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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
1) Use antibiotic resistance data obtained from the Collaboration on Animal Health and Food Safety Epidemiology (CAHFSE) and the National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS) programs and poultry studies to identify sources, reservoirs and amplifiers of resistant food borne and commensal bacteria, as well as the path of dissemination of these resistant bacteria in food producing animals and poultry. Results may be used for risk assessment and in developing mitigation strategies. .
2) Map the spread of antimicrobial resistance throughout the US using molecular epidemiology and population genetic studies of antimicrobial resistant bacterial isolates, including participation in USDA VetNet. .
3) Analyze and differentiate antimicrobial resistance mechanisms, both phenotypically and genotypically, and rapidly identify resistant strains.

1b.Approach (from AD-416)
Under current funding, this research is designed to be conducted by a team of five scientists, each focusing on one particular organism or area. Each SY will design a specific research plan maximizing collaborations within the Unit structure. Although independent research will be conducted, a majority of experiments will be interactive, minimizing the need to repeat experimental samplings, particularly in the field. This research format will also maximize acquisition of data which will provide insight of the interaction between bacterial populations within the host and/or environment, particularly those interactions involving food borne zoonotic and commensal bacteria. Three SYs will focus on the molecular aspects of AR, particularly in Campylobacter, Salmonella and commensal bacteria (E. coli and enterococci). Critical to the molecular research will be epidemiologic studies provided by the CAHFSE program and ecologic (field and environment) studies which will not only provide a source of isolates for the molecular studies, but will also determine prevalence and dissemination of AR attributes within production settings, the environment, and among bacterial populations. Another significant source of isolates will be available from the NARMS program. These isolates will be well characterized to the serotype level and antimicrobial resistance phenotype. Additionally, all isolates will have been subjected to PFGE analysis to determine relatedness among isolates. Specific genotypic characterization will be conducted. Pathogenic studies involving bacterial strains collected from the CAHFSE and the NARMS programs, as well as those which have been genetically modified in the laboratory, will provide information regarding virulence (or lack thereof) associated with the acquisition of AR. Additionally, transfer of resistance genes may be studied under these environments.

3.Progress Report
The mission of the Bacterial Epidemiology and Antimicrobial Resistance Unit is to study antimicrobial resistance (AR) in zoonotic food borne pathogens and commensal bacteria. To identify and detect food-borne pathogens as well as gain an understanding of the molecular epidemiology among those pathogens, several methods were developed in FY09. These included a rapid high-throughput multiplex PCR for Salmonella identification which can identify the top 50 serotypes, a multiplex polymerase chain reaction assay specific for Salmonella serovars Typhimurium and Heidelberg which can detect as few as 10 colony forming units (CFU) in 10 grams of cheese or meat in less than two days, and a multiplex PCR for Listeria speciation patterns specific for six species of Listeria including Listeria monocytogenes. A new real-time polymerase chain reaction (PCR) assay for identification of Campylobacter was faster and more sensitive than the standard PCR. Microarrays for AR, virulence gene detection, and plasmid detection in food-borne pathogens were also developed. Additional microarrays were developed for genotyping Campylobacter and Listeria which will be helpful for source tracking of these pathogens. Multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA) were also adapted during FY09 for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) and enterococci. Several studies addressed the problem of multi-drug resistance (MDR). Genes in MDR Salmonella and E. coli from animals encoding resistances to aminoglycosides, beta-lactams, chloramphenicol, sulfanilamide, tetracycline, and three mobile elements were detected. MDR was also detected in E. coli and enterococci isolated from dogs and cats. As part of the Unit’s mission, the animal arm of the National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS) is located in the BEAR-RU. In FY09, antimicrobial susceptibility was determined for approximately 4000 Salmonella, 500 Campylobacter, 3000 generic E. coli, and 1800 Enterococcus. Antimicrobial susceptibility patterns from the animal arm of NARMS were complemented by molecular typing information in the form of Pulsed-Field Gel Electrophoresis (PFGE) generated by USDA VetNet. VetNet serves to combine information on AR of isolates with genetic attributes to obtain specific characteristics for the isolates. Over 2000 Salmonella isolates submitted to the animal arm of NARMS were submitted to VetNet in FY09 and the VetNet database was used by FSIS and the CDC to assist in the investigation of several Salmonella-related food-borne outbreaks including those from fresh produce and meat.

