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:
This project is new commencing in October of fiscal year 2012. During the year, high-throughput sequencing was employed for whole-genome DNA sequencing of Salmonella enterica serotype Heidelberg. DNA sequences from all isolates are presently being analyzed to identify gene differences between isolates from various animal sources. For Campylobacter, whole-genome DNA microarrays were used to identify a total of 142 most significant variable genes from Campylobacter jejuni from 95 geographically diverse cattle, chicken and human C. jejuni isolates. Eighty-eight percent of the significant variable genes belonged to genomic prophage and hypervariable regions. Identification of genetic diversity among the Salmonella and Campylobacter isolates studied to date is necessary to identify and 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 Polymerase Chain Reaction (PCR) assay for Salmonella and Campylobacter for poultry source attribution. Multi-drug resistance (MDR) was evaluated in Salmonella and generic Escherichia coli from animals. PCR and microarrays were used to detect antimicrobial resistance (AR) genes and plasmids on which the genes are harbored. The DNA microarray, developed by an ARS scientist, includes a total of 1267 probes; 775 probes detect AR genes and 487 probes 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 characterize MDR E. coli and MDR S. Typhimurium. For MDR E. coli, AR genes were detected consistent with the MDR phenotypes of all isolates, and plasmid replicon typing identified one or more replicon types in all isolates tested. Class I integrons containing AR genes were also detected in some of the isolates. MDR S. Typhimurium, isolated from healthy cattle, poultry, and swine and resistant to at least five antimicrobials (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline) were selected for analysis. The distribution of AR and plasmid genes detected separated most of the isolates into two groups. Results showed significant linkage for the associations between plasmids, phage type, and animal source for those groups. In addition to whole-genome sequencing, high-throughput sequencing was also used to sequence plasmids isolated from MDR 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 AR genes contained on the plasmids as well as genes which may be responsible for mobility of AR genes, segments of plasmids or entire plasmids. These studies directly relate to Objective 2 of the project plan which aims to determine unique characteristics of high priority serotypes of AR food borne bacteria and those of highly resistant or multi-resistant genotypes with novel phenotypes.
Glenn, L.M., Lindsey, R.L., Frank, J.F., Meinersmann, R.J., Englen, M.D., Cray, P.J., Frye, J.G. 2011. Analysis of antimicrobial resistance genes detected in multidrug-resistant salmonella enterica serovar typhimurium isolated from food animals. Microbial Drug Resistance. 17(3):407-418.