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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 & Antimicrobial Resistance Research

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.


4. Accomplishments


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.

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.

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.

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.

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(3):732-739.

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.

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.

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.

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.

Last Modified: 10/17/2017
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