1. Development of microarrays for the detection of antimicrobial resistance genes in bacteria. Objective: Develop a technique for identifying and tracking the different genes responsible for antimicrobial resistance and virulence in bacteria. A DNA microarray was developed to detect 775 resistance genes and has been supplemented to detect 498 additional genes from the IncA/C and IncI plasmids that carry multi-drug resistance (MDR). This technique has identified resistance and virulence genes in a variety of bacteria, including Campylobacter, Salmonella, E. coli, Enterococcus, Staphylococcus (including MRSA), Listeria and Clostridium, a fundamental component of the Unit’s research. Analyses of bacteria co-cultured from swine fecal samples have identified genes shared between Campylobacter coli, E. coli, Enterococcus, and Salmonella. The microarray has also been used by FDA to study resistance genes in Salmonella isolated from turkeys and by Walter Reid Army Institute of Research to identify resistance genes in MDR Acinetobacter baumannii isolated from soldiers wounded in Iraq and Afghanistan. The array is currently being used to study the prevalence of MDR IncA/C plasmids in Salmonella and E. coli isolated from animals.

2. Development of a rapid high-throughput molecular technique to determine the serotype of Salmonella enterica. Objective: Develop a multiplex polymerase chain reaction (PCR) Salmonella serotyping technique for high-throughput analysis. The multiplex PCR assay can identify the top 50 serotypes isolated which represent 85% of all clinically isolated Salmonella and has been adapted to a high-throughput platform by incorporation of capillary analysis of the multiplex PCR products. This allows the determination of up to 90 isolates in 24 hours with very little hands on time at a cost of $1.50/sample as compared to several days and ~$40.00 for traditional serotyping. The technique requires little training, no specific anti-sera, and works in standard DNA sequencing instruments and could replace traditional serotyping for most Salmonella isolates. The technique is currently being tested by several state public health laboratories in the U.S. and also by the Public Health Agency of Canada.

3. Analysis of Campylobacter jejuni whole genome DNA microarrays: significance of prophage and hypervariable regions for discriminating isolates. Objective: C. jejuni is a major cause of bacterial gastroenteritis worldwide. Improved typing techniques for tracing the sources of this ubiquitous pathogen are essential for reducing human illness caused by C. jejuni. DNA microarrays were analyzed for the presence of significantly variable genes useful for genotyping C. jejuni. Statistical analyses of whole genome data from 95 geographically diverse cattle, chicken and human C. jejuni isolates identified a total of 142 most informative (i.e. significantly variable) genes. Of this total, 125 (88%) belonged to genomic prophage and hypervariable regions. The significance of genomic prophage and hypervariable regions for isolate discrimination is emphasized by these results. These genes should prove useful in the development of a more efficient microarray-based genotyping system 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. Development of a microarray for genotyping Listeria monocytogenes. Objective: A whole-genome DNA microarray was developed as a means of genotyping Listeria monocytogenes. Antimicrobial resistant L. monocytogenes isolates were tested against a Listeria specific whole-genome DNA microarray. The whole-genome DNA microarray provided an efficient and effective means for genotyping and performing comparative genomics of Listeria species. Listeria monocytogenes is a common foodborne pathogen that exhibits a high mortality rate (> 25%). The whole-genome DNA array provides a method for genotyping L. monocytogenes to further understand the epidemiology, genetic basis of antimicrobial resistant strains, and comparative genomics between Listeria species.

5. Development of a multiplex Polymerase Chain Reaction (PCR) for speciation of Listeria. Objective: To develop a multiplex PCR method for rapid speciation of Listeria. The multiplex reaction used conserved and variable regions of the iap gene to distinguish the six species of Listeria. The reaction produced distinct banding patterns that were easily interpreted. Listeria monocytogenes is a common foodborne pathogen that exhibits a high mortality rate (> 25%). Biochemical methods, such as API strips, can be costly, time-consuming, and prone to contamination. The speciation reaction is rapid (approximately 2 hours), less expensive, and can be highly effective, especially when combined with preliminary biochemical testing.

6. Sensitive and rapid molecular detection assays for Salmonella enterica serovar Typhimurium and Heidelberg in food. Objective: Develop high-throughput techniques for rapid identification of highly prevalent Salmonella enterica serotypes Typhimurium and Heidelberg to prevent their transmission to humans via contaminated food. A multiplex polymerase chain reaction (mPCR) assay specific for serovars Typhimurium and Heidelberg was developed and used to screen over 200 Salmonella strains. The assay was adapted to Real-Time PCR and could detect as few as 10 colony forming units (CFU) in 10 grams of cheese or meat in less than two days. This method can rapidly screen food for contamination with Salmonella, and can identify serovars Typhimurium and Heidelberg. The assay can be used to prevent transmission to humans resulting in improved food safety and consumer confidence.

