molecular epidemiology and emergence of worldwide

epidemic clones of Neisseria meningitidis in Taiwan. BMC Infect. Dis., 

6: 25. 

Hoang LM, Thomas E, Tyler S (2005). Rapid and fatal meningococcal 

disease due to a strain of Neisseria meningitidis containing the 

capsule null locus. Clin. Infect. Dis., 40: 38-42. 

Hu X (1991). Study on periodically prevalent feature for epidemic 

cerebrospinal meningitis in China. J. Chin. Epidemiol., 12: 136-139. 

Jordens JZ, Williams JN, Jones GR, Heckels JE (2002). Detection of 

meningococcal carriage by culture and PCR of throat swabs and 

mouth gargles. J. Clin. Microbiol., 40: 75-79. 

Maiden MC, Bygraves JA, Feil E (1998). Multilocus sequence typing: a 

portable approach to the identification of clones within populations of 

pathogenic microorganisms. Proc. Natl. Acad. Sci. USA., 95: 3140- 

3145. 

Shao Z, Li W, Ren J (2006). Identification of a new Neisseria 

meningitidis serogroup C clones from Anhui province, China. Lancet, 

367: 419-423. 


Taha MK (2000). Simultaneous approach for non-culture PCR-based 

identification and serogroup prediction of Neisseria meningitidis. J. 

Clin. Microbiol., 38: 855-857. 

Yang J, Zhang X, Xu X (2007). Genotypic analysis of serogroups other 

than A, B or C of Neisseria meningitidis in China. Scand. J. Infect. 

Dis., 39: 819-821. 

Yang L, Shao Z, Zhang X, Xu L, Peng J, Xu X, Liang X, Qi Y, Jin Q 

(2008). Genotypic characterization of Neisseria meningitidis 

serogroup B strains circulating in China. J. Infect., 56: 211-218. 

Zhang TG, He X, Chen LJ, He JG, Luo M, Yang J, Shao ZJ, Sun MP 

(2006). Laboratory confirmation of a suspicious meningococcal 

meningitis death case. J. Microbiol., 44: 457-460. 

Zhang X, Shao Z, Yang E (2007). Molecular characterization of serogroup 

C Neisseria meningitidis isolated in China. J. Med. Microbiol., 

56: 1224-1229.



DOI: 10.5897/AJMR11.536 


Short Communication 

Genotyping of Hepatitis C virus (HCV) in infected 

patients from Saudi Arabia 

Mohammed Ali M. Marie 

Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, 

P. O. Box 10219 Riyadh 11433, Kingdom of Saudi Arabia. E-mail: mmarie@ksu.edu.sa. Tel: +96614696022. 


A molecular study was carried to investigate the prevalence of Hepatitis C virus (HCV) genotypes in 

HCV infected population of Saudi Arabia. A total of HCV-positive clinical specimens (serum, EDTA 

plasma) with viral loads above 2,000 IU/ml were collected for genotyping. Genotyping of 358 samples 

revealed four different genotypes including 1 (1a and 1b), 2a, 3 (3a) and 4. The most prevalent genotype 

was 4 with rate of 74.2% followed by genotype 3 (11.7%) and 1a (3.9%). The predominance of HCV 

genotype 4 in our population confirms the predominance of HCV genotype 4 in Saudi Arabia and most 

of the Arab countries in the Middle East. 

Key words: Hepatitis C virus (HCV), genotypes, Saudi Arabia, Riyadh. 

INTRODUCTION 

Hepatitis C virus (HCV) infection is a global public health 

problem. World Health Organization (WHO) estimates up 

to 3% of the world's population to be infected with HCV 

and there are approximately 180 million individuals are 

thought to be infected. On average, 80% of acutely infected 

individuals develop a chronic infection (Lavanchy, 

2009; Ghany, 2009). HCV has a positive-sense singlestranded 

RNA genome of flavivirus and about 9.6 kb 

containing one long open reading frame (ORF) with 

untranslated regions at both ends (Choo et al., 1989). So 

far, six major genotypes (HCV-1 to HCV-6) have been 

described, each containing multiple subtypes (for 

example, 1a, 1b, etc.) (Tokita et al., 1995). The genotype 

of the HCV strain appears to be an important determinant 

of the severity and aggressiveness of liver infection, as 

well as patient response to antiviral therapy (Zein, 2000). 

HCV genotypes display significant differences in their 

global distribution and prevalence, making genotyping a 

useful method for determining the source of HCV transmission 

in an infected localized population (Hnatyszyn, 

2005). 

Abbreviations: HCV, Hepatitis c virus; WHO, world health 

organization; ORF, open reading frame; RNA, ribonucleic acid; 

PCR, polymerase chain reaction; EDTA, 

ethylenediaminetetraacetic acid; HCC, hepatocellular 

carcinoma. 

HCV genotype 1, 2 and 3 are distributed worldwide and 

their relative prevalence varies from one geographic area 

to another, whereas genotype 4 is predominantly prevalent 

in the Middle East and Africa, genotype 5 in South 

Africa and genotype 6 in Southeast Asia (Zein, 2000; 

Gish and Lua, 1997). According to the recent studies, 

genotype 4 is predominant in Egypt, 4 and 1 in Kuwait 

and Syria, and genotype 1 in Lebanon, Iraq and Iran 

(Osaba, 2002; Pacsa et al., 2001; Zali et al., 2000). Saudi 

Arabia shows an intermediate endemicity for HCV. 

