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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Research Project #440948

Research Project: Analysis of Genetic Factors that Increase Foodborne Pathogen Fitness, Virulence, and Antimicrobial Resistance Transfer, to Identify Interventions against Salmonella and Campylobacter in Food Animals

Location: Food Safety and Enteric Pathogens Research

2023 Annual Report

Objective 1: Identify genetic features of Salmonella outbreak isolates that influence fitness and impact prevalence in food animals. Sub-objective 1a: Characterize unique genetic features of outbreak-associated Salmonella. Sub-objective 1b: Evaluate colonization, dissemination and persistence of human outbreak-associated Salmonella in turkeys and/or swine. Objective 2: Identify mechanisms of AMR gene transfer to food borne pathogens in poultry microbiota and test novel mitigation strategies to limit AMR gene mobility. Sub-objective 2a: Identify commensal members of the microbiota harboring AMR and contributing to AMR transfer in young birds. Sub-objective 2b: Test the efficacy of microbiota modulation at hatch to reduce AMR HGT. Objective 3: Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals. Sub-objective 3a: Test efficacy of dual-purpose recombinant attenuated Salmonella vaccine(s) (RASV) encoding Campylobacter antigens to reduce intestinal colonization of Campylobacter and Salmonella in turkeys. Sub-objective 3b: Evaluate the efficacy of in-feed treatments to reduce intestinal colonization of Campylobacter and/or Salmonella in turkeys.

The goal of this project is to address research gaps in high priority, food safety research areas involving the most common causes of bacterial foodborne illness in the United States, Salmonella and Campylobacter. Limiting Salmonella and Campylobacter colonization as well as AMR transfer in food-producing animals can reduce foodborne pathogen carriage into the human food chain, decrease environmental contamination, diminish the cost of meat product recalls to producers, and limit AMR transmission. Experiments are planned to: 1) investigate genetic features and fitness factors that contribute to the emergence of Salmonella outbreak isolates associated with food animal products, 2) identify, characterize and displace commensal members of the poultry microbiome involved in transfer of antimicrobial resistance (AMR) to foodborne pathogens, and 3) develop and/or test non-antibiotic intervention strategies to reduce human foodborne pathogens at the first critical control point in the food animal production chain, namely on-farm colonization. Focusing on the ’who, when and how’ questions of these crucial food safety issues will support the advancement of applicable tools for targeted mitigations to control foodborne pathogens and AMR transmission, thereby providing the public with a safer food supply.

