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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
2011 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 (Mark Englen), Salmonella (Jonathan Frye) and commensal bacteria (E. coli and enterococci; Charlene Jackson). Critical to the molecular research will be epidemiologic studies provided by the CAHFSE program (Joseph Bailey ) and ecologic (field and environment) studies (Paula Fedorka-Cray) 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 (Englen, Frye, and Jackson). Pathogenic studies (Fedorka-Cray) 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
Genes conferring resistance to antimicrobials, vehicles carrying those resistance genes, and the genetic relationship between the studied bacterial isolates were determined. Polymerase Chain Reaction and microarrays were used to detect antimicrobial resistance genes and plasmids on which the genes are harbored. The DNA microarray, developed by an ARS scientist, was expanded to include a total of 1267 probes. It contains 775 probes to detect antimicrobial resistance (AR) genes and 487 probes to 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 detect AR genes and plasmid replicon types in multi-drug resistant (MDR) E. coli and MDR S. Typhimurium. Significant associations, determined using linkage disequilibrium, were found between replicon type and AR phenotype in MDR E. coli. Coli E1 plasmids, encoding kanamycin resistance, were widespread in Salmonella and were also linked to MDR. The DNA microarray was also utilized to study AR in S. Javiana, Acinetobacter, and MDR E. coli from companion animals. Research on MDR E. coli from companion animals is on-going and will include analysis of plasmids. MDR plasmids from bacteria of interest are being sequenced using high-throughput sequencing to determine if they are the primary source of AR. This research will identify plasmid types that are present in AR bacteria, resistance genes that they harbor, and possibly how the plasmids are being disseminated among the resistant isolates.

In the first report of food-borne bacteria from the Collaboration on Animal Health, Food Safety, and Epidemiology (CAHFSE) program, AR genes from Salmonella, generic E. coli, Campylobacter, and Enterococcus co-isolated from the same swine fecal sample were identified using the DNA microarray. Common resistance genes were found in Salmonella and E. coli indicating that these bacteria either share a common source for resistance genes or horizontal exchange of AR genes between these bacteria has occurred.

A study on dairy cattle conducted in conjunction with the National Animal Health Monitoring System (NAHMS) found that resistant enterococci were prevalent in dairy cattle fecal samples and that enterococci may be a reservoir of resistance for other bacteria.

The National Antimicrobial Resistance Monitoring System (NARMS) completed the 2009 Executive Report which summarized AR data from Salmonella, Campylobacter, and E. coli recovered in 2009 from food animals, retail meats, and human clinical cases. The animal arm of NARMS determined antimicrobial susceptibility patterns for over 1,300 Salmonella, 550 Campylobacter, 2,100 generic E. coli and 1,800 Enterococcus isolates. These cultures originated from the following studies: Food Safety and Inspection Service-Hazard Analysis and Critical Control Points System (FSIS-HACCP), FSIS-Baseline, NAHMS Sheep, Regional Dairy Quality Management Alliance (RDQMA) and On-Farm Poultry. Of those, all Salmonella and 475 Campylobacter were submitted to VetNet in FY11.


4.Accomplishments
1. Multi-drug resistance in Escherichia coli. Characterization of bacterial plasmids (similar to small chromosomes) is necessary because genes encoding important traits such as antimicrobial resistance are frequently carried on plasmids. ARS scientists at Athens, Georgia, analyzed the plasmid types, antimicrobial phenotypes, and genetic relationships of multi-drug resistant (MDR) Escherichia coli. A total of 15 different plasmid types were detected, all isolates carried multiple plasmid types and extensive genotypic diversity was observed. This study demonstrates that E. coli from animal sources are highly variable genotypically and are reservoirs of a diverse array of plasmids carrying antimicrobial resistance. This work will significantly aid in the design of monitoring strategies by Federal agencies and researchers on the spread of MDR plasmids and determine their threat to human and animal health.

