bacterial diversity in potable drinking water of mahabubnagar district

Bioscience Discovery Vol 02, No. t, lan.Z}tL ISSN 2229-3469
ABSTRACT
BACTERIAT DIVERSITY IN POTABTE DRINKING WATER OF
MAHABUBNAGAR DISTRICT (A.P.) INDIA
Paian Kumar pindi and p Raghuveeryadav
Department of Microbiology, Palamuru university, Mahabubnagar-50g001, lndia
Email: pavankumarpindi@gmail.com
Received:28 Oct 2010 Revised:05 Dec. 2010
Bacterial quality is probably the most important consideration in assessing
drinking water. The study is employed based on 16s rRNA gene approach to characterize the
bacterial community structure in ground water. A polyphasic approach involving cultivation,
direct viable counts, 16s rRNA based phylogenetic classification was applied for the
characterization of the bacterial population in a public drinking water. A total number of
19 out of 77 bacterial strains were identified identically from i places of Mahabubnagar
district, A.B lndia. Water for human consumption is required to be free from any pathoge-nic
bacteria. The 165 rRNA identity of the bacterial strains revealed that high numbeis of
Aeromonos, Psuedomonas, Enterobacter, Bacillus, Acinetobocterwere present. overall, the
results suggest that these bacterial groups are amongst potentially active bacteria in
drinking water. The highest diversity was found in ground water collected from Hanwada
and least in Bomraspet mandal.
Key words: Bacterial diversity, ground water, morphology, 165 rRNA gene
INTRODUCTION
Drinking water is an important resource
all round the world. A very little an attempt was
made on identifying of culturable bacteria in
potable drinking water. Drinking water can be
obtained from various sources, like surface water
or ground water. Due to the filtration of the water
from the soil, ground water contains a low number
of bacteria and nutrients. The monitoring and
identification of bacteria present in drinking water
have primarily been achieved using a plating and
isolation strategy. The majority of these culturable ,
bacteria described in drinking water networks are
included in the phylum proteobacteria and to a,
lesser extent in the phyla Actinobacteria,
Firmicutes, and Bacteriodetes (LeChevallier et a/.
1980 and 1987; Norton and Lechevallier 2000;
Olson and Nagy 1984; payment ef a/. 19g8). Some
of the commonly detected genera include
Pseudomonas, Caulobacter, Aeromonas,
Acinetobacter, and Bacillus. Over all, a common
perception is that pseudomonas is the most
abundant bacterial organism in water source.
Most studies concerning the quality of
drinking water deal with the analysis of the DOC
(Brauch et a l. t982; Bru chet et a l. L99t;Jah nel et
o/. 1998) or the determination of the viable counts
of different bacteria in the drinking water,
focusing on the culturable and pathogenic
bacteria.
The present study describes the
identification of the culturable bacterial
population in ground water of seven mandals of
Mahabubnagar district, A.p., lndia. Based on the
morphological characters and 165 rRNA gene
sequences the diversity of the bacteria in the
ground water was determined during the month
of September,2010.
MATERIAISANDMETHODS
Source of samples
Ground water samples were collected from seven
sites from different mandals of Mahabubnagar
district, A.P., lndia dur:ing the September, 2010.
[g!.er samples were collected from drinking water
supplying tanks in sterilized 1 Litre water bottles.
The pH of the samples was measured immediately
after sampling.
Thirteen drinking water samples were
collected froni different government hospitals,
Mahabubnagar district, Andhra pradesh, lndia on
22d and 23d November, 2009. Fifty five strains
were isolated from these 7 drinking water sources
on nutrient agar medium at 37"C. For isolation of
bacteria, 100 pl of the water sample was plated
on Nutrient agar medium and incubated at 37'C

Pavon Kumar Plndi and P Raghuveer yodav
for 2-days. Based on the different colony
morphology from each sample, a total of 77 strains
were selected and characterized in the present
study.
