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United States Department of Agriculture

Agricultural Research Service

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Research Project: Molecular Approaches for the Identification and Characterization of Antimicrobial Resistance in Foodborne Pathogens

Location: Bacterial Epidemiology and Antimicrobial Resistance

2012 Annual Report

1a.Objectives (from AD-416):
1. Identify and characterize potential genetic markers within and across genera of the high priority foodborne organisms for poultry attribution. With current priorities, the organisms should include Salmonella and Campylobacter.

2. Determine unique characteristics of high priority serotypes of antimicrobial–resistant foodborne bacteria and those of highly resistant or multi-resistant genotypes with novel phenotypes.

3. Evaluate the role of innovative chemical and/or biological treatments, such as arsenicals, prebiotics, or ammonium compounds and how they affect the prevalence and type of resistant pathogens or resistance genes.

1b.Approach (from AD-416):
In the previous project plan, studies were initiated in order to provide a basic understanding of the development, prevalence, dissemination, and persistence of antimicrobial resistance. Molecular methods were developed for rapid identification of bacterial species and antimicrobial resistance genes. The goal of the proposed project plan is to characterize antimicrobial resistant foodborne pathogens and commensals from the extensive National Antimicrobial Resistance Monitoring System (NARMS) bacterial culture collection and other foodborne bacterial culture collections using molecular tools.

For Objective 1, genomic sequencing will be performed initially on bacterial isolates from the NARMS program to identify potential genes which can be used as genetic markers. Genetic markers will differentiate bacteria from poultry from the other major food animal sources. This information is critical for tracing bacterial sources in foodborne outbreaks or contamination of environmental areas.

For Objective 2, unique characteristics of high priority serotypes or subtypes of antimicrobial–resistant foodborne bacteria will be examined in detail using genomic sequencing, microarray analysis, and PCR. Isolates that exhibit high levels of resistance, multi-drug resistant genotypes or novel phenotypes will be given priority. Vehicles for the dissemination of resistance genes (e.g. plasmids, transposons, and integrons) will be given special focus. We will detect new or emerging antimicrobial resistance in foodborne bacteria which is essential for understanding development of antimicrobial resistance.

In Objective 3, target bacterial populations identified from the above objectives will be tested for resistance to non-antimicrobial chemicals or solutions (biocides) commonly used in poultry or poultry processing. Resistance levels of isolates will be assessed followed by genetic analysis of the genes encoding the resistance. This will include bacterial conjugation to determine if the genes may be transferred within and between bacteria as well as cloning and characterization of the resistance genes. These validation studies will provide important data on resistance to commercially based non-antimicrobials in poultry processing.

3.Progress Report:
This project is new commencing in October of fiscal year 2012. During the year, high-throughput sequencing was employed for whole-genome DNA sequencing of Salmonella enterica serotype Heidelberg. DNA sequences from all isolates are presently being analyzed to identify gene differences between isolates from various animal sources. For Campylobacter, whole-genome DNA microarrays were used to identify a total of 142 most significant variable genes from Campylobacter jejuni from 95 geographically diverse cattle, chicken and human C. jejuni isolates. Eighty-eight percent of the significant variable genes belonged to genomic prophage and hypervariable regions. Identification of genetic diversity among the Salmonella and Campylobacter isolates studied to date is necessary to identify and characterize potential genetic markers as stated in Objective 1 of the project plan. The identified markers will ultimately be used to develop a genetic marker Polymerase Chain Reaction (PCR) assay for Salmonella and Campylobacter for poultry source attribution.

