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United States Department of Agriculture

Agricultural Research Service

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2012 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:
This Project Plan was completed November 3, 2011, and a new Project Plan was approved and implemented beginning September 13, 2011. Over the life of the project (2006-2011), the animal arm of the National Antimicrobial Resistance Monitoring System–Enteric Bacteria (NARMS) determined antimicrobial susceptibility patterns for over 37,000 isolates of Salmonella, Campylobacter, generic Escherichia coli and Enterococcus. Antimicrobial resistance (AR) patterns from the animal arm of NARMS were complemented by molecular subtyping using Pulsed-Field Gel Electrophoresis (PFGE) as part of USDA VetNet; Salmonella and Campylobacter from NARMS were submitted to VetNet. Molecular characterization included DNA microarrays developed by ARS scientists for AR, virulence gene detection, and plasmid detection. The DNA microarray contains a total of 1267 probes; 775 probes detect AR genes and 487 probes detect two different plasmid replicon types. The microarray was used to elucidate AR mechanisms for Salmonella, E. coli, Campylobacter, and Enterococcus from poultry, swine, and cattle; AR mechanisms and virulence of enterococci from retail food items were also determined. A rapid high-throughput multiplex polymerase chain reaction (PCR) for identification of the top 50 Salmonella serotypes was also developed. Utilizing bacterial isolates from the Collaboration on Animal Health and Food Safety Epidemiology program, potential sources of antimicrobial resistance was determined. AR genes from Salmonella, 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 AR genes or horizontal exchange of AR genes between these bacteria has occurred. The genetic source of multi-drug resistance (MDR) was investigated using replicon multiplex (PCR) and the plasmid microarray. The replicon typing multiplex PCR detects 18 plasmid replicon types found in the Enterobacteriaceae and were used to detect AR genes and plasmid replicon types in 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. E. coli isolates were genetically diverse by plasmid type, antimicrobial resistance, and genetic typing using PFGE. The DNA microarray was also utilized to study AR in S. Javiana, Acinetobacter, and MDR E. coli from companion animals. MDR E. coli isolates from companion animals were resistant to up to ten antimicrobials while enterococci isolates from companion animals were resistant to up to eight antimicrobials. Together, this research identified mechanisms of resistance, plasmid types present in AR bacteria, and studied how the plasmids may be disseminated among the resistant isolates.

4. Accomplishments
1. Identification of metabolic pathways that allow Salmonella to be a successful pathogen. Salmonella enterica serovar Typhimurium is a food borne zoonotic pathogen that causes gastroenteritis in humans. To establish an infection, Salmonella must compete successfully with the host microflora in the intestine during colonization. Salmonella is unique in that it can utilize molecular hydrogen, an abundant molecule in the host intestine, as an energy source for growth. However, very little is known about how Salmonella uses hydrogen. In collaborative studies, an ARS scientist from Athens, GA used microarray analysis to identify gene expression changes during exposure to hydrogen gas in Salmonella. Genes up-regulated by hydrogen included those that encoded proteins involved in the transport of amino acids and sugars. Genes involved in carbon conservation were also up-regulated and strains with deletion of two specific genes showed reduced hydrogen-dependent growth compared to the wild type. Hydrogen stimulates the expression of genes involved in nutrient and carbon acquisition and in carbon-conserving pathways, linking carbon and energy metabolism to sustain hydrogen-dependent growth. Salmonella strains unable to use hydrogen in this manner have significantly reduced virulence and survivability in mouse infection assays. Because hydrogen metabolism enables Salmonella to compete with the host flora and establish an infection, this pathway could be a target of drug or probiotic development to prevent infections with Salmonella. This research is of interest to industry involved in drug and food development to combat diseases caused by food borne pathogens.

2. Gene expression in Salmonella in response to acidic conditions in the host digestive tract. In the United States, Salmonella enterica serovar Kentucky is the most common type of Salmonella found in chickens while the number of Salmonella serovar Enteritidis isolated from chickens varies from year to year. Interestingly, while neither serovar cause disease in poultry, Enteritidis appears to result in more human disease while Kentucky only rarely causes disease in humans. The reasons for this are not well understood. In order to colonize the host, Salmonella must survive the low pH in animal digestive tracts. Studies of S. Kentucky isolated from chickens showed it responded differently to acid than other serovars. To explore this, gene expression of S. Kentucky was compared to S. Enteritidis. These strains were tested by ten minute exposures to hydrochloric acid (pH 4.5) or to acetic acid (pH 5.5) in rich growth media. Microarray analysis indicated that more genes were turned on or off in S. Kentucky than in S. Enteritidis under these conditions. Overall, it appeared that the responses to acid by S. Kentucky and S. Enteritidis are similar, but differences exist in the scope and magnitude of the responses. These responses could explain the difference in prevalence of serovar Kentucky and Enteritidis in chickens. Further investigations could identify host responses in chickens that could be adjusted to reduce the levels of dangerous pathogens like S. Enteritidis in chickens. This information is useful for the poultry industry which can use this research to improve food safety by reducing the risk of Salmonella infection to humans via poultry.

