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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

2013 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:
During the year, high-throughput sequencing was employed for whole-genome DNA sequencing of over 200 Salmonella enterica of various serotypes. DNA sequences from all isolates are being analyzed to identify gene differences between isolates from food animals, humans, and retail meat. Identification of genetic diversity among the Salmonella isolates is necessary to 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 PCR assay for Salmonella for poultry source attribution. Studies of genetic differences among Campylobacter were not performed as the scientist leading Campylobacter research retired during the fiscal year. We have also begun to define genetic differences among Enterococcus cecorum associated with outbreaks of enterococcal spondylitis among broilers which causes hind limb paralysis. This is of interest to the poultry industry as outbreaks can result in mortality ranging from 10-15%. High-throughput sequencing was used to sequence plasmids from multi-drug resistant 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 antimicrobial resistance genes contained on the plasmids and genes which may be responsible for mobility of resistance genes, segments of plasmids or entire plasmids. To determine the role of plasmids in dissemination of antimicrobial resistance, replicon typing of plasmids from multi-drug resistant enterococci was initiated. The plasmid replicon typing multiplex PCR detects 19 plasmid replicon families found in enterococci. These studies relate to Objective 2 of the project plan. Also relating to Objective 2, prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) was evaluated. Although MRSA is primarily a human health issue, food animals are an important source of infection and considered a food safety issue in Europe and some parts of North America. We isolated and characterized MRSA from swine herds on-farm, at lairage, on carcass swabs and retail pork and beef. In other studies, MRSA was present in marine environments including recreational water and marine mammals. Together these studies showed that MRSA is present in many sources all which may be important for dissemination and persistence of MRSA. Evaluation of resistance to biocides of food borne bacteria was initiated. Biocides commonly used in poultry production were selected and dilutions of each biocide were initially tested against a common laboratory strain of Salmonella. After appropriate ranges of dilutions were determined, twenty additional multi-drug resistant Salmonella isolates from food animals were selected and are presently being tested on the panel. After this phase, isolates of E. coli and Enterococcus will also be selected and tested using the panel. Expected results include development of a biocide susceptibility panel that will be used to test biocide resistance of food borne bacteria. This research addresses Objective 3 of the project plan.

1. Antimicrobial resistance genes in multi-drug resistant Salmonella enterica from animals, retail meats, and humans in the U.S. and Canada. The major foodborne pathogen, Salmonella enterica can be resistant to multiple antimicrobials (multi-drug resistant, MDR) and can cause human infections that would be difficult to treat. To determine the risk of MDR infections of humans, a better understanding of the genetics of MDR in Salmonella isolated from animals, food, and humans is necessary. MDR S. enterica strains frequently found in animals, retail meat, and human infections in the U.S. (ARS, CDC, and FDA) and Canada (Health Canada) (n=56) were analyzed by ARS researchers in Athens, GA using a microarray developed at ARS. This microarray detects hundreds of MDR genes in a single assay. Genes encoding resistance to multiple classes of antimicrobials including streptomycin, penicillins, chloramphenicol, sulfamethoxazole, tetracycline, and trimethoprim were detected. Similar resistance genes were detected regardless of serovar, source, or location. The presence of similar resistance genes suggests that MDR Salmonella may have collected specific resistance genes which may indicate multiple origins of MDR in Salmonella from food animals, retail meat, and human infections. The Editor of Microbial Drug Resistance selected the paper reporting this data as a highlighted article due to the number of requests for the paper and the impact of the data on the field of research. This research provides critical information to both federal and state public health agencies for combatting antimicrobial resistance in humans and animals.

2. The intestinal fatty acid propionate inhibits Salmonella invasion of host cells. Infection with Salmonella can cause gastroenteritis which can be severe in young, old, or immune compromised people. The infection begins when Salmonella invades the cells lining the intestinal wall. To do this, Salmonella makes invasion proteins which are produced by the expression of genes located in Salmonella Pathogenicity Island 1 (SPI1). SPI1 is a cluster of genes grouped together in the bacterium’s chromosome and are required to cause disease. The short chain fatty acid propionate can be abundant in the intestine of animals and has been shown to stop the production of invasion proteins by reducing SPI1 gene expression and synthesis of the invasion proteins. In this study, propionate was the only short chain fatty acid that stopped invasion; therefore it could be used to control infection. This control required a specific gene, hilD, located in SPI1. It was also determined that blocking the conversion of propionate to the high energy metabolic intermediate compound propyionyl-CoA was responsible for the effects. How Salmonella bacteria cause disease is important for understanding human and animal infections and is critical for scientists as they develop new ways to block infection.

