Minisymposium Antibiotic resistome in the food chain - Deutsch ...

Supported by: 

Minisymposium 

Antibiotic resistome 

in the food chain 

Jena, 10. (after lunch) -11. (until lunch) October 

Organizers: Hans Krügel // Valery Danilenko // HP Saluz // RM Rodicio // R Ehrlich 

// Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany 

// Vavilov Institute for General Genetics, Moscow, Russia 

// University of Oviedo // Alere Techn. (Clondiag) 

In the framework: Program Internationalising Science and Research, 

Federal Ministry Education and Research, DLR, Bonn, Germany 

http://www.deutsch-russisches-wissenschaftsjahr.de/ru/




Invitation 

Our meeting is dedicated to the general problem of growing antibiotic resistance 

and the possible impact due to use of antibiotics in the food chain, from manure 

to soil influencing bacterial ecology. Commensals and zoonotic bacteria contaminate 

usually without harm many fresh products, while others are indigenous part 

of the production technology of e.g. milk or meat products. We invite speakers 

and audience interested in antibiotics, mechanisms of resistance, evolution of 

genes, integrons and plasmids as well as in the development of molecular diagnostic 

technology. 

Topics 

Selected antibiotics and resistance 

Soil, manure, ecology 

Zoonotic, commensal and probiotic bacteria 

Antibiotic resistance genes, integrons, plasmids 

Rapid PCR and array diagnostic technology




Participants 

Anne Bleicher 

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll 

Institute, Jena, Germany 

Sabine Brantl 

Friedrich-Schiller-University, Jena, Germany 

Reinhard Breitling 

Jena Bioscience GmbH, Jena, Germany 

Valery Danilenko 

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 

Russian Federation 

Axel Dost 

Klinik für Kinder & Jugendmedizin Universitätsklinikum Jena, Germany 

Ralf Ehricht 

ALERE TECHNOLOGIES GmbH, Jena, Germany 

Bernd Giese 

Food GmbH Jena, Analytik – Consulting, Germany 

Michael Grün 


Ingrid Hänel 

FLI, Jena, Germany 

Christian Hoischen 

Leibniz Institute for Age Research, FLI, Jena, Germany 

Helmut Hotzel 


Yvonne Hupfer 

Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll- 


Melanie Jahn 

Analytik Jena | Biometra, Jena, Germany 

Sven Jechalke 

Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut, Federal 

Research Centre for Cultivated Plants, Braunschweig, Germany 

Hans Krügel 



M Kurzai 

Klinik für Kinder & Jugendmedizin Universitätsklinikum Jena, Germany 

Stefan Monecke 

Institute for Medical Microbiology and Hygiene, Technical University of Dresden, 

Dresden, Germany 

Grit Mrotzek 



Jens Müller 


Elena Poluektova 

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 

Russian Federation 

M.Rosario Rodicio 

Laboratory of Microbiology (Department of Functional Biology), University of 

Oviedo, Oviedo, Spain 

Ivan Rychlik 

Veterinary Research Institute, Hudcova 70, 621 00, Brno, Czech Republic. 

Hans Peter Saluz 



Uta Schmidt 

Institut für Umweltmedizin, Erfurt, Germany 

Gisbert Schumann 

ALERE TECHNOLOGIES GmbH, Jena, Germany 

Stefan Schwarz 

Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt- 

Mariensee, Germany 

Roman Siddiqui 

TMF – Technologie- und Methodenplattform vernetzte medizinische Forschung, 

Berlin, Germany 

Kornelia Smalla 

Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut, Federal 

Research Centre for Cultivated Plants, Braunschweig, Germany 

Alexei Sorokin 

The Genome Analysis Team ANALGEN, TGU MICALIS, INRA Jouy-en-Josas, France 

Rudolf Stadermann 

Health Care JBS, Unterwellenborn, Germany 

Rainer Stumm 

Institut für Umweltmedizin, Erfurt, Germany 

Melanie Trenkman, 


Alexander Tretyakov 



Gisela Werner 

WERNER BioAgents, Jena, Germany 

Walter Werner 

WERNER BioAgents, Jena, Germany




October 10/11, 2011 // Jena, Germany 

Hotel ”Schwarzer Bär” Jena 

Programme Monday, October, 10. 

12:30 Registration 

13:15 Opening address 

13:20 Resistance derivatives of virulence plasmids specific of non-typhoidal serotypes 

of Salmonella enterica. 