7. Analysis of antimicrobial resistance genes found in MDR Salmonella and Escherichia coli isolated from animals throughout the eleven years of the National Antimicrobial Resistance Monitoring System (NARMS) testing. Objective: Identify antimicrobial resistance genes responsible for multi-drug resistance (MDR) phenotypes in Salmonella and E. coli, determine how they are related, and how they have changed over the history of NARMS. Genes in MDR Salmonella and E. coli encoding resistances to aminoglycosides, beta-lactams, chloramphenicol, sulfanilamide, tetracycline, and three mobile elements were detected in the majority of isolates. MDR bacteria in food animals may represent a risk to human health. The data collected in this study identified resistance genes responsible for MDR isolates and described how it has changed and developed over the 1997-2007 period. This data is useful in understanding how MDR develops in animals and how it could be prevented in the future.

8. Mechanisms of antimicrobial resistance in enterococci isolated from dogs and cats. Objective: The contribution of dogs and cats as reservoirs of antimicrobial resistant enterococci remains largely undefined. This is increasingly important considering the possibility of transfer of bacteria from companion animals to the human host. In this study, antimicrobial resistant enterococci from dogs and cats from veterinary clinics were screened for the presence of resistance genes. Five tetracycline resistance genes were detected with tet(M) accounting for approximately 60% of tetracycline resistance; erm(B) was also widely distributed among 96% of the erythromycin resistant enterococci. Five aminoglycoside resistance genes were also detected among the kanamycin resistant isolates with the majority of isolates containing aph(3”)-IIIa. The bifunctional aminoglycoside resistance gene, aac(6’)-Ie-aph(2”)-Ia, was detected in gentamicin resistant isolates and ant(6)-Ia in streptomycin resistant isolates. Results from this study indicate that healthy dogs and cats are a source of antimicrobial resistant enterococci that harbor resistance genes that can be transferred to the human host.

9. Antimicrobial susceptibility testing and species determination of Campylobacter isolates from the Food Safety Inspection Service Young Chicken Baseline Study. Objective: To determine the species and antimicrobial resistance profile of Campylobacter isolates from chicken carcass rinses. The Food Safety Inspection Service conducted a nationwide baseline study to estimate the prevalence of Campylobacter on broiler carcasses. Over 4500 isolates from this study were processed for species determination and antimicrobial susceptibility testing. The majority of isolates were identified as either C. jejuni (68%) or C. coli (32%). One C. lari isolate was also identified. Tetracycline resistance appeared most often among the isolates (44%). About a fifth of the isolates (21%) were resistant to the fluoroquinolone, ciprofloxacin. Resistance was also seen to azithromycin, erythromycin, clindamycin, and telithromycin. This data can be used in the development of risk-based sampling programs and regulatory decisions. Additionally, it shows the prevalence of antimicrobial resistance among Campylobacter found on chicken carcasses from federally inspected slaughter plants.

10. Analysis of a commercial real-time polymerase chain reaction (PCR) assay for the identification of Campylobacter coli and Campylobacter jejuni. Objective: Evaluate a new real-time PCR assay for identifying C. coli and C. jejuni against a gel-based PCR method. The ability to accurately and quickly identify Campylobacter spp. is very important for epidemiological studies. A total of 1,211 Campylobacter isolates from various sources were identified in parallel using a commercially available real-time PCR assay and a traditional, gel-based method. The majority (87%) of isolates were identically identified by both methods. Some (9%) isolates were identified in a mixed culture by either one or the other method; however, mixed cultures were identified more often using real-time PCR than the gel-based PCR method. Results are obtained from the real-time assay in about 90 minutes while the traditional method requires three to four hours. The real-time assay requires less manual labor and gives results in less than half the time. Additionally, its greater sensitivity allows the detection of fewer cells.