2. Similar resistance genes in different bacteria isolated from swine. Different bacterial species can exchange resistance genes in laboratory animal experiments. To determine if this happens in the environment, Salmonella, E. coli, Campylobacter and Enterococcus bacteria were isolated from swine feces collected on-farm from 2003 to 2006 by the Collaboration on Animal Health and Food Safety Epidemiology (CAHFSE). DNA microarrays developed by ARS scientists at Athens, Georgia, were used to detect hundreds of antimicrobial resistance genes in the isolates. Statistical analysis determined that Salmonella and E. coli isolated from the same fecal sample had identical genes detected at a significant level. This indicated that Salmonella and E. coli may have a common source for acquiring antimicrobial resistance genes or may naturally exchange resistance genes in the swine environment. The data suggests that antimicrobial resistance elements in E. coli may serve as a reservoir for resistance in important pathogens such as Salmonella. This finding is important for agencies such as the Food Safety and Inspection Service (FSIS) who monitor food for Salmonella contamination and the Food Drug and Administration who regulate the use of antimicrobials on-farm. Further studies are under way to determine if this exchange is also found in poultry and other animals and to determine if antimicrobial use on-farm has an impact on this phenomenon.

3. Antimicrobial resistance in Salmonella Typhimurium isolated from animals. Antimicrobial resistance (AR) in bacteria may prevent successful treatment of foodborne infections, especially if the bacteria are multi-drug resistance (MDR). The serotype with the most MDR isolates collected from animals by the National Antimicrobial Resistance Monitoring System (NARMS) is Salmonella serotype Typhimurium. To determine the genetics responsible for MDR, representative animal isolates from healthy cattle, poultry, and swine resistant to at least five antimicrobials were selected for study from each year of NARMS from 1997-2007. Microarray analysis of these isolates detected MDR genes as well as plasmids that can move resistance between bacteria. Isolates were evenly divided into two groups by the MDR and plasmid genes detected. Members of one group were definitive phage type DT104 with AR genes found in an integron and was isolated mostly from swine. The other group had MDR plasmid genes detected from a distinctive plasmid group and was isolated mostly from cattle. These results indicated that MDR in Salmonella Typhimurium is caused by two different genetic mechanisms each of which is associated with a different animal source. Further studies may enable attribution of particular isolates from human infections to an animal source based upon on its MDR genetics, a major goal for regulatory agencies such as the Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDC) and the Food Safety and Inspection Service (FSIS).

4. Microarray detection of antimicrobial resistant plasmids in bacterial pathogens. Analysis of antimicrobial resistance (AR) and plasmid genes in bacterial pathogens is a critical component to understanding the development of AR and AR infections in humans. ARS scientists at Athens, Georgia, developed microarrays for the detection of multi-drug resistant (MDR) plasmids in bacteria. The DNA microarrays can detect over1200 antimicrobial resistance and plasmid genes commonly found in these bacteria. This technique allows the rapid identification of genetic elements responsible for widespread MDR. In addition, the technology has been transferred to other scientists to study important bacterial pathogens. These include Walter Reed Army Institute for Research investigation of MDR Acinetobacter isolates from wounded soldiers returning from Iraq; and the Food and Drug Administration’s (FDA) study of MDR Salmonella serotype Javiana isolated from animals and humans. These studies will allow the spread of MDR plasmids in Salmonella to be monitored and determination of their threat to human and animal health.

5. ColE1 plasmids in Salmonella enterica serovars. Antimicrobial resistance (AR) may prevent successful treatment of bacterial infections. Many Salmonella carry ColE1 plasmids encoding kanamycin resistance (KANR), however little is know about its genetics or its relationship to multi-drug resistance (MDR). A set of 102 KANR Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR for ColE1 plasmids. ColE1 positive isolates were further studied by restriction mapping and sequencing. All seven ColE1 positive Salmonella animal isolates were serovar Typhimurium, resistant to five additional antimicrobials (MDR), and were also phage type DT104. Sequence analysis revealed genotypes of ColE1 plasmids differing by the number of insertion sequences (IS903). The finding that these plasmids are widespread and linked to MDR requires close monitoring of its prevalence and transmission among bacteria isolated from food animals and humans. These studies provide an understanding of the genetics causing MDR in Salmonella which is a major concern for the Food and Drug Administration (FDA), USDA Food Safety and Inspection Service (FSIS), and the Centers for Disease Control and Prevention (CDC).