3. Analysis of antimicrobial resistance genes in environmental soil bacteria. Acinetobacter baumannii-calcoaceticus complex (ABC) causes wound infections in many combat casualties. An increase in ABC strains which are multi-drug resistant (MDR) has complicated treatment of these wounds in U.S. personnel participating in Operation Iraqi Freedom. In this study, DNA patterns from 298 ABC isolates were characterized to determine how closely they matched on a genetic level. Pulsed Field Gel Electrophoresis (PFGE) was used to classify them into 67 distinct PFGE types (PFTs). Microarray analysis of DNA from isolates detected the presence of several antimicrobial resistance genes some of which indicated resistance to the antimicrobial imipenem. Imipenem resistance (IR) in MDR isolates is of particular concern as it is one of the last drugs available to treat MDR ABC infections. DNA Southern blot analysis demonstrated that the IR specific gene was plasmid-borne or both plasmid and chromosomally located. A plasmid carrying the IR specific gene inserted into a susceptible ABC strain conferred IR to the recipient showing that the plasmid contained the resistance and could be transferred to other bacteria. The distribution of the IR-ABC with specific PFTs implied nosocomial spread in military treatment facilities. This study has determined the genes causing IR and MDR in ABC infections and how it is spread in the hospital environment. It also demonstrated that resistance developed in these ubiquitous soil bacteria which could be easily transferred to other bacteria including food borne pathogens. This information is particularly useful to the U.S. military and military scientists in order to protect the U.S. military from bacterial infections in war zones.

4. 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.

5. Source tracking in surface and groundwater using multiple indicators. Bacterial contamination caused by discharge of human and animal waste is a serious challenge to maintaining and preserving the quality of water resources. ARS scientists in Athens, Georgia, in collaboration with Environment Canada and the Universiti Teknologi MARA, applied a multi-indicator approach to identify sources of fecal contamination in Fraser Valley, a province of British Columbia, Canada. Poultry waste generated from the industry in this area is used as fertilizer and spread onto the fields thus creating a potential source of surface and groundwater pollution. Sterol analysis, Enterococcus Bacterial Source Tracking, and chemometric analysis were used as pollution source identifiers to determine if fecal bacteria in the environment originated from humans, livestock, or wildlife. Fecal contamination was detected in 100% of surface water and 15% of groundwater sites tested. Contribution from the poultry industry to surface water pollution was detected at nine sampling locations. Human fecal pollution was also detected at four surface water and one groundwater location. An ability to ascribe sources when confronted with a complex pollution situation is essential for planning management actions and implementing best management practices. Researchers can use this information to further efforts to protect and preserve surface and ground water quality from the impacts of human and agricultural activities.

Review Publications
Lamichhane-Khadka, R., Frye, J.G., Porwollik, S., Mcclelland, M., Maier, R.J. 2011. Hydrogen-Stimulated carbon acquisition and conservation in salmonella enterica serovar typhimurium. Journal of Bacteriology. 193(20):5824-5832.

Joerger, R.D., Sartori, C., Frye, J.G., Turpin, J.B., Schmidt, C., Mcclelland, M., Porwollik, S. 2012. Gene expression analysis of Salmonella enterica Enteritidis NalR and Salmonella enterica Kentucky 3795 exposed to HCl and acetic acid in rich medium. Foodborne Pathogens and Disease. 9(4):331-337.

Huang, X., Chahine, M.A., Frye, J.G., Cash, D.M., Lesho, E.P., Craft, D.W., Lindler, L.E., Nikolich, M.P. 2012. Molecular analysis of imipenem-resistant Acinetobacter baumannii isolated from US service members wounded in Iraq, 2003–2008. Epidemiology and Infection. 140(12):2302-2307.

Furtula, V., Jackson, C.R., Osman, R., Chambers, P. 2012. Use of Enterococcus, BST and sterols as indicators for poultry pollution source tracking in surface and groundwater. In Oosthuizen, J., editors. Environmental Health-Emerging Issues and Practice. Rijeka, Croatia: InTech-Open Access. p. 57-78.

Last Modified: 2/23/2016
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