M.Rosario Rodicio 

Laboratory of Microbiology (Department of Functional Biology), University of Oviedo, Oviedo, Spain 

13:50 Ultrarapid Microarray based Genotyping 

Ralf Ehricht 

ALERE Technologies GmbH, Jena 

14:20 Antibiotic resistance in S. aureus strain of animal origin, and its impact on 

medicine 

Stefan Monecke 1/2 , Anna C. Shore 3 , Stefan Schwarz 4 , Ralf Ehricht 2 

1Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany, 2Alere Technologies 

GmbH, Jena, Germany, 3Microbiology Research Unit, Dublin Dental University Hospital, University of Dublin, Trinity College, 

Dublin, Ireland, 4Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany 

14:50 Characterization of Methicillin-resistant Staphylococcus aureus Isolates 

from Food and Food Products of Poultry Origin 

Andrea T. Feßler1 , Kristina Kadlec1 , Melanie Hassel2 , Tomasz Hauschild1 , Christopher Eidam1 , Ralf Ehricht3 , Stefan Monecke3,4 

and Stefan Schwarz1 1 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany, 2 Landesuntersuchungsamt 

Rheinland-Pfalz, Koblenz, Germany, 3 Alere Technologies GmbH, Jena, Germany, 4 Institute for Medical Microbiology and 

Hygiene, Faculty of Medicine „Carl Gustav Carus“, Technical University of Dresden, Germany 

15:20 Culture independent characterisation of changes in gut microbiota of hens 

in a consequence to antibiotic therapy 

Ivan Rychlik 

Veterinary Research Institute, Brno, Czech Republic. 

15:50 - 16:20 Coffee break offers discussion time with tea/coffee/cake and cookies 

16:20 Fate and effect of veterinary medicines entering soil via manure 

Kornelia Smalla 

Institut für Epidemiologie und Pathogendiagnostik, Julius Kühn-Institut (JKI) Bundesforschungsinstitut für 

Kulturpflanzen,Braunschweig, Germany 

16:50 Microbial and molecular tracing of antibiotic resistance genes in the food 

chain 

Anne Bleicher, Jens Müller1 , Hans Krügel 

Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute Jena, Germany , 1Food GmbH Jena, 

Analytik – Consulting, Germany




17:20 Helmut Hotzel, 


17:50 MobiLab- The portable laboratory for on-site pathogen detection 

Melanie Jahn, Melanie Trenkman, 


18:20 Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses 

Grit Mrotzek, Haryanti, Isti Koesharyani, Alexander N. Tretyakov, Ketut Sugama, Hans Peter Saluz 

Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute Jena, Germany 

13:50 Ultrarapid Microarray based Genotyping 

Ralf Ehricht 

ALERE Technologies GmbH, Jena 

19.00-22.00 Get together “Spiegelsaal” Hotel Schwarzer Bär 

Programme Tuesday, October, 11. 

09:00 Studies of genomes of Bacillus cereus group strains related to food safety 

Alexei Sorokin, 

The genome analysis team ANALGEN, TGU MICALIS, INRA Jouy-en-Josas, France 

09:30 Toxin-antitoxin genetic systems of Lactobacilli 

E.U. Poluektova, 

Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation 

10:00 Kinases, their inhibitors and bacterial antibiotic resistance 

Valery Danilenko, 

Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation 

10:30 Coffee break offers discussion time with tea/coffee/sandwiches 

11:00 Nourseothricin – past, present and future 

Reinhard Breitling, 

Jena Bioscience GmbH, Jena, Germany 

11:30 Functional Analysis of Cervimycin C resistance in Bacillus subtilis : a promoter 

up mutation and increased mRNA stability of gene bmrA 

12:30 fini 

Hans Krügel, 

Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany




Functional Analysis of Cervimycin C resistance in Bacillus subtilis: 

a promoter up mutation and increased mRNA stability of gene bmrA 

Hans Krügel // Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany. 

hans.kruegel@hki-jena.de 

Due to the ecologic separation of the producing 

organisms antibiotics find sensitive bacteria 

in new ecologic communities, like human 

or veterinary pathogens and commensals. To 

overcome the increasing resistance development, 

new compounds are screened. One such, Cervimycin 

C, belongs to a complex of compounds produced 

by Streptomyces tendae and consists of a tetracyclic 

polyketide decorated with trideoxysugar chains, 

solely active against Gram-positive bacteria (Herold 

et al., 2005). To analyse the molecular response we 

propagated Bacillus subtilis in the presence of increasing 

CmC concentrations. Two independent Cervimycin 

C (CmC) resistant clones of Bacillus subtilis 

were identified each carrying two mutations in the intergenic 

region preceding the ABC transporter gene 

bmrA. In the double mutant, real-time PCR revealed 

an increased amount of bmrA mRNA with increased 

stability. Accordingly, isolation of membrane proteins 

yielded a strong band at 64 kDa corresponding to 

BmrA. We show that one mutation optimized the -35 

box sequence conferring resistance to 3 µM CmC, 

while the +6 mutation alone had no effect, but increased 

the potential of the strain harboring the -35 

mutation to grow at 5 µM CmC. Transcriptional fusions 

revealed an elevated bmrA promoter activity 

for the double mutant. EMSAs confirmed a 30-fold 

higher binding affinity of RNA polymerase for this 

mutant compared to the wild-type, and the effect 

was due to the -35 box alteration of the bmrA promoter. 