6. Plasmid-mediated quinolone resistance among non-Typhi Salmonella. Over the last two decades, the number of non-Typhi Salmonella (NTS) exhibiting plasmid-mediated decreased susceptibility to ciprofloxacin, a type of fluoroquinolone, has increased. Because patients have experienced treatment failure from infections caused by these particular strains, further studies were performed to determine their presence in human, food animal, and retail meat samples. In collaboration with scientists from the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA), over 4,000 NTS isolates from the National Antimicrobial Resistance Monitoring System were screened for decreased susceptibility to fluoroquinolones. Six isolates, all from human samples, were identified that harbored the plasmid-mediated genes responsible for the decreased susceptibility to fluoroquinolones. This suggests meat and food animals are not significant sources of this particular NTS phenotype. Continued monitoring is warranted, however, because ciprofloxacin is used in the treatment of invasive Salmonella infections in adults. This information is particularly useful for Federal agencies such as the CDC and the FDA as well as physicians as they continue to monitor Salmonella infections in humans.

7. Minimum Inhibitory Concentrations of azithromycin against Salmonella enterica. Due to increasing resistance to traditional antimicrobial agents, the use of azithromycin for treating invasive Salmonella infections is rising. However, as this use is fairly new, no clinical azithromycin breakpoints have been established by either U.S. or European authorities. One component needed in defining resistance breakpoints is the distribution of minimum inhibitory concentrations (MICs) among the specific bacteria. In this study, almost 700 non-Typhi Salmonella isolates from food animals, humans, and retail meats were tested for antimicrobial susceptibility to azithromycin; 72 Salmonella Typhi isolates from humans were also tested. The MICs ranged from 1-32 µg/mL with the highest MICs of 32 µg/mL originating from human isolates. As further clinical data will be needed before a specific azithromycin breakpoint can be established, an epidemiological cut-off value of = 16 µg/mL is proposed.

8. Optimized isolation and prevalence of Clostridium difficile from healthy food animals. Clostridium difficile is an anaerobic, spore-forming bacterium that can cause a wide range of disease in humans. Although generally thought to be hospital-acquired, community-acquired infections have been increasingly reported along with bacterial detection in retail meats. Further research is needed to determine if there is a potential for these contaminated meats to be a potential source of foodborne illness. ARS researchers in Athens, Georgia, compared two methods for isolating C. difficile from food animal feces: the single alcohol shock (SS) and double alcohol shock (DS). A little over 250 fecal samples from swine, dairy cattle, and beef cattle were processed using both methods. The DS method generated significantly more positive samples from the swine feces while the SS method was significantly better for recovery from beef cattle feces. No significant difference was observed between the methods for isolation from dairy cattle feces. The highest prevalence of C. difficile was seen in the swine samples, followed by beef cattle and dairy cattle, respectively. This suggests food animals harbor C. difficile and that isolation methods may not have universal application across animal types. This information will be particularly important for physicians and researchers as they monitor the spread of C. difficile to determine the threat to human health.

9. Antimicrobial resistant Escherichia coli from companion animals. The contribution of dogs and cats as reservoirs of antimicrobial resistant Escherichia coli remains largely undefined. This is increasingly important considering the possibility of transmission of bacteria from companion animals to the human host. ARS scientists in Athens, Georgia, screened dogs and cats from veterinary clinics for the presence of E. coli. E. coli was isolated from swabs of nasal, oral, rectal, abdomen, and hindquarter areas and although rectal swabs yielded the most E. coli from both dogs and cats, the organism was distributed evenly among the other body sites sampled. E. coli isolates from both dogs and cats were resistant to all antimicrobials tested except three. Among the resistant isolates, 21 resistance patterns were observed, where 18 patterns represented multidrug resistance (MDR). This study is useful for veterinarians and pet owners demonstrating that dogs and cats are a common source of antimicrobial resistant E. coli which may be transferred to humans from various sites on the animal’s body.

