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United States Department of Agriculture

Agricultural Research Service

Research Project: EPIDEMIOLOGY, ECOLOGY, AND MOLECULAR GENETICS OF ANTIMICROBIAL RESISTANCE IN PATHOGENIC AND COMMENSAL BACTERIA FROM FOOD ANIMALS
2007 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
See attached sheets.


4.Accomplishments
1. Title: Antimicrobial susceptibility testing of food-borne pathogens by the animal arm 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. Accomplishment: Since 1996, the animal arm of NARMS has been based in the USDA-ARS at the RRC in Athens, GA. Salmonella was selected as the sentinel organism and antimicrobial resistance in over 45,000 Salmonella isolates has been determined. Campylobacter, generic E. coli and Enterococcus were added in subsequent years resulting in over 4,000, 7,000 and 4,000, respectively, antimicrobial susceptibility patterns for each of those bacteria. Veterinary Diagnostic Laboratories serve as diagnostic sentinel sites. Impact: 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. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)

2. Title: Swine and dairy studies from the Collaboration in Animal Health Food Safety and Epidemiology (CAHFSE). Objective: To enhance overall understanding of pathogens that pose a food-safety risk and to routinely monitor critical diseases in food-animal production. In response to growing surveillance needs, USDA-ARS, APHIS, and FSIS collectively developed CAHFSE, which will enhance our understanding of pathogens that pose a food-safety risk by tracking them from farm to plant. The goal is to collect information on the prevalence of resistant bacteria and risk factors that may be used to develop mitigation strategies. Accomplishment: From the APHIS National Animal Health Monitoring System (NAHMS) samples, a total of 7788 on-farm swine fecal samples from 130 farms were analyzed for Salmonella and about one-fourth of these were analyzed for Campylobacter, E. coli, and enterococci. From the APHIS dairy study, a total of approximately 500 fecal and environmental samples were analyzed for Salmonella with one-fourth of these sampled for the other bacteria. The incidence of Salmonella, E. coli, and enterococci was consistent with previous years, while the level of Campylobacter was almost 50% less than in previous years. The predominant serotypes of Salmonella from swine samples were S. Derby, S. Typhimurium 5(-), S. Heidelberg, and, S. Mbandaka. Antimicrobial resistance profiles of each of the isolates were determined. Impact: Results and participating farms will determine the direction that CAHFSE will pursue in the coming years so that USDA will be able to identify and implement mitigation strategies for animal health and food safety issues in a timely manner thereby averting adverse economic, animal well-being, and public health consequences. Further, it will provide comprehensive science-based answers regarding animal health and public health, it will serve as a model for future surveillance efforts on a national level, and it will complement information obtained from both the National Antimicrobial Resistance Monitoring System (NARMS) and USDA VetNet programs. (National Program Component 1.1, Problem Statement 1.1.2: Epidemiology)

3. Title: 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. Accomplishment: VetNet has two functioning databases including the NARMS Salmonella and Campylobacter databases. The Salmonella database contains over 13,000 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. Impact: The establishment of USDA VetNet enhances the mission of the agriculture and public health communities in surveillance and investigation of food borne illness outbreaks. (National Program Component 1.1, Problem Statement 1.1.1: Methodology and 1.1.2: Epidemiology)

4. Title: Determining the effectiveness of HACCP-Based Inspection Models Project (HIMP) compared to traditional USDA-FSIS inspection on microbiological quality of commercial processed chicken: Objective: The U.S. poultry industry would like more plants to use HIMP inspection and consumer groups are questioning whether any plants should use HIMP; therefore, at FSIS’s request, this study was designed to compare the Salmonella, Campylobacter, and generic E. coli on processed carcasses from HIMP and traditionally inspected plants. Accomplishment: Salmonella incidence and numbers of Campylobacter and E. coli on processed broilers from all 16 plants in the U.S. using HIMP inspection were found to be equal or lower than from traditionally inspected plants. Impact: FSIS now has the data to support continued or increased use of HIMP inspection systems. (National Program Component 1.1, Problem statement 1.1.2: Epidemiology)