In vitro transcription experiments substantiated 

the results of the EMSA. EMSAs in the presence of 

heparin indicated that the mutations did not influence 

the formation and/or stability of open complexes. Halflife 

measurements demonstrated that the +6 mutation 

stabilized bmrA mRNA ≈two-fold. Overall, we found 

that an ABC-transporter confers antibiotic-resistance 

by cumulative effects of two mutations in the promoter 

region. Such fast adaptation to antibiotic stress limits 

the application value of the new drug, but beside their 

more or less pronounced medical value many antibiotics 

may become important tools for elucidating the 

biology of microorganisms. 

FEMS Microbiol Lett. 2010 Dec;313(2):155-63. Krügel H, Licht A, Biedermann 

G, Petzold A, Lassak J, Hupfer Y, Schlott B, Hertweck C, Platzer 

M, Brantl S, Saluz HP.




Nourseothricin – past, present and future 

Reinhard Breitling // Jena Bioscience GmbH, Jena, Germany 

Nourseothricin is a aminoglycoside glycopeptide 

antibiotic belonging to the Streptothricin 

family. It was described in the early 1960th as a secondary metabolite of the nystatin producer 

Streptomyces noursei. 

Due to the antibacterial effect, Nourseothricin was 

applied in the 1980th as an ergotropic agent for 

growth promotion of agricultural animals. In connection 

with this application plasmid–based resistance 

to Streptothricin was searched for and observed 

in territories where Nourseothricin was feeded to 

pigs. Resistance plasmids of different incompatibility 

groups were found in E. coli from pigs and from 

man. Hybridization to bacterial Streptothricin resistance 

gene probes was also observed with plasmids 

isolated a long time before the application of streptothricins. 

The streptothricin resistance determinants 

were found to be linked to other resistance genes 

like streptomycin/spectinomycin- and trimethoprimresistances 

on bacterial transposons. Moreover, 

streptothricin resistance genes were found worldwide 

also in numerous other bacteria including Aerococcus, 

Acinetobacter, Burkholderia, Citrobacter, 

Enterobacter, Klebsiella, Morganella, Proteus, Pseudomonas, 

Psychrobacter, Salmonella, Serratia, Shigella, 

Vibrio and more. They were localized usually 

on class 2 integrons and found in clinical isolates of 

man, at agricultural animal sites and in meet prod- 

ucts. Ergotropic use of Nourseothricin was stopped 

after 1989. 

The advent of Nourseothricin started in the 1990th 

with its application as selection tool in molecular 

genetics when the resistance genes of bacteria and 

Streptomyces self-resistance genes, both encoding 

streptothricin N-acetyltransferases, were employed 

as antibiotic selection markers for generation of recombinant 

organisms. The range of hosts in which 

Nourseothricin selection was established has been 

growing extraordinarily since that time and includes 

at present Gram-positive and Gram-negative bacteria, 

yeast and filamentous fungi, protozoa and microalgae 

as well as plant cells. 

These broad applications were due to the advantages 

of Nourseothricin as low or no background at low selection 

concentrations, no cross-resistance with other 

aminoglycosid or therapeutic antibiotics, not used in 

human or veterinary medicine, therefore, no conflict 

with regulatory requirements and the physico-chemical 

properties as long-term stability (10 years) and 

high solubility in water (1 g/L). 

Presently, Nourseothricin is used in about 50 recombinant 

host-vector systems and the employment of a 

permanently increasing number of species for genetic 

engineering will further extend its application.




MobiLab | The portable laboratory for on-site pathogen detection 

Melanie Jahn, Melanie Trenkmann // Analytik Jena AG, Germany 

The demand of fast and reliable pathogen diagnostics 

is increasing rapidly due to pandemics 

like H1N1 in year 2009. Next to viral 

infection diseases also the quality control of various 

foodstuffs rises, as media consistently refer about 

cases of contaminations, where persons incur serious 

food poisonings. 

The most frequent infection in relation to this is 

caused by Salmonella. Diseases, which are affected 

by these bacteria (salmonellosis) belongs to zoonosises, 

because human and animals can be infected 

as well. Especially infections by food products often 

occur, because Salmonella mostly appear in eggs, 

poultry products and minced meat. Thus the observation 

of this pathogen is essential for consistent 

quality of food products. Therefore the requirement 

for a molecular pathogen diagnostics under field 

conditions and hence a robust and fast result analysis 

become necessary. 