3. Pathogenicity of Dodecyltrimethylammonium chloride (DTAC) resistant Salmonella. Salmonella infection causes a self-limiting gastroenteritis in humans. The prevalence of antimicrobial and multidrug-resistant Salmonella has increased worldwide since the 1980’s. However, the effect of antimicrobial resistance on the development of illness after ingesting Salmonella strains is not well-described. Differences in gene expression between a parental strain capable of causing disease and four modified Salmonella strain were screened. The modified Salmonella strains was the same parental strain exposed to the antimicrobial sanitizer dodecyltrimethylammonium chloride (DTAC), until the strains were less susceptible to DTAC [Reduced Susceptibility Strains (SRS)]. DATC is commonly found in soaps, shampoos, and sanitizers. Scientists at ARS in Athens, GA analyzed gene expression using a microarray technique. Two bacterial genes which showed differences in expression were fimbriae (hair like structures on the bacteria’s surface) responsible for adhesion to host cells or associated with host cell invasion; a third gene with differences in expression was responsible for a protein which is important for bacterial growth inside host cells. Transmission electron microscopy determined that the fimbriae were absent in the four SRS strains, but abundant in the virulent parental strain. All four SRS strains had a significantly reduced ability to invade host cells in tissue culture as compared to the parental strains. These studies demonstrated that the development of resistance to antimicrobial sanitizer in these strains also resulted in reduced ability to grow in host cells. Therefore, the use of these sanitizers may result in decreased likelihood of Salmonella causing severe illness. These observations should be considered when developing antimicrobial treatments for sanitizing work areas (such as processing facilities) contaminated with Salmonella.

4. Prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) of porcine origin. Methicillin-resistant Staphylococcus aureus (MRSA) primarily causes human disease and, until recently, food animals had not been considered as important sources of infection. Among food animals, pigs have been implicated as one source of potential infections to humans including farmers, slaughterhouse workers, and veterinarians who are in frequent contact with MRSA colonized pigs. Livestock-associated MRSA (LA-MRSA) has been reported among pigs, pig farmers, and the environment in a number of European countries and North America. In collaboration with Ohio State University, ARS researchers in Athens, GA determined the occurrence and persistence of MRSA among swine herds on-farm, at lairage, on carcass swabs and retail pork. Pork samples from the same groups of swine were collected at retail market. The overall prevalence of MRSA in pigs was 3% on-farm and 11% at holding pens at slaughterhouses. MRSA was detected in 2% of the carcass swabs and 4% of the retail pork samples. LA-MRSA was detected in all farm to retail samples, and 18% of the MRSA were identified as LA-MRSA. Contamination of retail meat with LA-MRSA and other strains potentially impacts public health as retail meat could be an important source in MRSA dissemination.

5. Prevalence of Methicillin-resistant Staphylococcus aureus from retail meat and humans in Georgia. Staphylococcus aureus is a Gram-positive bacterium that can be commonly found on the skin or in the nasal passages of most humans and animals. It has been implicated in a number of diseases in humans ranging from minor skin infections to more serious infections such as pneumonia. Additionally, staphylococcal food poisoning, characterized by vomiting and diarrhea, is a leading cause of foodborne illness in the U.S. Food sources of S. aureus have recently expanded to include retail meat products from food-producing animals including swine, poultry, and cattle; there is increasing interest in the presence of S. aureus, specifically methicillin-resistant S. aureus (MRSA), in retail meat products. In this study, ARS researchers from Athens, GA isolated staphylococci from retail pork and retail beef in Georgia and MRSA from the products were compared to human MRSA from the same geographic area. MRSA were recovered from 3% of retail pork, 4% of retail beef, and 50% of human samples and some MRSA were multidrug resistant. Using molecular analysis, three retail beef MRSA were identical to MRSA known to cause human infections. This study serves as an indication that staphylococci and MRSA are present on retail meat. This information is especially useful for consumers and personnel who handle raw meat as safe handling methods should be followed to avoid being exposed to MRSA.