Multi-drug resistance (MDR) was evaluated in Salmonella and generic Escherichia coli from animals. PCR and microarrays were used to detect antimicrobial resistance (AR) genes and plasmids on which the genes are harbored. The DNA microarray, developed by an ARS scientist, includes a total of 1267 probes; 775 probes detect AR genes and 487 probes detect two different plasmid replicon types, Inc A/C from several different bacteria and Inc HI1 from Salmonella Typhi. The plasmid replicon typing multiplex PCR detects 18 plasmid replicon types found in the Enterobacteriaceae. These technologies were used to characterize MDR E. coli and MDR S. Typhimurium. For MDR E. coli, AR genes were detected consistent with the MDR phenotypes of all isolates, and plasmid replicon typing identified one or more replicon types in all isolates tested. Class I integrons containing AR genes were also detected in some of the isolates. MDR S. Typhimurium, isolated from healthy cattle, poultry, and swine and resistant to at least five antimicrobials (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline) were selected for analysis. The distribution of AR and plasmid genes detected separated most of the isolates into two groups. Results showed significant linkage for the associations between plasmids, phage type, and animal source for those groups. In addition to whole-genome sequencing, high-throughput sequencing was also used to sequence plasmids isolated from MDR Salmonella, Escherichia coli and Enterococcus from food animals. Twelve plasmids were successfully sequenced and analysis of the DNA sequence is in progress. Analysis of the plasmid sequence is expected to reveal AR genes contained on the plasmids as well as genes which may be responsible for mobility of AR genes, segments of plasmids or entire plasmids. These studies directly relate to Objective 2 of the project plan which aims to determine unique characteristics of high priority serotypes of AR food borne bacteria and those of highly resistant or multi-resistant genotypes with novel phenotypes.

1. Antimicrobial resistance mechanisms of Salmonella enterica serovar Typhimurium. Antimicrobial resistance (AR) in food borne bacteria is a concern for both animal and human health. This is especially true when multi-drug resistance (MDR) occurs in foodborne pathogens such as Salmonella enterica. MDR has been found in Salmonella enterica serovar Typhimurium isolated from animals as part of the National Antimicrobial Resistance Monitoring System (NARMS). Some of these bacteria are resistant to five or more antimicrobials including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT resistance phenotype). To investigate the genes responsible for this MDR, S. Typhimurium isolated from healthy cattle, poultry, and swine and resistant to at least ACSSuT were selected between the years 1997 to 2007 (n=33). AR and plasmid gene content of these isolates were evaluated using microarray analysis. The distribution of genes detected separated most of the isolates into two groups. Those in group A (n=15) were often a specific type called definitive phage type DT104 and were isolated mostly from swine. Isolates in group B (n=16) had many Inc A/C plasmid genes detected and were isolated mostly from cattle. Further analysis demonstrated significant linkage for the associations between plasmids, phage type, and animal source for these groups. This study identified the two major genetic elements responsible for MDR in S. Typhimurium isolated from healthy food animals. This information clearly defines the genetics behind MDR in this important pathogen and will enable the scientific community to determine the sources of these genetic elements and develop procedures to reduce their prevalence in the food chain.

2. Antimicrobial resistance mechanisms of commensal Escherichia coli isolated from chickens. Multi-drug resistant (MDR) commensal bacteria in food animals are a potential source of MDR genes for pathogenic bacteria and could present a hazard to animal and human health. To determine the genetic cause of resistance, 32 MDR Escherichia coli isolates from poultry were examined (four isolates from each year 2000-2007). Microarray analysis was used to detect antimicrobial resistance (AR) genes and mobile genetic elements, such as plasmids, known to be associated with the spread of MDR. AR genes were detected consistent with the MDR phenotypes of all isolates, and a large number of IncA/C plasmid genes were detected in 27 of the isolates, indicating the likely presence of this plasmid known to carry MDR genes. Testing for 18 plasmid replicon types associated with MDR in Enterobacteriaceae detected one or more replicon types in all 32 isolates and confirmed the presence of IncA/C in the 27 isolates. Class I integrons were also assayed for and detected in 25 of the isolates. The class I integrons detected ranged in size from ~1000 to 3300bp and sequence analysis identified AR genes in those integrons. The class I integrons, IncA/C plasmids, and MDR-associated plasmids found in the isolates indicated the importance of these genetic elements in the accumulation and spread of AR genes in the microbial community associated with poultry. This study is important for federal regulatory agencies as identifying the source of MDR elements in food animals is an important step towards developing interventions to reduce resistant bacteria which are potential hazards to human health.