5. Title: Verification of the effectiveness of USDA-FSIS HACCP procedures for determining microbial condition of broiler chickens: Objective: Since 1996, FSIS has sampled broiler chickens for Salmonella as part of the HACCP rules. Because of Federal Express pick-up times, most samples have been taken from the early morning shift. Some groups have questioned if this sampling time would bias results (lower Salmonella early in day compared to potentially higher incidence later in the day). In addition, questions have been raised about effectiveness of different rinse volumes for sampling. Therefore, this study was undertaken to determine if time-of-day or different rinse volumes affected microbial recovery from commercially processed broiler chickens. Accomplishment: Time-of-day that broiler carcasses were taken did not affect incidence of Salmonella. The house that the birds came from was shown to affect recovery. Numerically, but not statistically more Salmonella were recovered from 400 than 100 mL rinse samples. Campylobacter recovery was low, but numerically, but not statistically higher with 100 mL than 400 mL rinse. Impact: FSIS is assured that there is no bias in their sampling time and that the 400 mL rinse used in sampling is appropriate. (National Program Component 1.1, Problem statement 1.1.1: Methodology)

6. Title: Identification of Salmonella serotype using multiplex PCR. Objective: Develop a rapid molecular method for identifying the most prevalent Salmonella serotypes isolated from animals. Accomplishment: The Salmonella are separated into over 2,500 different types by serotyping with anti-sera to specific surface structures. This typing method takes several days, requires highly trained staff and expensive reagents and often cannot type up to 10% of the Salmonella tested. A new typing technique was developed that detects genes specific for Salmonella serotypes by multiplex PCR. Impact: This assay can identify the top 20 serotypes isolated from animal samples which represent 70% of all Salmonella isolated from animals. The technique can be completed in less than five hours, requires no specialized training, no specific anti-sera, and uses inexpensive reagents. (National Program Component 1.1, Problem Statement 1.1.1: Methodology)

7. Title: Development of a universal antimicrobial resistance microarray. Objective: Develop a technique for identifying and tracking the different genes responsible for antimicrobial resistance and virulence in bacteria. Accomplishment: A DNA microarray to detect 100 resistance genes was successfully tested and has now been expanded to detect 775 resistance and virulence genes simultaneously. Impact: This technique can identify resistance and virulence genes in any bacteria, including those studied by the BEARRU. This will help to establish the source, spread and epidemiology of those genes, a fundamental component of the Unit’s research. Analysis of bacteria collected by the Collaboration in Animal Health Food Safety and Epidemiology (CAHFSE) with this technique has identified common genes in different species of bacteria co-cultured from the same animal. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)

8. Title: Cephalosporin resistance in Salmonella from cattle. Objective: Previous studies had detected increased resistance to cephalosporins in Salmonella isolated from cattle. Therefore, a follow up study was done to characterize the increase in resistance. Accomplishment: Approximately 3,984 cattle isolates were assayed for resistance to beta-lactams. Ninety-seven were found to have reduced susceptibility to ceftriaxone; however none were found to have an Extended Spectrum Beta-lactamase (ESBLs) phenotype or to have genes encoding ESBL enzymes. Impact: ESBLs are a major problem in Europe and Asia; therefore, the lack of ESBLs in cattle isolates from the U.S. is a very significant finding indicating that this phenotype has yet to emerge in the U.S. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)

9. Title: Antimicrobial resistance of Salmonella serotype Enteritidis. Objective: To develop techniques for the identification of Salmonella Enteritidis strains that easily colonize chicken eggs versus those that do not. Accomplishment: Three strains of S. Enteritidis were compared to S. Heidelberg, S. Newport, and S. Typhimurium for growth in the presence of 240 serially diluted antibiotics in a high-throughput phenotype microarray. Impact: Egg-contaminating strain of S. enteritidis were found to be resistant to beta-lactam antibiotics as a class and strains of monomorphic S. enteritidis PT13a could be differentiated from dimorphic S. Enteritidis PT4, S. Typhimurium, S. Heidelberg, and S. Newport by a group of 36 antibiotics. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)