11. Prevalence of antimicrobial resistant Escherichia coli from dogs and cats. Objective: The contribution of dogs and cats as reservoirs of antimicrobial resistant E. coli remains largely undefined. This is increasingly important considering the possible transfer of bacteria from companion animals to the human host. In this study, dogs and cats from veterinary clinics were screened for the presence of E. coli. A total of 317 E. coli were isolated from nasal, teeth, rectal, belly and hindquarter sites of 155 dogs and 121 cats. Approximately 19.2% and 24.4% of E. coli from dogs and cats, respectively, were resistant to antimicrobials. The highest level of resistance was to ampicillin for both groups of animals. Multi-drug resistance (MDR) (resistance >2 antimicrobials) was observed in 36 isolates which were resistant to as few as two and as many as ten antimicrobials. Results from this study indicate that healthy dogs and cats are a source of antimicrobial resistant E. coli and may act as a reservoir of antimicrobial resistance that can be transferred to the human host.

12. Detection and prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) from retail meat. Objective: Recently, methicillin-resistant Staphylococcus aureus (MRSA) have been found in swine in a number of European countries. These MRSA have also been found in the swine farmers, their families, and veterinarians who have had contact with those farms and in retail pork products. The spread of MRSA originating from animals appears to be a source of MRSA in humans and may have the potential to threaten human health. From the study, 3% (3/100) of pork products were positive for MRSA. Two pork chop samples (pork loin chops and boneless chops) and one pork rib sample were MRSA positive. Of the nine antimicrobial resistance genes tested, only ermA conferring erythromycin resistance was detected in one MRSA from pork while ermA, ermC, and tetK, conferring tetracycline resistance, was found in human MRSA isolates. Genetic patterns of the isolates were different indicating that MRSA from pork products and humans were not genetically related. Because none of these studies have been performed in the U.S. and the U.S. imports a number of swine from Canada, the extent of MRSA in swine and pork products as well as in the human population remains unknown. This information will be useful in evaluating the extent of MRSA from animal sources.

13. Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) from companion animals. Objective: The contribution of companion animals as reservoirs of MRSA remains largely undefined. This is increasingly important considering the possibility of transfer of bacteria from companion animals to the human host. In this study, MRSA from companion dogs and cats were isolated and characterized. MRSA were isolated from various sites on ill and healthy animals. All isolates were of the same staphylococcal chromosome cassette (SCC) mec (methicillin) type. Susceptibility of MRSA isolates was performed to determine the range of resistance; all isolates exhibited the same resistance pattern among the 18 antimicrobials tested. Also, a new multi-locus sequence type (MLST) was detected among the MRSA isolates. MRSA causes severe to fatal infections among high risk populations and healthy individuals. Sources of community-acquired MRSA infections vary and the current study shows that companion animals may harbor the organism.

14. Antimicrobial susceptibility testing of food-borne pathogens by the animal arm of the National Antimicrobial Resistance Monitoring System (NARMS). Objective: To conduct antimicrobial susceptibility testing on Salmonella, Campylobacter, E. coli and Enterococcus from animal and environmental sources. Since 1996, the animal arm of NARMS has been based in the USDA-ARS at the Russell Research Center in Athens, GA. Salmonella was selected as the sentinel organism and antimicrobial resistance in over 54,000 Salmonella isolates has been determined. Campylobacter, generic E. coli and Enterococcus were added in subsequent years resulting in over 6,000, 18,000 and 10,000, respectively, antimicrobial susceptibility patterns for each of those bacteria. Veterinary Diagnostic Laboratories serve as diagnostic sentinel sites. This is the only national program for surveillance of resistant bacteria in animals in the U.S. and provides critical information regarding the prevalence and distribution of antimicrobial resistant bacteria in on-farm animals, from diagnostic submissions, and from raw product collected from federally inspected slaughter and processing plants.

15. Molecular typing of Salmonella and Campylobacter using pulsed-field gel electrophoresis (PFGE) by USDA VetNet. Objective: USDA VetNet was established in 2003 and was modeled after PulseNet USA, the national molecular subtyping network for food-borne disease surveillance. The objectives of USDA VetNet are to use PFGE to subtype zoonotic pathogens submitted to the animal arm of the National Antimicrobial Resistance Monitoring System (NARMS), compare USDA VetNet and PulseNet PFGE patterns, and to use the comparative data for surveillance and investigation of food-borne illness outbreaks. Whereas PulseNet subtypes seven food borne disease-causing bacteria: E. coli O157:H7, nontyphoidal Salmonella, Shigella, Listeria monocytogenes, Campylobacter, Yersinia pestis, and Vibrio cholerae, VetNet, at present, subtypes nontyphoidal Salmonella serotypes and Campylobacter from animals including diagnostic specimens, healthy farm animals, and carcasses of food-producing animals at slaughter. VetNet has two functioning databases including the NARMS Salmonella and Campylobacter databases. The Salmonella database contains over 17,000 Salmonella isolates, while the Campylobacter database contains over 900 Campylobacter isolates. Both databases contain the PFGE Tagged Image File Format (TIFF) images, demographic information, and the antimicrobial resistance profiles assigned by NARMS. In the future, veterinary laboratories will be invited to participate in VetNet. The establishment of USDA VetNet enhances the mission of the agriculture and public health communities in surveillance and investigation of food borne illness outbreaks.