Currently available detection systems require proof 

testing of samples in a qualified laboratory by trained 

personnel, whereby manpower, time and money are 

enormous. The sample material is sent to distant 

laboratories and has to be incubated for at least 24 

hours. Afterwards a PCR test or rather a real-time 

PCR test for detection is performed. Mainly for the 

minced meat processing industry this waiting time of 

incubation can be too long, as the result of a possible 

contamination is just known after the meat is already 

at costumer site or even consumed. 

Beyond that the tests presuppose expensive devicerelated 

requirements as well as cost-intensive reagents. 

In meat companies a direct on-site verification 

is not possible so far. Therefore Analytik Jena AG has 

developed an innovative device platform, the Mobi- 

Lab, to reduce waiting times for results and to offer an 

economically priced sample analysis. 

The MobiLab contains all necessary device components 

for whole pathogen detections, means a temperature-controlled 

thermal shaker for samples lysis, 

a magnet trap and a rapidPCR thermal cycler for specific 

amplification. Further laboratory equipment is not 

necessary. After sample taking the extraction process 

takes place by using approved chemistry based on 

magnetic particles, resulting in ultra pure DNA. Afterwards 

the amplification is carried out in a patented 

disposable cartridge, which allows an easy handling 

and avoid any possible contamination of all following 

samples. Finally the test result becomes visible on a 

high specific lateral flow strip, integrated in the cartridge, 

and allows a clearly Yes / No statement.




Antibiotic resistance in S. aureus strain of animal origin, and its impact 

on medicine 

Stefan Monecke 1/2 , Anna C. Shore 3 , Stefan Schwarz 4 , Ralf Ehricht 2 // 1 Institute for Medical Microbiology and Hygiene, Technical University of 

Dresden, Dresden, Germany, 2 Alere Technologies GmbH, Jena, Germany, 3 Microbiology Research Unit, Dublin Dental University Hospital, University of 

Dublin, Trinity College, Dublin, Ireland, 4 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany 

In recent years, methicillin-resistant Staphylococcus 

aureus (MRSA) have become a global problem. 

In addition to the long-known healthcareassociated 

strains, novel strains have also emerged 

outside of hospitals, in the community as well as in 

livestock. Domestic animals can serve as reservoir 

for both, new strains and novel resistance genes. 

Three examples shall be discussed to emphasise 

this problem. 

ST398-MRSA-V is an emerging strain that is often 

found in livestock, although this strain is also increasingly 

isolated from human patients. It was first 

discovered in the Netherlands, affecting farmers and 

pigs from their farm. Further investigations showed 

its presence in a high proportion of pigs as well as 

in farm personnel, veterinarians and veterinary students. 

ST398-MRSA-V has been identified in several 

European countries, the USA and Australia and 

it has also been observed in cattle, horses, dogs and 

poultry as well as in retail meat of different domestic 

animals. 

Another problematic issue is the recent emergence 

of CC130 and ST425 strains from humans and livestock, 

respectively, which harbour a novel type of 

SCCmec element, SCCmec XI. It harbours a highly 

deviant allele of mecA that cannot be detected by 

commercial mecA PCRs. In addition, assays for the 

detection of modified penicillin binding protein, PB- 

P2a, often fail to identify these MRSA strains. 

Another example is the emergence of the multiresistance 

gene cfr. It confers resistance to phenicols, 

lincosamides, streptogramin, pleuromutilins and oxazolidinones, 

including linezolid. The cfr gene has 

mainly been associated with coagulase-negative 

staphylococci and methicillin-susceptible S. aureus 

from animals, and only a few cfr-positive MRSA have 

been described so far. Recently it was detected in a 

case of an infection of an Irish patient with PVL+ ST8- 

MRSA-IV “USA300”, a hypervirulent epidemic MRSA 

strain as well as in a veterinary isolate of ST398-MR- 

SA-V. 

These examples show a risk for a zoonotic transmission 

of MRSA and of resistance markers. As this might 

evolve to become a true public health threat, close 

surveillance of MRSA in livestock is warranted.




Toxin-antitoxin genetic systems of Lactobacilli 

E.U.Poluektova // Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation 

The following questions will be discussed: 

identification, properties, types, possible 

functions of toxin-antitoxin (TA) systems in 

different groups of microorganisms; identification 

and characterization of TA systems of mazEF and 

relBE types in the strains of Lactobacillus plantarum, 

L.fermentum, L.casei, L.rhamnosus from the collection 

of our laboratories (46 strains) and from the 

GenBank; two-genes and solo TA systems; the relationship 

of TA genes and mobile genetic elements; 

possible use of TA systems as strain specific genetic 

markers.