10. Title: Genotyping of Campylobacter jejuni from U.S. cattle and poultry by comparative genome indexing, pulsed-field gel electrophoresis and flaA gene sequencing. Objective: Comparative genome indexing (CGI) using whole-genome DNA microarrays was evaluated as a means of genotying Campylobacter jejuni relative to two standard methods, pulsed-field gel electrophoresis (PFGE) and flaA short variable region sequencing (flaA SVR typing). Accomplishment: The highest discrimination among the isolates was provided by CGI. The congruence of CGI for C. jejuni strains typed as identical by PFGE indicates the potential value of CGI as a more informative substitute for PFGE or flaA SVR. Impact: Campylobacter jejuni is the most common causative agent of bacterial gastroenteritis in the world. CGI is a new genome-sequence based technique for typing bacterial pathogens and has recently been applied to C. jejuni. The development of CGI as a molecular typing tool for C. jejuni offers a highly effective and informative means of further understanding the epidemiology and population genetics of this ubiquitous pathogen. (National Program Component 1.1, Problem Statement 1.1.1: Methodology)

11. Title: Antimicrobial resistance and virulence factors of Enterococcus faecalis isolated from retail food. Objective: The contribution of enterococci to food-borne illnesses via dissemination by retail food remains undefined. This study was conducted to determine prevalence and associations of antimicrobial resistance and virulence factors of Enterococcus faecalis isolated from retail food items. Accomplishment: While the highest rates of resistance were observed for lincomycin and bacitracin, low rates of resistance is less than or equal to 40 were found for chloramphenicol, ciprofloxacin, erythromycin, gentamicin, nitrofurantoin, penicillin, and tylosin. A number of virulence determinants were present in the retail food enterococci including aggregation protein, toxins, and sex pheromones which facilitate the conjugation process. Statistical associations were found between some virulence factors and antimicrobial resistance. Impact: The presence of virulence determinants in enterococci from retail food suggests that enterococci from these sources may be more pathogenic than previously believed and may pose an increased hazard to humans susceptible to enterococcal infection. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)

12. Title: Prevalence of streptogramin resistance in enterococci from animals. Objective: Previous reports suggest that streptogramin resistance among enterococci is common and streptogramin resistance can be attributed to the use of virginiamycin in animals. In this study, prevalence and mechanisms of streptogramin resistance commonly found in the staphylococci were investigated in the enterococci. Accomplishment: From 2000-2004, enterococci were isolated from poultry carcass rinsates, fruits, vegetables, retail meats, and environmental rinsates or from swine and cattle fecal samples collected on-farm. Enterococcus isolates were identified to species and then analyzed for antimicrobial susceptibility. One enterococcal isolate was positive for two streptogramin resistance genes which were previously only identified in Staphylococcus aureus. Other tests of analysis suggest that the genes may be linked. Impact: This is the first report of two streptogramin resistance genes identified in enterococci which are commonly found in staphylococci. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)

13. Title: Determining the incidence of Clostridium difficile in swine and dairy herds: Objective: Clostridium difficile causes a severe colitis which is typically thought to be nosocomial. However, there is some indication that some portion of the infections may be community acquired. Fecal and environmental samples from the National Animal Health Monitoring System (NAHMS) swine and dairy samples were analyzed to determine incidence rate and to compare the strains from these sources to those causing human illness. Accomplishment: Approximately six percent of swine fecal and 1% of dairy environmental samples were found to contain C. difficile. Further studies are being conducted to determine the relatedness of these animal associated strains to human disease strains. Impact: If animal related strains are the same or closely related to human disease strains, there are potentially very great implications to human health. If they are not related, then the concerns being raised by public health agencies will be substantially alleviated. (National Program Component 1.1, Problem statement 1.1.2: Epidemiology)


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of active CRADAs and MTAs6
Number of web sites managed4
Number of non-peer reviewed presentations and proceedings103

Review Publications
Bahnson, P.B., Cray, P.J., Ladely, S.R., Mateus-Pinella, N.E. 2006. Herd-level risk factors for salmonella enterica subsp. enterica in U.S. market pigs. Veterinary Microbiology. 76(3-4):249-262.