6.Technology Transfer


Review Publications
Frye, J.G., Fedorka-Cray, P.J., Jackson, C.R., Rose, M. 2008. Analysis of salmonella enterica with reduced susceptibility to the third-generation cephalosporin ceftriaxone isolated from U.S. cattle during 2000-2004. Microbial Drug Resistance. 14(4):251-258.

Woods, D.F., Reen, F., Gilroy, D., Buckley, J., Frye, J.G., Boyd, E. 2008. Rapid multiplex PCR and Real-Time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C. Journal of Clinical Microbiology. 46(12):4018-4022.

Marschall, J., Tibbetts, R.J., Dunne, W., Frye, J.G., Fraser, V.J., Warren, D.K. 2009. Presence of the KPC carbapenemase gene in Enterobacteriaceae causing bacteremia, and the correlation with in vitro carbapenem susceptibility. Journal of Clinical Microbiology. 47(1):239-241.

Leader, B.T., Frye, J.G., Hu, J., Cray, P.J., Boyle, D.S. 2009. High-throughput Molecular Determination of Salmonella enterica Serovars Use of Multiplex PCR and Capillary Electrophoresis Analysis. Journal of Clinical Microbiology. 47(5):1290-1299.

Akkina, J.E., Hogue, A.T., Angulo, F.J., Johnson, R., Petersen, K.E., Saini, P.K., Cray, P.J., Schlosser, W.D. 1999. Epidemiologic aspects, control, and importance of multiple-drug resistant salmonella typhimurium dt104 in the united states. Journal of Veterinary Medicine. 214 (6) P. 790-798

Pradhan, A., Van Kessel, J.S., Karns, J.S., Wolfgang, D.R., Hovingh, E., Nelen, K.A., Smith, J.M., Whitlock, R.H., Fyock, T., Ladely, S.R., Cray, P.J., Schukken, Y.H. 2009. Dynamics of endemic infectious diseases of animal and human importance on three dairy herds in the northeastern United States. Journal of Dairy Science. 92:1811-1825.

Richardson, L.J., Cox Jr, N.A., Bailey, J.S., Berrang, M.E., Cox, J.M., Buhr, R.J., Cray, P.J., Harrison, M.A. 2009. Evaluation of TECRA® broth, Bolton broth and direct plating for recovery of Campylobacter spp, from broiler carcass rinsates from several commercial processing plants. Journal of Food Protection. 72(5):972-977.

Bailey, J.S., Cray, P.J., Richardson, L.J., Cox Jr, N.A., Cox, J.M. 2008. Detection of Campylobacter from broiler carcass rinse samples utilizing the TECRA Visual Immunoassay (VIA). Journal of Rapid Methods and Automation in Microbiology.16(4):374-380.

Zou, W., Frye, J.G., Chang, C., Liu, J., Cerniglia, C.E., Nayak, R. 2009. Microarray Analysis of Antimicrobial Resistance Genes in Salmonella enterica from Preharvest Poultry Environment. Journal of Applied Microbiology. 107(3):906-914.

Pittenger, L.G., Englen, M.D., Parker, C., Frye, J.G., Quinones, B., Horn, S.T., Son, I., Cray, P.J., Harrison, M.A. 2009. Genotyping Campylobacter jejuni by comparative genome indexing: an evaluation with pulsed-field gel electrophoresis and flaA SVR sequencing. Foodborne Pathogens and Disease. 6(3):337-349.

Davis, J.A., Jackson, C.R. 2009. Comparative antimicrobial susceptibilty of Listeria monocytogenes, L. innocua and L. welshimeri. Microbial Drug Resistance. 15(1):27-32.

Tibbetts, R., Frye, J.G., Dunne, W. Detection of KPC-2 in a Clinical Isolate of Proteus mirabilis and First Reported Description of Carbapenemase Resistance Caused by a KPC Beta-Lactamase in P. mirabilis. Journal of Clinical Microbiology. 46(9):3080-3083.

Last Modified: 11/28/2015
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