3. Identification of genes that differentiate Campylobacter jejuni isolated from cattle, chickens and humans. Campylobacter is a leading cause of food borne illness in humans and improving our understanding of the epidemiology of this organism is essential. The objective of this study was to identify the genes that were most significant for discriminating isolates of Campylobacter jejuni by analyzing whole genome DNA microarrays. Statistical analyses of whole genome data from 95 geographically diverse cattle, chicken and human C. jejuni isolates identified a total of 142 most significant variable genes. Of this total, 125 (88%) belonged to genomic prophage and hypervariable regions. The significance of genomic prophage and hypervariable regions in determining the overall genetic diversity of C. jejuni is emphasized by these results. These genes should prove useful to food borne illness tracing programs, such as PulseNet, in the development of genotyping systems for C. jejuni as well as a means to further our understanding of the epidemiology and population genetics of this major food borne pathogen.

4. Enterococci from retail meat and produce. Although enterococci are considered opportunistic pathogens, they can be reservoirs of antimicrobial resistance. This is increasingly important considering food borne illnesses from meat and produce. The objective of the present study was to use band-based molecular typing methods to determine if genetically related enterococci were found among different stores, food types, or years. Different enterococcal species were prevalent on fruits, vegetables, and meat from retail grocery stores. Specific species, such as Enterococcus casseliflavus from fruits and vegetables, were predominant on certain food products, but were also found in lower numbers on other food items. The majority of enterococcal isolates from the retail food items were resistant primarily to bacitracin, flavomycin, and lincomycin. Resistance of enterococci to penicillin, salinomycin, and nitrofurantoin was low and none of the isolates were resistant to linezolid or vancomycin. Enterococcal isolates with identical banding patterns were identified from different stores, food types, and of different species. These data suggest that foods commonly purchased and consumed from grocery stores are a source of genetically related antimicrobial resistant enterococci that can be transferred to the human population. This research will be useful to policy makers and researchers to better understand bacterial contamination of meat and produce in order to contain and eliminate food borne outbreaks.

Review Publications
Glenn, L.M., Lindsey, R.L., Frank, J.F., Meinersmann, R.J., Englen, M.D., Cray, P.J., Frye, J.G. 2011. Analysis of antimicrobial resistance genes detected in multidrug-resistant salmonella enterica serovar typhimurium isolated from food animals. Microbial Drug Resistance. 17(3):407-418.

Pittenger, L.G., Frye, J.G., Mcnerney, V., Reeves, J., Haro, J.H., Cray, P.J., Harrison, M.A., Englen, M.D. 2012. Analysis of Campylobacter jejuni whole-genome DNA microarrays: Significance of prophage and hypervariable regions for discriminating isolates. Foodborne Pathogens and Disease. 9(5):473-479.

Glenn, L.M., Englen, M.D., Lindsey, R.L., Frank, J.F., Turpin, J.E., Berrang, M.E., Meinersmann, R.J., Cray, P.J., Frye, J.G. 2012. Analysis of antimicrobial resistance genes detected in multiple-drug-resistant Escherichia coli isolates from broiler chicken carcasses. Microbial Drug Resistance. 18(4):453-463.

Jackson, C.R., Spicer, L.M., Barrett, J.B., and Hiott, L.M. 2012. Application of multiplex PCR, Pulsed-Field Gel Electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci. In: Magdelden, S., editors. Gel Electrophoresis. Rijeka, Croatia: InTech-Open Access. p. 269-298. Available:

Huang, X., Frye, J.G., Chahine, M.A., Glenn, L.M., Ake, J.A., Su, W., Nikolich, M.P., Lesho, E.P. 2012. Characteristics of plasmids in multi-drug-resistant enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq. PLoS One. 7(7):1-8.e40360. doi: 10.1371/journal.pone.0040360.

Last Modified: 4/18/2014
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