Rolon, A., Bailey, J.S., Hofacre, C.L., Holt, P.S., Wilson, J.L. 2007. Intestinal humoral immune response and resistance to salmonella challenge of progeny from breeders vaccinated with killed antigen. International Journal of Poultry Science. 6(6):417-423.

Bailey, J.S., Rolon, A., Hofacre, C.L., Holt, P.S., Wilson, J.L., Cosby, D.E., Richardson, L.J., Cox Jr, N.A. 2007. Humoral and mucosal-humoral immune response to a salmonella vaccination program in broiler breeders. International Journal of Poultry Science. 6(3):172-181.

Bailey, J.S., Rolon, A., Hofacre, C.L., Holt, P.S., Wilson, J.L., Cosby, D.E., Richardson, L.J., Cox Jr, N.A. 2007. Resistance to Challenge of Breeders and Their Progeny with and without Competitive Exclusion Treatment to Salmonella Vaccination Programs in Broiler Breeders. International Journal of Poultry Science. 6(6):386-392.

Barb, C.R., Barrett, J.B. 2005. Neuropeptide Y modulates growth hormone secretion but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture. Domestic Animal Endocrinology. 29:548-555.

Burtnick, M.N., Downey, J., Brett, P.J., Boylan, J.A., Frye, J.G., Hoover, T.R., Gherardini, F.C. 2007. Insights into the complex regulation of rpoS in Borrelia burgodorferi. Molecular Microbiology. 65(2):277-293.

Cox Jr, N.A., Richardson, L.J., Buhr, R.J., Cray, P.J., Bailey, J.S., Wilson, J.L., Hiett, K.L. 2006. Natural presence of campylobacter spp. in various lymphoid origins bodies of commercial broiler breeder hens . Avian Diseases. 50(3):450-453.

Foley, S.L., White, D.G., Mcdermott, P.F., Walker, R.D., Rhodes, B., Cray, P.J., Simjee, S., Zhao, S. 2006. Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources. Journal of Clinical Microbiology. 44(10):3569-3577.

Frye, J.G., Cray, P.J. 2007. Prevalence, Distribution and Characterization of Ceftiofur Resistance in Salmonella enterica Isolated from Animals in the U.S. from 1999 to 2003. International Journal of Antimicrobial Agents. 30(2):134-142.

Garland, J.E., Frye, J.G., Gray, J.T., Berrang, M.E., Harrison, M.A., Cray, P.J. Transmission of salmonella enterica serotype typhimurium in poultry with and without antimicrobial selective pressure. Journal of Applied Microbiology. 101(6):1301-1308

Jackson, C.R., Cray, P.J., Barrett, J.B., Hiott, L.M., Woodley, T.A. 2007. Prevalence of streptogramin resistance in enterococci from animals: Identification of vatD from animal sources in the United States. International Journal of Antimicrobial Agents. 30(1):60-66.

Jackson, C.R., Cray, P.J., Wineland, N., Tankson, J.D., Barrett, J.B., Douris, A., Pearson Gresham, C., Hall, M.C., Mcglinchey, B., Price, M.V. 2007. Introduction to usda vetnet: status of salmonella and campylobacter databases from 2004 through 2005. Foodborne Pathogens and Disease. 4(2):241-248.

Kim, S., Frye, J.G., Hu, J., Cray, P.J., Gautom, R., Boyle, D.S. 2006. Multiplex pcr based method for identification of common clinical serotypes of salmonella enterica subsp. enterica. Journal of Clinical Microbiology. 44(10)3608-3615.

Losinger, W.C., Traub-Dargatz, J.L., Garber, L.P., Cray, P.J., Ladely, S.R., Ferris, K.E., Morgan, K. 2002. Factors associated with fecal shedding of salmonella spp by horses on us operations. Brazalian Archive of Medicine Veterinaria and Zootecnia.54(2):109-116.

Mcgowan, L., Cray, P.J., Jackson, C.R., Barrett, J.B., Hiott, L.M. 2006. Prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats. Journal of Food Protection. 69(12):2976-2982.