Fast short-fragment PCR for rapid and sensitive detection of shrimp 

viruses 

Grit Mrotzek, Haryanti, Isti Koesharyani, Alexander N. Tretyakov, Ketut Sugama, Hans Peter Saluz 

We developed a fast short-fragment PCR 

method for the detection of white spot 

syndrome virus (WSSV), infectious hypodermal 

and hematopoietic necrosis virus (IHHNV), 

and monodon baculovirus (MBV)*. Fast two-temperature 

(95°C denaturation and 60°C annealing/ 

extension) PCRs were performed in 5 µl-10 µl volume 

samples in miniaturized microplates using a fast 

Peltier thermal cycler. 40 cycles were completed in 

25-30 minutes. Rapid high-resolution agarose gel 

electrophoresis of 70 bp -150 bp PCR fragments was 

performed in 10 minutes. High sensitivity of PCR 

product detection (50 pg-100 pg) was obtained using 

ultra sensitive dyes such as GelStar® and a gel documentation 

system equipped with a blue-light transilluminator. 

This novel method is faster and more sensitive 

than its TaqMan® real-time PCR counterparts. 

* Mrotzek G, Haryanti, Koesharyani I, Tretyakov AN, Sugama K, Saluz 

HP. Fast short-fragment PCR for rapid and sensitive detection of 

shrimp viruses. J Virol Methods. 2010 Sep;168(1-2):262-6.




Characterization of Methicillin-resistant Staphylococcus aureus Isolates 

from Food and Food Products of Poultry Origin 

Andrea T. Feßler1 , Kristina Kadlec1 , Melanie Hassel2 , Tomasz Hauschild1 , Christopher Eidam1 , Ralf Ehricht3 , Stefan Monecke3,4 and 

Stefan Schwarz1 // 1 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany, 2 Landesuntersuchungsamt 

Rheinland-Pfalz, Koblenz, Germany, 3 Alere Technologies GmbH, Jena, Germany, 4 Institute for Medical Microbiology and Hygiene, Faculty of Medicine 

„Carl Gustav Carus“, Technical University of Dresden, Germany 

During a survey of fresh chicken and turkey 

meat as well as chicken and turkey meat 

products for the presence of methicillinresistant 

Staphylococcus aureus (MRSA) isolates, 

32 (37.6%) of 85 samples collected during 2009 in 

Rhineland-Palatinate/Germany were MRSA-positive. 

One MRSA isolate per positive sample was 

further characterized using various molecular typing 

methods including a diagnostic DNA microarray 

(StaphyType, Alere Technologies, Jena, Germany). 

Twenty-eight of these MRSA isolates belonged to the 

clonal complex (CC) 398 which is widespread among 

food-producing animals. These CC398 isolates carried 

SCCmec elements of types IV or V, exhibited 

spa types t011, t034, t899, t2346 or t6574 and either 

the known dru types dt2b, dt6j, dt10a, dt10q, dt11a, 

dt11v, dt11ab or the novel dru types dt6m, dt10as, 

dt10at. In addition, two MRSA ST9 isolates with a 

type IV SCCmec cassette, spa type t1430 and dru 

type dt10a as well as single MRSA ST5 and ST1791 

isolates with a type III SCCmec cassette, spa type 

t002 and dru type dt9v were identified. All but two 

isolates were classified as multiresistant. A wide variety 

of resistance pheno- and genotypes were detected. 

All isolates were negative for the major virulence 

factors such as Panton-Valentine leukocidin, 

toxic shock syndrome toxin 1 or exfoliative toxins. In 

contrast to the MRSA CC398 isolates, the four ST9, 

ST5 or ST1791 isolates harbored the egc gene clus- 

ter for enterotoxin G, I, M, N, O and U genes. Although 

the relevance of contamination of fresh poultry meat 

or poultry products with MRSA is currently unclear, the 

presence of multiresistant and - in part enterotoxinogenic 

- MRSA emphasizes the need of further studies 

to elucidate possible health hazards for consumers.




Resistance derivatives of virulence plasmids specific of non-typhoidal 

serotypes of Salmonella enterica 

M. Rosario Rodicio // Laboratory of Microbiology (Department of Functional Biology), University of Oviedo, Oviedo, Spain 

Non-typhoidal serotypes of Salmonella enterica 

are one of the leading causes of bacterial 

food-borne disease. Most human infections 

are confined to the small intestine and associated 

with inflammatory diarrhoea. However, bacteria can 

also spread beyond the intestine causing focal or 

systemic infections, particularly in infants, the elderly 

and immuno-compromised hosts. Although 

usually not required, antimicrobial therapy is mandatory 

for the control of invasive infections and for patients 

with recognized risk factors. A limited number 

of non-typhoidal serotypes carry virulence plasmids 

taht are actively evolving through acquisition of new 

genetic information. Of particular interest is the capture 

of genes conferring resistance to antimicrobials, 

which can result from cointegration with resistance 

plasmids but is more frequently mediated by mobile 

genetic elements, such as insertion sequences, integrons 

and transposons. These hybrid virulence-resistance 

plasmids have been found in Typhimurium 

and its monophasic 4,5,12:i:- variant, Enteritidis and 

Choleraesuis, which rank among the most common 

and/or invasive non-typhoidal serotypes. The epidemiology 

of these plasmids, the mechanisms underlying 

their emergence and evolution, as well as 

the consequences for both the pathogens and their 

hosts, will be presented.