Genomic DNA isolation
The genomic DNA from the bacterialcells
was obtained using a modification of the method
described by Sambrock et o/. (1989). The bacterial
cells from pure cultures were harvested by
centrifugation (12,000 rpm)for 2 min, and the cell
pellets mixed with 600 il of lysis buffer (L0 mM
Tris-HCl, 1 mM EDTA [pH 7.5],0.S% SDS, and 100
ig/ml proteinase K) and incubated at 37" C for t h.
After the addition of 100 it 5 M NaCt, and 80 il
CTAB/NaCI, the samples were incubated at 65" C
for 10 min. The samples were cooled to room
temperature, followed by extraction of the aqueous
phase with an equal volume of chloroform:
isoamyl alcohol (24l. l, v/v), and then with an
equal volume of phenol: chloroform: isoamyl
alcohol (25: 24:- L, vfvl, which was centrifuged at
12,000 rpm and 4oC for 10 min. lsopropanol (0.6
x) was mixed with the aqueous phase, and
centrifuged at 12,000 rpm and 4 C for 10 min.
After removal of the aqueous phase, the DNA
pellets were washed with 70% ethanol, and
centrifuged at 12,000 rpm and 4 C for 10 min, The
DNA pellets were dried under vacuum, and then
dissolved in TE buffer (10 mM Tris-HCl, and L
mMEDTA [pH z.s] ).
2.2. 165 rRNA gene sequencing and phylogenetic
anatysis
For 165 rRNA gene sequencing, DNA was prepared
using the Mo Bio microbial DNA isolation kit (Mo
Bio Laboratories lnc., Solano Beach, CA, USA) and
sequenced as described previously (Lane 1991).
The resultant almost complete sequence of the
165 rRNA gene contained 1502 nucleotides. The
165 rRNA gene sequence of the isolate was
subjected to BLAST sequence similarity search
(Altschul et a/. 1990) and EzTaxon (Chun ef o/.
2007) to identify the nearest taxa. The entire
related 165 rRNA gene sequences were
downloaded from the database (http://
www.ncbi.nlm.nih..sov) aligned using the
CLUSTAL_X program (Thompson et at. L9971.
Table 1. Bacterial abundanoe in potable drinking water samples collected from Mahabubnagar.
Place ofthe Number of
collection lsolates
Nameof the lsdate
Vapur
05 Acinetobader junii, Aeromonos hydrophilo, psuedomianas
otitidis, Bacillus cereus, E,coli
Koilkonda
o4
Methylobocteri um calcooceticus, Aeronmonos p unctato, Bo ci ll us
infontis. E.coli
Kodangal
03
Hanwada
07
Bomraspet
Maddur
Kosgi
01
02
06
RESUTTSAND DISCUSSION
Overall, the results suggest that these
bacterial groups are mesophilic and could grow
in the temperature range of 20 to 40.C with
optimum growth temperature is 37.C. All the
strains could grow without NaCl in the medium
and the tolerance to NaCl varied from tto 2%,. All
strains could grow either in the pH range of 6-g,
A total of 77 bacterial strains werb recovered
from the 7 drinking water sources (Table 1).
C it robact er Freu nd i i, Ba ci ll us c e re us, Boc i lt us ni o be nsis,
Acinetobacter ursingii, Acinetobacter junii, Aeromonos
hydrophila, Psuedomonos olcaligenes, pseudomonas otitidis,
' Lysi n! bacil t us fu sifor mi s, E. m li,
Citrobacter sp
De lfti a I ocu stri s, B rev ibo cter iu m sp.
Bocillus sp, Pseudomonos aerugirtosa, Citrobocter Freundii ,
A fth robocte r sp. Acin etobocte r E.coli
33
Morphological and taxonomic analysis of all the
77 strains isolated from water samples indicated
that 19 strains were identical. The nearest
phylogenetic neighbor of all 77 isolated strains
were identified following BLAST analysis of the
165 rRNA gene sequence. BLAST analysi; indicated
that four strains belonged to th€ genus Bacillus ,
three strains belonged to the genus ps uedomonos,
each two strains belonged to the genus

Bioscience DiscoveryVol02, No. 1, Ian.2011
Acinetobocter , Aeromonos , each one strain to the
Brevibacterium sp. Citrobacter Freundii, "Delftia
locustris, Lysinibocillus fusiformis, E.coli,
M e thy I o b o cte ri u m co I co a ceficus. Tota I i.9 d ifferent
taxa were identified based on the BTAST analysis
oul of 77 strains.