Smith, D., Altekruse, S.F., Bailey, J.S. 2007. Assessment of post-Hurricane Katrina recovery in poultry slaughter establishments. Journal of Food Protection. 70(6):1498-1501.

Son, I., Englen, M.D., Berrang, M.E., Cray, P.J., Harrison, M.A. 2007. Antimicrobial resistance of arcobacter and campylobacter from broiler carcasses. International Journal of Antimicrobial Agents. 29(4):451-455.

Son, I., Englen, M.D., Berrang, M.E., Cray, P.J., Harrison, M.A. 2007. Prevalence of arcobacter and campylobacter on broiler carcasses during processing. International Journal of Food Microbiology. 113(1):16-22.

White, P.L., Naugle, A.L., Jackson, C.R., Cray, P.J., Rose, B.E., Pritchard, K.M., Levine, P., Saini, P.K., Schroeder, C.M., Dreyfuss, M.S. 2007. Salmonella Enteritidis in Meat, Poultry, and Pasteurized Egg Products Regulated by the U.S. Food Safety and Inspection Service, 1998 through 2003. Journal of Food Protection. 70(3):582-591.

Zhao, S., Cray, P.J., Friedman, S., Mcdermott, P.F., Walker, R.D., Qaiyumi, S., Foley, S.L., Hubert, S.K., Ayers, S., English, L. 2005. Characterization of salmonella typhimurium of animal orgin obtained from the national antimicrobial resistance monitoring systems (narms). Foodborne Pathogens and Disease. 2(2):169-181.

Feder, I.E., Gray, J.T., Pearce, R., Fratamico, P.M., Bush, E., Porto, A., Wallace, F.M., Cray, P.J., Luchansky, J.B. 2007. Testing of swine feces obtained through the national animal health monitoring system's swine 2000 study for the presence of escherichia coli 0157:h7. Journal of Food Protection. Vol. 70(6):1489-1492.

Dunowska, M., Morley, P.S., Traub-Dargatz, J.L., David, M.A., Patterson, G., Frye, J.G., Hyatt, D.R., Dargatz, D.A. 2007. Comparison of salmonella enterica serotype infantis isolates from a veterinary teaching hospital. Journal of Applied Microbiology. 102(6):1527-1536.

Englen, M.D., Hill, A.E., Dargatz, D.A., Ladely, S.R., Cray, P.J. 2007. Prevalence and antimicrobial resistance of campylobacter in u.s. dairy cattle. Journal of Applied Microbiology. 102(6):1570-1577.

Davies, P.R., Hurd, H.S., Funk, J.A., Cray, P.J., Jones, F.T. The role of contaminated feed in the epidemiology and control of salmonella enterica in pork production. Foodborne Pathogens and Disease. 1(4):202-215. 2004.

Miller, W.G., Englen, M.D., Kathariou, S., Wesley, I.V., Wang, G., Pittenger, L.G., Siletz, R.M., Muraoka, W.T., Cray, P.J., Mandrell, R.E. 2006. Identification of host-associated alleles by multilocus sequence typing of campylobacter coli strains from food animals. Microbiology. 152(1):245-255.

Cox Jr, N.A., Richardson, L.J., Buhr, R.J., Northcutt, J.K., Cray, P.J., Bailey, J.S., Fairchild, B.D., Mauldin, J.M. 2006. Natural occurrence of campylobacter spp., salmonella serovars and other bacteria in unabsorbed yolks of market age commercial broilers. Journal of Applied Poultry Research. 15(4):551-557.

Musgrove, M.T., Jones, D.R., Northcutt, J.K., Cox Jr, N.A., Harrison, M.A., Cray, P.J., Ladely, S.R. 2006. Antimicrobial resistance in salmonella and escherichia coli isolated from commercial shell eggs. Poultry Science. 85:1665-1669.

Morley, P.S., Apley, M.D., Besser, T.E., Burney, D.P., Cray, P.J., Papich, M.G., Traub-Dargatz, J.L., Weese, J. 2005. Antimicrobial drug use in veterinary medicine. Journal of Veterinary Internal Medicine. V. 19. P. 617-629

Last Modified: 10/25/2014
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