Microbial and molecular tracing of antibiotic resistance genes in 

the food chain 

Anne Bleicher, Jens Müller1 , Hans Krügel // Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute Jena, Germany 

, 1Food GmbH Jena, Analytik – Consulting, Germany 

The resistance situation of microbial populations 

in the food chain was monitored by 

random sampling of bacterial isolates from 

different foods, animal and sewage samples. The resistance 

profile of complex as well as pure cultures 

has been recorded for 41 substances belonging to 

fourteen antibiotic classes. The breakpoint values 

according to the European Committee on Antimicrobial 

Susceptibility Testing (EUCAST) were assayed 

in a microtiter plate assay. 

The bacterial strains collected displayed a multidrug 

resistance phenotype against antibiotics relevant in 

human therapy and animal farming. The occurrence 

of cross-resistances to veterinary/clinical substances 

could be reported for fluoroquinolones, amphenicoles 

and cephalosporines in bacteria isolated from 

sheep nose, animal feed and sewage. The novel ßlactam 

antibiotic meropenem was shown to be inactive 

against a Pseudomonas putida from sewage 

and an Enterococcus faecium from sheep nose, both 

species incriminated in nosocomial infections. 

Genetic resistance determinants tetA, tetB, bla- 

TEM, bla-PSE, bla-ampC, sul1, sul2, aadA1 and 

aph1 were frequently detected in PCR using 24 specific 

primer pairs. Class 1 and 2 integrons harbouring 

two to three gene cassettes (dhfrA1, aadA1 and 

sat2) were detected in 13 % and 9 % of the strains, 

respectively. Class 1 integrons were mainly identified 

in sewage samples, whereas class 2 integrons were 

restricted to food samples. 

In 53 % of the isolates (86% Gram-negatives), the 

presence of low copy plasmids was indicated on agarose 

gels after alkaline lysis procedure. For five Gram 

negative strains, their resistance-conferring nature 

has been proven by conjugation experiments to E. coli 

recipients, showing self-transmission rates as high as 

10-2. 

In general, for antibiotics which experience a broad 

use both in veterinary and human therapy, such as 

tetracyclines, sulfonamides and penicillins, resistance 

was observed frequently in various microorganisms. 

On the other hand, when veterinary used substances 

were different from clinical relevant drugs, such as 

for fluoroquinolones, resistance occurred only rarely. 

The microorganisms examined in this study integrated 

not as many resistance genes in their integrons as 

reported for clinical isolates. The resistance profile of 

isolates apparently harbouring plasmids seems to be 

more diverse compared to bacteria without plasmids. 

Emerging problems such as vancomycin-resistant enterococci 

could not be detected in any of the samples.




Ultrarapid Microarray based Genotyping 

RALF EHRICHT // Alere Technologies GmbH, Jena 

Microarray technology enables to screen in 

a rapid and parallel manner for diverse interactions 

between different types of molecules. 

This allows one to analyse different markers 

within a single sample. Microarrays with covalently 

attached oligonucleotides are used, e.g., for the partial 

or complete genotyping of full bacterial or viral 

genomes. This allows determining the presence or 

absence of complete genes or gene clusters as well 

as single point mutations within isolates of a given 

target species. Furthermore, a discrimination of different, 

related species within a genus is possible. 

The application of this technology in its different 

formats is often time consuming and limited due to 

hands on time, sample preparation, price, potential 

of automatisation and parallelisation, software analysis 

and availability in high number of units in perfect 

quality. The ArrayStrip / ArrayTube platform in combination 

with the ArrayMate, a reading and analysing 

device is an easy, fast and economic set of tools for 

diverse microarray based applications. These tools 

can be manufactured in mass production in high 

quality; and they can be combined with ultrarapid 

isothermal amplification technologies as well as with 

an alternative precipitation staining detection principle 

and an automated image analysis. 

Figure 1.) ArrayTube, ArrayStrip and ArrayMate reader 

Using different types of microarray based assays examples 

for species discrimination of Chlamydia and 

antibiotic resistance gene detection in E.coli and S. 

aureus are presented. Complete assay time ranges 

from a few hours down to 30 min using the isothermal 

Recombinase Polymerase Amplification (RPA) reaction. 

Figure 2.): Schematic comparison between RPA and PCR 

Literature: http://www.clondiag.com/technologies/publications.php




Studies of genomes of Bacillus cereus group strains related to 

food safety. 