Coli form is the name of a test adopted in
1914 by the Public Health Service for the
Enterobocterioceae family. lt is the commonly-used
bacterial indicator of sanitary quality of foods
and water. Coli form bacteria live in soil or
vegetation and in the gastrointestinal tract of
animals. Coli forms enter water supplies from the
direct disposal of waste into streams or lakes or
from runofffrom wooded areas, pastures, feedlots,
septic tanks, and sewage plants into streams or
groundwater. Coli forms are not a single type of
bacteria, but a grouping of bacteria that includes
many strains, such as E. coli. Water sample from
Hamada shown more contamination and less
found in Bomraspet mandal.
ln the present study species belong to the
genera Aero monos, M ethylobode ri u m, Bqcill us and
Pseudomonas were isolated which is supported
by the different studies where several novel species
which belong to the genera Aeromonos,
Methylobocterium (Lee et a/. 2009), Bocillus(Zhang
et o1.20061 and Pseudomonos were reported from
drinking water systems. Aeromonas spp. and
Aeromonos hydrophila found in diverse aquatic
environments like wellwater and heavily polluted
ISSN 2229-3469
waters and survives easily in waters polluted by
various disinfectants and chemicals.
ln the present study species belong to the genus
Acinetobacter from Vepur, Hanwada and Kosgi,
Bocillus cereus from Vapur and Kodangal and
Pseudomonos oeruginoso from Kosgi, Aeromonos
hyd ro ph i I o |/'uys et o/. 2@21, B a c i t I us cereus (Suth a r
et a1.2008b1, Cupriavidus pauculus (Vandamme et
a/. 1999) and Pseudomonos aeruginoso (Suthar ef
o/. 2008b). Bacillus infantis which cause sepsis was
isolated from clinical specimens (Ko et o/. 2006)
which are opportunistic pathogens were isolated.
coNcrustoN
More information about the microorganisms and
their metabolism is important to determine the
techniques, which should be applied before
ground water can be used as drinking water in
order to take precautions to avoid health problems
at problematic places. The results from this study
further improve our understanding of the
molecular diversity and bacterial population
dynamics of drinking water microbial
communities. Moreover, these results provide the
sequence foundation for the development of
molecular assay that target active drinking water
bacteria. The public must also recognize that
public drinking water is not frequently monitored
by health laboratories for acceptable quality. The
supplemental treatment measures will require
careful in-plant monitoring and maintenance to
prevent in water quality enhancement.
TMRATURECITED
Altschul S F, Cish W, Mitler W Myers E W and Lipman D J. 1990. Basic local alignment search tool.
Moleculor Biolog'y 215: 403-410.1
Brauch H J, Volker E and Frick B R . 1982. Uberlegungen zur charakterisierung irga4ischer
Wasserinhaltstoffe uber ihre adsorbierbarkeit an Aktivkohle. Veroffentlichungen des Bereichsund der
Lehrstuhle fur Wasserchemie und der DVGW Forschungasstelle am Engler-Bunte-lnstitut der Universitat
Karlsruhe, Heft 20, 43-62.
Bruchet A, Legrand M F, Arpino P and Dilettato D.1991. Recent methods for the determination of
volatile and non-volatile organic compounds in natural and purifiled drinking water. Fournol'of
ch ro motog ra phy 562: 469-480.
Chun J, Lee J H, Jung Y, Kim M, Kim $ Kim B K and Um Y W. 2007. EzTaxon: a web-based tool for the
identification of prokaryotes based on 165 ribosomal RNAgene sequences. tntJ Syst EvotMicrobiotsT:
2259-226L.