Seroprevalence rates ranging from 0.9 to 5% have been 

reported among children and adults, respectively 

(Shobokshi et al., 1999). Some studies have indicated 

the predominance of genotype 4 in this region (Mellor et 

al., 1995; Al-Faleh et al., 1995). Hence HCV subtype 

distribution is needed to provide clues for studying 

epidemiology, the mode of transmission, and response to 

treatment. The objective of this study was to determine 

the distribution of HCV genotypes among the patients 

attending different hospitals and polyclinics in Riyadh, 

Saudi Arabia. 


This was a descriptive study conducted from January 2008 to 

December 2010 from different hospitals as well as polyclinics at 

Advanced Cell laboratory, Riyadh, Saudi Arabia. The latter is a 

reference laboratory serving a number of hospitals and polyclinics 

in Riyadh. Patients positive for anti-HCV antibodies were referred

Table 1. Genotype distribution of 358 HCV positive patients. 

Nationality Total 

HCV genotype 

Mohammed 2389 

1 1a 1b 2a 3 3a 4 

Saudi 208 - 6 7 5 8 - 182 

Egyption 98 13 - - - 8 - 79 

Pakistan 33 - - - - 26 7 - 

Others 17 - 8 5 - - - 4 

Total (%) 358 (100) 13 (3.6) 14 (3.9) 12 (3.3) 5 (1.4) 42 (11.7) 7 (1.9) 265 (74.2) 

Table 2. HCV Genotype among male and female. 

Gender 

1 1a 1b 

HCV genotype 

2a 3 3a 4 

Total (%) 

Male 13 3 2 1 36 7 178 240 (67) 

Female 0 11 10 4 6 0 87 118 (33) 

to the reference laboratory for detection of HCV RNA level and for 

genotyping. The study was designed to include patient's demographics 

(age, sex and nationality) as well. Detection of HCV 

ribonucleic acid (RNA) was carried out on a 500 ul sample of each 

serum sample positive for anti-HCV antibodies, using a commercial 

polymerase chain reaction (PCR) -based test (Taqman amplicor, 

Roche, USA) and following manufacturer’s instructions. Internal 

control supplied by the manufacturer was added to each specimen, 

as an extraction and amplification control. HCV positive clinical 

specimens (serum, ethylenediaminetetraacetic acid) (EDTA) plasma 

with viral loads >2,000 IU/ml were selected for genotyping using 

Versant HCV genotype assay (LiPA) 2.0 (Innogenetics, Siemens 

Healthcare Diagnostics, USA). This kit allows an improved and 

more accurate distinction between HCV genotype 1 and subtypes c 

to l of genotype 6 as well as between subtypes a and b of genotype 

1. Data was analyzed using SPSS-14 version. A p Value of < 0.5 

was considered significant for statistical analysis. 

RESULTS 

Genotype distribution in 358 HCV-positive patients is 

shown in Table 1. Overall, HCV genotype 4 was the most 

predominant genotype (74.2%) followed by genotype 3 

(11.7%) and 1a (3.9%). Differences in genotype distribution 

were statistically significant (p


Saudi Arabian patients can be attributed to many factors. 

These include the expatriates from different nationalities 

resided in Saudi Arabia for quite some time and participated 

in blood donation. 

According to the World Health Organization, 180 million 

individuals in the world are infected with HCV and this is 

a growing global problem. The development of an 

effective vaccine remains the ideal way to combat HCV 

infection. In addition to the implications for clinical outcome 

of infection, and for treatment, genotyping of HCV 

also has major implications for HCV vaccine development. 

Recent data suggest that for a vaccine to be fully 

protective it should contain a range of deferent envelope 

proteins corresponding to the common genotypes in 

particular geographic regions. Vaccines for use in the 

Middle East should, therefore, not be based only on 

genotype 4 sequences; other genotypes such as 1a and 

1b are also equally important. Finally, genotyping of HCV 

may be a useful epidemiological marker particularly in 

establishing suspected unconventional routes of HCV 

transmission such as vertical, intraspousal or interfamilial 

transmission (Alfaresi, 2011). 


The author would like to thank all the staff at Research 

center, college of applied medical sciences, King Saud 

University for their valuable support. 

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Hossain A, El-Hazmi M (1995). Profile of hepatitis C virus (HCV) and 

the possible modes of transmission of the virus in Gizan, area of 

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UPCOMING CONFERENCES 

7th Conference of the World Mycotoxin Forum and 

XIIIth IUPAC International Symposium on Mycotoxins and Phycotoxins, 

Rotterdam, Netherlands, 5 Nov 2012 

13th International Congress on Yeasts, Madison, USA, 

26-30 Aug 2012

Conferences and Advert 

August 2012 

13th International Congress on Yeasts, Madison, USA, 26 Aug 2012 

September 2012 

6th International Congress on Biocatalysis, Hamburg, Germany, 2 Sep 2012 

9th International Congress on Extremophiles, Sevilla, Spain, 10 Sep 2012 

October 2012 

Actinobacteria within Soils: Capacities for Mutualsim, Symbiosis and Pathogenesis, 

Muenster, Germany, 25 Oct 2012 

November 2012 

7th Conference of the World Mycotoxin Forum and XIIIth IUPAC International 

Symposium on Mycotoxins and Phycotoxins, Rotterdam, Netherlands, 5 Nov 2012 

December 2012 

Marine Microbiology and Biotechnology: Biodiscovery, Biodiversity and 

Bioremediation, Cork, Ireland, 19 Dec 2012 

International Conference on Pathology and Microbiology, Bangkok, Thailand, 26 Dec 

2012

African Journal of 

Microbiology Research 

Related Journals Published by Academic Journals 

■ Journal of Cell and Animal Biology 

■ African Journal of Biotechnology 

■ Biotechnology and Molecular Biology Reviews 

■ African Journal of Microbiology Research 

■ African Journal of Biochemistry Research 

■ Journal of Bacteriology Research

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