6. Methicillin-resistant Staphylococcus aureus clones from whales, humans, and the environment. Staphylococcus aureus is a major cause of nosocomial infections in the United States. Studies have shown that humans shed S. aureus onto surfaces in their environment (including water) and that this is a source of spread to other individuals and in animals including marine mammals. In 2011, a live mass stranding of 26 short-finned pilot whales occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria recovered from the whales during the routine clinical care and necropsy of a deceased whale included Methicillin-Sensitive S. aureus (MSSA) and MRSA. Samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal to investigate potential sources or reservoirs of MSSA and MRSA. MRSA were clonally related from multiple environmental locations as well as from humans and animals. Genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. These studies serve as an indication that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.

7. Human-associated methicillin-resistant Staphylococcus aureus from a subtropical recreational marine beach. Staphylococcus aureus is an opportunistic pathogen that can also be commonly found on the skin and in nasal passages. Reports of S. aureus detected in marine environments have occurred since the early 1990’s. In this study, S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, were characterized in order to investigate possible sources and to identify the risks to bathers of exposure to these organisms. Over a period of 17 months, 1001 water and 36 intertidal sand samples were collected by either bathers or investigators at a subtropical recreational beach. Methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were isolated, identified, and characterized using molecular methods. Both MSSA and MRSA were isolated from the sources and some MRSA were genetically related to the community-associated MRSA (CA-MRSA). There was a significant correlation between the daily average number of bathers and all S. aureus in the water. These results indicate that CA-MRSA found in waters and sand at a subtropical recreational beach were clonally related or highly similar, and the presence of S. aureus in these waters correlated with the daily average number of bathers in the water. This report directly addresses humans bathing in a marine environment and S. aureus in the water and supports the concept that humans are a potential direct source for S. aureus in marine waters. These results are important for evaluating sources of environmental contamination and in designing strategies to reduce microbial contamination of the environment.

8. Antimicrobial resistant Enterococcus from poultry and environmental samples. Fraser Valley in the province of British Columbia is considered the poultry capital of Canada. The poultry waste generated from the industry is used as fertilizer and spread onto the fields thus creating scattered sources of surface and groundwater pollution. ARS scientists in Athens, GA have collaborated with Environment Canada to investigate antimicrobial resistance of Enterococcus from different environmental compartments including litter from two farms, 12 surface and 28 groundwater sites in an area of intensive poultry production and litter application. Overall, 14% of litter isolates, 42% of surface water isolates and 0% of groundwater isolates were resistant to one antibiotic. Statistical analyses was applied to the resistance data set for source tracking and indicated that poultry was a possible source of the surface water contamination observed. Data presented in this study will assist both Canadian and U.S. poultry producers in assessing the relative contribution of poultry waste to contamination of water sites with antimicrobial resistant bacteria and in designing strategies to reduce microbial contamination of the environment.

9. Genetic relatedness of enterococci poultry and the environment. The potential for contamination of surface and groundwater due to poultry waste used as fertilizer on fields was investigated. ARS scientists in Athens, Georgia have collaborated with Environment Canada to assess the genetic relationship of enterococci from surface and groundwater to enterococci isolated from poultry sources in Fraser Valley, a province of British Columbia, Canada. Using two molecular analysis methods, enterococci from layer litter and surface and groundwater were compared. Enterococci were isolated from all three sources, but overall grouping was independent of source by both molecular methods. Although enterococci from litter and water sources were grouped together using both methods, water isolates could not be definitively identified as originating from poultry litter. These results suggested that although poultry waste was used as fertilizer, a variety of hosts may be contributing to fecal contamination especially in aquatic environments. Researchers can use this information for source tracking environmental contamination from poultry and other potential sources of contamination and in designing strategies to reduce microbial contamination of the environment.