Progress Report
Publicly available whole genome sequence (WGS) data provides a rich dataset to perform genome comparisons that could identify unique sequences and fitness factors contributing to the emergence and persistence of human foodborne outbreak-associated Salmonella isolates. In support of “Sub-objective 1A, Characterize unique genetic features of outbreak-associated Salmonella” and in collaboration with the USDA Food Safety Inspection Service (FSIS), investigation of genomes of multidrug-resistant (MDR) Salmonella enterica serovar Infantis (S. Infantis) associated with chicken and turkey products revealed variation in the size and genetic composition of a large extrachromosomal DNA containing numerous genes for antibiotic resistance, metal tolerance, and bacterial virulence. Current investigations are evaluating bacterial phenotypes associated with these genetic factors to determine if they contribute to enhanced fitness of the outbreak-associated S. Infantis isolates. Vaccination of turkeys against Salmonella can decrease colonization on the farm as well as reduce Salmonella entering the processing plant. Progress was made on addressing interventions against outbreak isolates of MDR Salmonella in food animals, supporting “Sub-objective 1B, Evaluate colonization, dissemination and persistence of human outbreak-associated Salmonella in turkeys and/or swine” and “Objective 3, Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals.” A commercial vaccine was tested for efficacy against MDR S. Infantis in turkeys. Groups of turkeys were vaccinated or mock-vaccinated and subsequently challenged with S. Infantis. After challenge, samples were collected to assess S. Infantis counts in the intestine and various tissues. Vaccination provided significant reduction in intestinal colonization and systemic dissemination of S. Infantis compared to mock-vaccinated turkeys. Data are in the final stages of analysis, and the results will provide valuable information to turkey producers on a mitigation strategy to reduce Salmonella during turkey production. Vaccination of food animals against Salmonella relies on priming the host immune system to reduce colonization when the animal is subsequently exposed to Salmonella in the environment. In support of “Objective 3, Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals”, a study partially funded by the U.S. Poultry and Egg Association was expanded to examine the impact of a commercial or an experimental Salmonella vaccine on turkey host responses to an outbreak isolate of MDR Salmonella enterica serovar Reading (S. Reading). Cytokine gene expression in the spleen of vaccinated turkeys inoculated with S. Reading was lower when compared to non-vaccinated turkeys. Cecal tonsil RNA was extracted and sequenced to measure gene expression patterns across the entire genome; analysis is ongoing to identify genes and pathways influenced by Salmonella or each of the vaccines. Antimicrobial resistance (AMR) in Salmonella heightens public health concerns, as it threatens the proper clinical management of severe salmonellosis in humans. The role of commensal bacteria in transferring AMR to Salmonella is not understood. In support of “Objective 2A, Identify commensal members of the microbiota harboring AMR and contributing to AMR transfer in young birds”, the barn environment was evaluated as a reservoir of microbes with antimicrobial resistance genes (ARG) that could be acquired by Salmonella enterica serovar Heidelberg (S. Heidelberg) in the chicken intestine. Because previous research suggested that Escherichia coli (E. coli) plasmids and ARG can be acquired by Salmonella, evaluation was focused on tetracycline resistant E. coli. While AMR E. coli was detected and genomic analysis revealed ARG on the E. coli chromosome or plasmids, transfer of resistance to Salmonella was not observed. These results indicate that the presence of ARG in E. coli alone does not predict the potential for AMR transfer and Salmonella acquisition in the turkey intestine. Understanding the context for ARG in the environment is necessary to determine the risk for transfer to foodborne pathogens. Multiple strategies are needed to reduce Salmonella colonization in poultry, including exploiting interactions between commensal bacteria and Salmonella to reduce colonization. The microbiota develops and becomes more diverse as birds get older, making it more difficult for Salmonella to colonize. Increasing the rate of microbiota development in poultry may reduce Salmonella colonization. In support of “Objective 3B, Evaluate the efficacy of in-feed treatments to reduce intestinal colonization of Campylobacter and/or Salmonella in turkeys”, a synthetic consortium of commensal bacterial was given to chicks after hatch to speed up microbiota development and exclude S. Heidelberg colonization. After giving birds the bacterial consortium, they were inoculated with S. Heidelberg. Cecal contents were sampled over the next 4 weeks to determine Salmonella colonization and microbiota composition. Birds that received the microbial consortium had significantly lower Salmonella loads than birds that didn’t receive the consortium. Ongoing research is exploring how specific changes to the microbial consortium composition may improve inhibition of Salmonella colonization. Campylobacter is a major food safety concern and is transmitted to humans mainly via contaminated poultry meat. Controlling Campylobacter at the source (i.e., poultry) is important because reducing the prevalence of Campylobacter in poultry will also reduce the risk of transmission of Campylobacter to humans. In support of “Objective 3B, Evaluate the efficacy of in-feed treatments to reduce intestinal colonization of Campylobacter and/or Salmonella in turkeys”, a comparison of the chicken gut microbiota between the Campylobacter-positive and Campylobacter-negative farms was conducted to determine if Campylobacter status influences the microbiota composition. Key differences were detected in the microbiota of Campylobacter-positive flocks, including the increase of specific bacteria when Campylobacter was present and bacterial species that were present when Campylobacter was absent. Overall, these findings may be valuable for developing an evidence-based approach to design tailored gut microbial communities to mitigate Campylobacter colonization in poultry to enhance food safety.

1. Overcoming the antibiotic resistance of Salmonella to improve the fight against a harmful human pathogen. The Centers for Disease Control and Prevention lists antibiotic resistant Salmonella on the serious threat level, and >10% of Salmonella are multidrug-resistant (resistant to three or more drug classes). Reduction of antimicrobial effectiveness paired with limited production of new antibiotics has created a critical need for options that make existing antibiotics effective against antibiotic-resistant bacteria. Resistance to tetracycline often occurs by Salmonella rapidly pumping the antibiotic out of the cell (i.e. efflux); therefore ARS researchers in Ames, Iowa, tested an efflux pump inhibitor (EPI) with tetracycline to determine if a synergistic reduction in resistance could be achieved in tetracycline-resistant Salmonella. The EPI reduced Salmonella resistance to tetracycline up to 32-fold in several tetracycline resistant Salmonella isolates, thereby reducing resistance in tetracycline-resistant Salmonella to levels that would no longer classify the isolates as ‘resistant’ to tetracycline. The data suggest that EPIs have the potential to contribute to novel treatment options that are effective against antibiotic resistance in highly relevant bacteria, including Salmonella. Research to determine the safety and effectiveness of EPIs in food animals will benefit veterinarians and producers battling antibiotic resistant bacteria.