10. Antimicrobial resistant enterococci from U.S. dairy cattle. The potential for transfer of antimicrobial resistant bacteria into the human population is cause for concern. The enterococci are a ubiquitous group of bacteria which may serve as reservoirs of antimicrobial resistance genes. ARS scientists in Athens, Georgia have collaborated with the Animal and Plant Health Inspection Service (APHIS) to study prevalence and antimicrobial resistance of enterococci in fecal samples from U.S. dairy operations participating in the 2007 National Animal Health Monitoring System (NAHMS) survey of dairy cattle health and management practices. Results from this study indicated that fecal samples from U.S. dairy cattle contain high numbers of antimicrobial resistant enterococci and may act as a reservoir of antimicrobial resistant enterococci that can be transferred to the human host via the food chain. This research will be useful to Federal agencies and researchers studying the role that food animals have in the dissemination and persistence of antimicrobial resistance in humans which may impact human health.


Review Publications
Karlsson, M.S., Howie, R.L., Rickert, R., Krueger, A., Tran, T., Zhao, S., Ball, T.A., Haro, J.H., Pecic, G., Joyce, K., Cray, P.J., Whichard, J., Mcdermott, P. 2010. Plasmid-mediated quinolone resistance among non-typhi Salmonella enterica isolates, USA. Emerging Infectious Diseases. 16(11):1789-1791.

Thitaram, S.N., Frank, J.F., Lyon, S.A., Siragusa, G.R., Bailey, J.S., Lombard, J.E., Haley, C.A., Wagner, B.A., Dargatz, D.A., Cray, P.J. 2011. Clostridium difficile from healthy food animals: Optimized isolation and prevalence. Journal of Food Protection. 74(1):130-133.

Lindsey, R.L., Frye, J.G., Thitaram, S.N., Meinersmann, R.J., Cray, P.J., Englen, M.D. 2011. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis. Microbial Drug Resistance. 17(2):157-163.

Jackson, C.R., Lombard, J.E., Dargatz, D.A., Cray, P.J. 2011. Prevalence, species distribution and antimicrobial resistance of enterococci isolated from U.S. dairy cattle. Letters in Applied Microbiology. 52(1):41-48.

Huang, X., Frye, J.G., Chahine, M.A., Cash, D.M., Barbe, M.G., Babel, B.S., Kasper, M.R., Whitman, T.J., Lindler, L.E., Bowden, R.A. 2010. Genotypic and Phenotypic Correlations of Multidrug-Resistant Acinetobacter baumannii-A. calcoaceticus Complex Strains Isolated from Patients at the National Naval Medical Center. Journal of Clinical Microbiology. 48(11):4333-4336.

Frye, J.G., Lindsey, R.L., Meinersmann, R.J., Berrang, M.E., Jackson, C.R., Englen, M.D., Turpin, J.B., Cray, P.J. 2011. Related antimicrobial resistance genes detected in different bacterial species co-isolated from swine fecal samples. Foodborne Pathogens and Disease. 8(6):663-679.

Davis, J.A., Jackson, C.R., Cray, P.J., Barrett, J.B., Brousse,Jr, J.H., Gutafson, J., Kucher, M. 2011. Anatomical Distribution and Genetic Relatedness of Antimicrobial Resistant E. coli from Healthy Companion Animals. Journal of Applied Microbiology. 110(2):597-604.

Chen, C., Lindsey, R.L., Strobaugh Jr, T.P., Meinersmann, R.J., Frye, J.G. 2010. Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serotypes. Applied and Environmental Microbiology. doi:10.1186/1471-2180-10-207.

Chen, C., Strobaugh Jr, T.P., Lindsey, R.L., Frye, J.G., Uhlich, G.A. 2011. Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars. Plasmid Journal. 65:246-252.

Last Modified: 12/25/2014
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