Alexei Sorokin // The genome analysis team ANALGEN, TGU MICALIS, INRA Jouy-en-Josas, France 

The genome analysis team ANALGEN, for 

which I am responsible in the TGU MICA- 

LIS at INRA Jouy-en-Josas, is specializing in 

analysis of bacterial genomes. Most of our activities 

were devoted to the analysis of genomes of the foodrelated 

pathogens from the Bacillus cereus group 

and also of lactic acid bacteria, like Lactococcus lactis, 

Streptococcus thermophilus and some other. In 

this presentation I shall briefly outline our activities 

related to the B. cereus group and a recent experience 

using the next generation sequencing (NGS) 

methodology (SOLiD platform). 

B. cereus group pathogens represent fourth by importance 

source of collective food poisoning in France. 

The importance of this problem increases with the 

broaden use of refrigerated food technologies and, 

probably, with the climate change. In relation to the 

latter, one very noxious strain, NVH391-98, was 

characterized recently. Its study and characterization 

of four other closely related isolates permitted 

to conclude that they represent a novel moderately 

thermophilic species (growth up to 55oC), Bacillus 

cytotoxicus, of the B. cereus group. B. cytotoxicus 

strains possess the genome, which is 1.5 Mb smaller 

than that of any other B. cereus group bacterium, and 

they are more sensitive to growth conditions. The importance 

of these bacteria for the food security is 

stressed by the fact that the first strain, NVH391-98, 

was isolated only in 1998 and already five strains are 

reported, four of which were isolated from food contamination. 

Recently we characterized a lambda-like 

temperate phage phBC391A2, residing in the genome 

of B. cytotoxicus NVH391-98 as a prophage, and having 

a potential for the generalized transduction. 

The B. cereus group contains also species those 

representatives are able to grow in cold (as low as 

5oC) – B. weihenstephanensis. It is evident that the 

ability to cold growth could be important for the problem 

of contamination of refrigerated foods. Since 

the genomic sequencing of a representative strain 

B. weihenstephanensis KBAB4 revealed that genetically 

it is rather similar to the mesophilic strains, the 

question arises, how easily a mesophilic pathogen 

could become psychrotolerant? An attempt to isolate 

spontaneous mutants able to grow in cold indicates 

that such adaptation should require some additional 

genes. Currently we are trying to develop experimental 

systems that would allow us to detect such genes. 

In this respect we started to characterize the phages 

of these bacteria and to apply NGS for characterization 

of mutations. Characteristics of a representative 

psychrotolerant temperate phage phBW2061 and 

some results of NGS application to the bacteria will 

be reported.




Culture independent characterisation of changes in gut microbiota 

of hens in a consequence to antibiotic therapy 

Ivan Rychlik, Petra Videnska, Helena Hradecka, Marcela Faldynova, Hana Havlickova, Frantisek Sisak. // Veterinary Research Insitute, 

Brno, Czech Republic 

Recent developments in massive parallel sequencing allow detailed, 

culture-independent, characterisation of mixed bacterial 

populations. One of typical mixed population is represented by 

microbial community in gut, changes in which can be easily monitored 

in a response to different interventions. One of the most aggressive 

interventions which induces significant perturbations in gut microbiota 

is antibiotic therapy. In this study we were therefore interested in the 

changes in fecal microbiota occurring in a response to tetracycline and 

streptomycin therapy of adult hens. In the first experiment, adult hens 

were treated for 7 days with antibiotics. During the antibiotic therapy 

and following 5 weeks, the presence of antibiotic resistance genes 

(strA, tetB, sul1 and sul2) was quantified by real time PCR. Fecal microbiota 

composition was determined by PCR amplification of 16S rRNA 

genes with universal bacterial primers followed by 454 pyrosequencing 

of mixed PCR products. In the second experiment, hens were treated 

with antibiotics for 2 days, the therapy was then interrupted for 12 days, 

and on day 14, the therapy was repeated for additional 2 days. After 

the repeated therapy, the birds were followed for additional 2 weeks. 

Feacal samples collected daily were processed exactly as in the first 

experiment. 

Both tetracycline and streptomycin therapy increased prevalence of 

antibiotic resistance genes in fecal microbiota 100 to 1000 times when 

compared with their prevalence before the therapy. In both experiments, 

maximal increase in the prevalence of antibiotic resistance genes in fecal 

microbiota was recorded 2 days post therapy, despite the fact that in 

the first experiment the birds were treated with the antibiotics for 7 days. 

Both tetracycline and streptomycin therapy selected the most for the 

appropriate resistance genes, i.e. streptomycin therapy increased the 

most prevalence of strA and tetracycline therapy resulted in the highest 

increase in tetB. However, a strong co-selection by each of the antibiotics 

was observed also for the antibiotic resistance genes that do protect 

host organism to such antibiotic. Repeated therapy in the second experiments 

caused considerably lower positive selection of targeted antibiotic 

resistance genes when compared with the first round of therapy. 