Review Publications
Hung, C., Garner, C.D., Slauch, J.M., Dwyer, Z.W., Lawhon, S.D., Frye, J.G., Ahmer, B.M., Altier, C. 2013. The intestinal fatty acid propionate inhibits Salmonella invasion through the post-translational control of HilD. Molecular Microbiology. 87(5):1045-1060.

Kautz, M.J., Dvorzhinskiy, A., Frye, J.G., Stevenson, N., Herson, D.S. 2013. Pathogenicity of Dodecyltrimethylammonium Chloride-Resistant Salmonella enterica. Applied and Environmental Microbiology. 79(7):2371-2376.

Jackson, C.R., Furtula, V., Farrell, E.G., Barrett, J.B., Hiott, L.M., Chambers, P. 2012. A comparison of BOX-PCR and pulsed-field gel electrophoresis to determine genetic relatedness of enterococci from different environments. Microbial Ecology. 64(2):378-387.

Frye, J.G. 2012. Application of microarray technology for microbial source tracking. In: Foley, S.L., Nayak, R., Johnson, T.J., Shukla, S.K., editors. Molecular Typing Methods for Tracking Foodborne Microorganisms. New York, NY: Nova Science Publishers. p. 319-340.

Guard, J.Y., Sanchez-Ingunza, R., Morales, C., Stewart, T.E., Liljebjelke, K., Van Kessel, J.S., Ingram, K.D., Jones, D.R., Jackson, C.R., Cray, P.J., Frye, J.G., Gast, R.K., Hinton Jr, A. 2012. Comparison of dkgB-linked Intergenic Sequence Ribotyping to DNA Microarray Hydridization for Assigning Sterotype to Salmonella enterica. FEMS Microbiology Letters. 337(1):61-72.

Plano, L.R., Shibata, T., Garza, A.C., Kish, J., Fleisher, J., Sinigalliano, C.D., Gidley, M.L., Withum, K., Elmir, S.M., Hower, S., Jackson, C.R., Barrett, J.B., Cleary, T., Davidson, M., Davis, J., Mukherjee, S., Fleming, L.E., Solo-Gabriele, H.M. 2013. Human-associated methicillin-resistant Staphylococcus aureus from a subtropical recreational marine beach. Microbial Ecology. 65(4):1039-1051.

Frye, J.G., Jackson, C.R. 2013. Genetic Mechanisms of antimicrobial resistance identified in Salmonella enterica, Escherichia coli, and Enteroccocus spp. isolated from U.S. food animals. Frontiers in Microbiology. 4(135):1-22.

Furtula, V., Jackson, C.R., Farrell, E.G., Barrett, J.B., Hiott, L.M., Chambers, P. 2013. Antimicrobial resistance in Enterococcus spp. isolated from environmental samples in the area of intensive poultry production. International Journal of Environmental Research and Public Health. 10(3):1020-1036.

Jackson, C.R., Davis, J.A., Barrett, J.B. 2013. Prevalence and characterization of Methicillin-resistant Staphylococcus aureus isolats from retail meat and humans in Georgia. Journal of Clinical Microbiology. 51(4):1199-1207.

Hower, S., Phillips, M.C., Brodsky, M., Dameron, A., Tamargo, M.A., Salazar, N.C., Jackson, C.R., Barrett, J.B., Davidson, M., Davis, J., Mukherjee, S., Ewing, R.Y., Gidley, M.L., Sinigalliano, C.D., Johns, L., Johnson Iii, F.E., Adebanjo, O., Plano, L.R. 2013. Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility. Microbial Ecology. 65(4):1024-1038.

Glenn, L.M., Lindsey, R.L., Folster, J.P., Pecic, G., Boerlin, P., Gilmour, M.W., Harbottle, H., Zhoa, S., Mcdermott, P., Cray, P.J., Frye, J.G. 2013. Antimicrobial resistance genes in multidrug-resistant Salmonella enterica isolated from animals, retail meats, and humans in the United States and Canada. Microbial Drug Resistance. 19(3):175-184.

Englen, M.D., Berrang, M.E., Meinersmann, R.J., Cray, P.J. 2010. Evaluation of two commercial real-time PCR assays for detecting Campylobacter in broiler carcass rinses. Journal of Food Safety. 30(2010):732-739.

Last Modified: 4/16/2014
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