FelfiildiT, Hedger Z, Vargha M and Mdrialigeti K. 2010. Detection of potentially pathogenic bacteria in
the drinking water distribution system of a hospital in Hungary. Clinical Microbiot lnfections. 16: 89-92.
34

Pavan Kumor Pindi and P Raghuveer Yadqv
Huys G, Kdmpfer P, Altwegg M, Kersters l, Lamb A, Coopman R, Ldthy H J, Vancanneyt M,
Janssen P and Kersters K. 1997. Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from
drinking water production plants and reservoirs. lnternationol Journol of Systemotic Bocteriology 47:
1165-1171.
JahnelJ B, Llieva P, Abbt-Braun G,!.n{,flimm.e! F. H. 1998. Aminosauren und Kohlenhydrate als
Strukturbestandteile von refraktaren organischen Saurdhl Vom Wasser 9O: 205-2t6.
KoKS,OhWS,LeeMVLeeJH,LeeH,PeckKR,teeNYandSongJH.2006.Bocillusinfontissp.nov.and
Bacillus idriensis sp. nov., isolated from a patient with neonatal sepsis. lnternotionolJournalof Systemotic
ond Evolutionary Microbiology 56: 254!-2544.
Lane D J. t99t. rcSn3S rRNA sequencing. ln Nucleic Acid Techniques in Bocterial Systemotics. Edited by
Stackebrandt, E. and Good Fellow, Wiley: Chichester M. pp 115-175.
leChevallier M W, Babcock T M and Lee R G. 1987. Examination and characterization of distribution
system biofilms. Appl. Environ. Microbiology.5S z 27L4-2734.
LeChevallier M W Seidler R J and Evans T M. 1980. Enumeration and characterization of standard
plate count bacteria in chlorinated and raw water supplies. Appl. Environ. Microbiol. 4O :922-930.
Lee S W, Oh H W Lee K H and Ahm T Y. 2009. Methylobacterium donkookense sp. nov., isolated from
drinking water. Journal of Microbiotogy. 47 :7t6-72O.
Norton C D and Lechevallier M W. 2000. A pilot study of bacteriological population changes through
potable water treatment and distribution; Appl. Environ. Microbiol.65:268-276,
Olson B H and Nagy t A. 1984. Microbiology of potable water. Adv. Appl. Microbiol. 30 : 73-t32.
Payment P, Gamache F and Paquette G. 1988. Microbiological and virological analysis of water
from two water filtration plants and their distribution systems. Con. J. Mirobiol.34 : 1304-1309.
Sambrook J, Fritschi E F and Maniatis T. 1989. Molecular cloning: a laboratory manual, Cold Spring
Harbor Laboratory Press, New York.
Suthar $ Singh S, Chhimpa V Bishnoi R K, Saharan l, Mittal N and Garg V K. 2008b. The problem of safe
drinking water in Northern Rajasthan, lndia. ln Puranik, etol. (Eds.), Proceeding 76th nationalsymposium
on environment. Hisar: Dept. of Environmental Science and Technology, G. J. U. Sci and Tech. pp. 491-
496.
Thompson J D, Higgins D G, Gibson T J, Plewniak F, Jeanmougin F and Higgins D G. 1997. The CLUSTAL_X
windows interface: flexible strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Research 252 48764882.
Vandamme P, GorisJ, Coenye T, Hoste B, Janssens D, Kersterc K, De Vos P and Falsen E. 1999. Assignment
of Centers for Disease Control group lVc-2 to the genus Rolstonia as Rolstonio poucula sp. nov.
I nte rnotionol Journa I of Syste motic Bocteriology 49:563-669.
Zhang T" Fan x, xanadai,i;;;;;a" y
"iJ
r.ig H H. 2006. Bocittus mocouensissp. nov., a tong-chain
bacterium isolated from a drinking water supply. lnternotionalJournol of Systemotic and Evolutionary
Microbiology 56: 349-353.
35