2. Enhancing swine feed with fiber promotes beneficial gut bacteria and limits Salmonella in pigs. In-feed, non-antibiotic products are desired by swine producers to enhance pig production and control infectious diseases. ARS researchers in Ames, Iowa, evaluated a commercially available in-feed prebiotic compound called beta-glucan and discovered that pigs fed beta-glucan in their diet had higher levels of beneficial gut microorganisms and lower amounts of Salmonella in their intestines compared to pigs fed a non-amended diet. The data suggest that swine producers who implement beta-glucan diet supplementation could provide modest improvements in pig gut health while limiting Salmonella colonization.

3. Developed a new model to preserve short-chain fatty acids in test samples and identify regions to target beneficial gut metabolites. Intestinal bacteria impact poultry health, including producing metabolites absorbed by birds. Short-chain fatty acids (SCFA) are an important class of beneficial metabolites; however, they are difficult to detect in tissues because SCFA are often lost during sample preparation. ARS researchers in Ames, Iowa, contributed to the development of a method (chemical derivatization) to stabilize SCFA in tissues before traditional sample preparation to improve SCFA detection. Tested on chicken intestinal tissues, this method improved the sensitivity of SCFA detection and allowed comparisons between tissues. Spatial metabolomic mapping techniques allow for the identification of intestinal regions critical for intestinal health. Identification of regions where SCFA are produced and absorbed improves our understanding of microbial functions and may suggest new targets for modulation to improve animal health and production. This technology will be useful to agriculture researchers and feed producers for testing interventions that modulate intestinal function in a targeted fashion.

Review Publications
van der Graaf-van Bloois, L., Duim, B., Looft, T.P., Veldman, K.T., Zomer, A.L., Wagenaar, J.A. 2023. Antimicrobial resistance in Campylobacter fetus: emergence and genomic evolution. Microbial Genomics. 9(3).
Forsman, T.T., Paulson, A.E., Larson, E.A., Looft, T.P., Lee, Y. 2023. On-tissue derivatization of volatile metabolites for matrix assisted laser desorption/ionization mass spectrometry imaging. Journal of Mass Spectrometry. 58(5). Article e4918.
Bearson, S.M., Trachsel, J.M., Bearson, B.L., Loving, C.L., Kerr, B.J., Shippy, D.C., Kiros, T.G. 2023. Effects of ß-glucan on Salmonella enterica serovar Typhimurium swine colonization and microbiota alterations. BMC Porcine Health Management. 9(7). Article 003024.
Kerr, B.J., Trachsel, J.M., Bearson, B.L., Loving, C.L., Bearson, S.M., Byrne, K.A., Pearce, S.C., Ramirez, S.M., Gabler, N.K., Schweer, W.P., Helm, E.T., De Mille, C.M. 2022. Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity. Journal of Animal Science. 100(11). Article skac282.
Warren, W.C., Rice, E.S., Meyer, A., Hearn, C.J., Steep, A., Hunt, H.D., Monson, M.S., Lamont, S.J., Cheng, H.H. 2023. The immune cell landscape and response of Marek's disease resistant and susceptible chickens infected with Marek's disease virus. Scientific Reports. 13. Article 5355.
Maki, J.J., Lippolis, J.D., Looft, T.P. 2022. Proteomic response of Turicibacter bilis MMM721 to chicken bile and its bile acids. BMC Research Notes. 15(1). Article e236.
Price, E.D., Dassanayake, R.P., Bearson, S.M. 2023. Increasing antimicrobial susceptibility of MDR Salmonella with the efflux pump inhibitor 1-(1-Naphthylmethyl)-piperazine. Biochemical and Biophysical Research Communications. 668:49-54.
Loving, C.L., Bearson, S.M., Bearson, B.L., Kerr, B.J., Kiros, T.G., Shippy, D.C., Trachsel, J.M. 2022. Effect of dietary ß-glucan on intestinal microbiota diversity and Salmonella vaccine immunogenicity and efficacy in pigs. Veterinary Microbiology. 278. Article 109648.
Zwirzitz, B., Oladeinde, A.A., Johnson, J., Zock, G., Milfort, M.C., Fuller, L.A., Ghareeb, A., Foutz, J., Teran, J., Woyda, R., Abdoa, Z., Looft, T., Plumblee Lawrence, J.R., Aggrey, S.E. 2023. Temporal dynamics of the cecal and litter microbiome of chickens raised in two separate broiler houses. Frontiers in Physiology.