Sequencing of 16S rRNA amplification product showed that common 

chicken microbiota consisted of representatives of 13 different phylla although 

representatives of Firmicutes, Bacteriodetes, Actinobacteria and 

Proteobacteria formed over 99 % of total microbial community. Major 

genuses present in healthy hens included representatives of Lactobacillus, 

Bacteriodes, Sporacetigenium, Streptococcus, Enterococcus, Bifidobacterium, 

Coprococcus and Faecalibacterium. Both streptomycin and 

tetracycline treatment decreased diversity of faecal microbiota to one half 

with tetracycline being more effective in gut microbiota destruction than 

streptomycin. After the tetracycline treatment, only representatives of Escherichia, 

Enterococcus and Lactobacillus increased in fecal microbiota 

while all other bacterial genuses were considerably suppressed. Effect of 

streptomycin, although also quite deleterious, when compared with that 

of tetracycline was slightly less dramatic. This conclusion is supported by 

the observation that in addition to taxons mentioned above, representatives 

of Turicibacter, Olsenella or Streptococcus survived administration 

of this antibiotic. Interestingly, although both antibiotic caused significant 

changes in fecal microbiota as early as 2 days after their administration, 

recovery of the original microbiota was quite fast and 12 days after cessation 

of the therapy, the fecal microbiota was essentially as complex as 

before the therapy. 

In this study we have shown one or two days after antibiotic therapy with 

either tetracycline or streptomycin, strong selection of clones harboring 

the antibiotic resistance genes was observed. This selection correlated 

with dramatic changes in the composition of faecal microbiota which 

were, however, surprisingly temporarial and less than 2 weeks post cessation 

of the therapy, the fecal microbiota was as complex as in nontreated 

birds.




Studies of genomes of Bacillus cereus group strains related to 

food safety. 

Alexei Sorokin // The genome analysis team ANALGEN, TGU MICALIS, INRA Jouy-en-Josas, France 

The genome analysis team ANALGEN, for 

which I am responsible in the TGU MICA- 

LIS at INRA Jouy-en-Josas, is specializing in 

analysis of bacterial genomes. Most of our activities 

were devoted to the analysis of genomes of the foodrelated 

pathogens from the Bacillus cereus group 

and also of lactic acid bacteria, like Lactococcus lactis, 

Streptococcus thermophilus and some other. In 

this presentation I shall briefly outline our activities 

related to the B. cereus group and a recent experience 

using the next generation sequencing (NGS) 

methodology (SOLiD platform). 

B. cereus group pathogens represent fourth by importance 

source of collective food poisoning in France. 

The importance of this problem increases with the 

broaden use of refrigerated food technologies and, 

probably, with the climate change. In relation to the 

latter, one very noxious strain, NVH391-98, was 

characterized recently. Its study and characterization 

of four other closely related isolates permitted 

to conclude that they represent a novel moderately 

thermophilic species (growth up to 55oC), Bacillus 

cytotoxicus, of the B. cereus group. B. cytotoxicus 

strains possess the genome, which is 1.5 Mb smaller 

than that of any other B. cereus group bacterium, and 

they are more sensitive to growth conditions. The importance 

of these bacteria for the food security is 

stressed by the fact that the first strain, NVH391-98, 

was isolated only in 1998 and already five strains are 

reported, four of which were isolated from food contamination. 

Recently we characterized a lambda-like 

temperate phage phBC391A2, residing in the genome 

of B. cytotoxicus NVH391-98 as a prophage, and having 

a potential for the generalized transduction. 

The B. cereus group contains also species those 

representatives are able to grow in cold (as low as 

5oC) – B. weihenstephanensis. It is evident that the 

ability to cold growth could be important for the problem 

of contamination of refrigerated foods. Since 

the genomic sequencing of a representative strain 

B. weihenstephanensis KBAB4 revealed that genetically 

it is rather similar to the mesophilic strains, the 

question arises, how easily a mesophilic pathogen 

could become psychrotolerant? An attempt to isolate 

spontaneous mutants able to grow in cold indicates 

that such adaptation should require some additional 

genes. Currently we are trying to develop experimental 

systems that would allow us to detect such genes. 

In this respect we started to characterize the phages 

of these bacteria and to apply NGS for characterization 

of mutations. Characteristics of a representative 

psychrotolerant temperate phage phBW2061 and 

some results of NGS application to the bacteria will 

be reported.




Actinobacteria as resistome: new test systems for biotargets directed 

synthesis of drugs 

Valery Danilenko // Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation




Fate and effect of veterinary medicines entering soil via manure 

Kornelia Smalla // Institut für Epidemiologie und Pathogendiagnostik, Julius Kühn-Institut (JKI) Bundesforschungsinstitut für 

Kulturpflanzen,Braunschweig, Germany




Zoonoses 

Helmut Hotzel // FLI, Jena, Germany




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