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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

2022 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
In FY22, progress was made in all three objectives under National Program 108. DNA sequence comparisons using publicly available whole genome sequence (WGS) data from multi-drug resistant (MDR) Salmonella isolates associated with human foodborne outbreaks could identify unique DNA sequences and fitness factors contributing to the emergence and pervasiveness of Salmonella outbreak isolates, as well as identify potential intervention targets. In support of “Objective 1A, Characterize unique genetic features of outbreak-associated Salmonella,” investigation of the human foodborne outbreak of MDR Salmonella enterica serovar Reading (S. Reading) associated with turkeys and turkey products revealed genetic variations between S. Reading strains isolated before the outbreak compared to during the outbreak. Many of the S. Reading isolates from the outbreak period were missing 40 genes and had 21 additional genetic variations (e.g., single nucleotide polymorphisms), with some of the genetic differences in genes involved in Salmonella virulence. Current studies are analyzing bacterial phenotypes associated with these genetic variations to determine if the genetic differences are contributing to enhanced Salmonella fitness, thereby providing a competitive advantage for the outbreak-associated S. Reading isolates. A recent human outbreak of multi-drug resistant (MDR) Salmonella enterica serovar Heidelberg (S. Heidelberg) resulted in 56 reported human illnesses and 17 hospitalizations across 15 states. Infection was determined to be due to contact with dairy beef calves. The calves infected with S. Heidelberg also showed signs of disease, and a loss of up to 65% of the calves occurred due to death from bacteremia. In support of “Objective 1A, Characterize unique genetic features of outbreak-associated Salmonella,” assays were performed to compare the cell invasion ability of two S. Heidelberg variants isolated during the outbreak. The highly virulent variant associated with death in calves was significantly more invasive compared to the less virulent variant. Current research is comparing the genome sequence of the two S. Heidelberg variants to each other and to other Heidelberg strains to identify genetic elements responsible for the elevated pathogenicity and the emergence of the human and bovine pathogen. The role of eggshell microbes in driving the succession of antibiotic resistance genes and horizontal transfer in newly hatched chicks is unknown. In support of “Objective 2A, Identify commensal members of the microbiota harboring AMR and contributing to AMR transfer in young birds,” we conducted two animal trials from eggs from different hatcheries to test the contribution of the eggshell microbiota in horizontal transfer of antibiotic resistance genes. Antibiotic resistant E. coli was detected on the eggshell and in chick intestines after hatch. The potential of E. coli to transfer antibiotic resistance genes on plasmids to Salmonella was investigated, although it was not detected in this study. Genome sequencing of the resistant E. coli strains confirmed that the antibiotic resistance genes were either located on chromosomes or on plasmids that could not transfer to the Salmonella used in the study. Analysis of the genomic data is continuing, but preliminary results suggest the genetic context of the antibiotic resistance donor and recipient impacts the risk for horizontal gene transfer to pathogens. Bacterial evolution can happen quickly and may lead to new biochemical properties such as antibiotic resistance or virulence factors. To understand how Campylobacter jejuni has changed over the last 60 years, we utilized a historic collection of ovine C. jejuni strains, dating back to the 1960s for genomic comparisons with contemporary strains isolated from ovine sources. In support of “Objective 2A, Identify commensal members of the microbiota harboring AMR and contributing to AMR transfer in young birds,” we sequenced the genomes of C. jejuni strains and conducted comparative genomic analysis with genomes of contemporary strains, available in public databases. The results show that some historic strains share a common lineage with contemporary isolates, while others are unique. Potential areas of horizontal gene transfer were detected and serve as useful markers to reconstruct lineages. Sequence analysis continues and is focused on antibiotic resistance and virulence gene acquisition over time. Salmonella can form a complex bacterial community matrix called a biofilm that adheres to a variety of surfaces and is resistant to diverse disinfectants and antibiotics. In support “Objective 3, Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals,” a study was performed to determine if a bovine antimicrobial peptide (AMP) could eradicate biofilms from different Salmonella strains. The AMP prevented Salmonella biofilm formation and was also able to kill Salmonella in the protective environment of an already formed biofilm. We are currently evaluating suitable manufacturing and delivery mechanisms of the AMP for food animal production and processing settings. The human foodborne pathogen Campylobacter mainly colonizes in the intestinal tract of poultry where they are intimately associated with the mucus layer of the intestinal tissues. Thus, mucus may be a key environmental cue for Campylobacter, so investigation of the microbe’s response to mucus exposure may identify protein targets in Campylobacter for development of interventions, particularly vaccine antigens. In support of “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,” proteins from two Campylobacter mucus-induced genes were purified and demonstrated to be immunoreactive with sera containing antibodies from C. jejuni-challenged turkeys. These mucus-induced target genes/proteins will be further investigated to expand our understanding of the turkey immune response to C. jejuni, which may enable assay development for detecting colonized animals as well as provide potential target antigens for development of novel mucosal vaccines against Campylobacter. Vaccination of food animals against Salmonella is a pre-harvest food safety intervention strategy that can decrease shedding of Salmonella on the farm as well as reduce Salmonella contamination entering the processing plant. Progress was made on two subordinate projects addressing vaccination against outbreak isolates of MDR Salmonella in food animals, supporting “Objective 1B, Evaluate colonization, dissemination and persistence of human outbreak-associated Salmonella in turkeys and/or swine” and “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.” A collaboration with Boehringer Ingelheim Animal Health USA, Inc., evaluated the Enterisol Salmonella T/C vaccine against the highly prevalent Salmonella enterica serovar I 4,[5],12:i:- in pigs, revealing significant reduction in Salmonella shedding and colonization of the swine intestinal tract. Under a project partially funded by the U.S. Poultry and Egg Association, protection provided by two Salmonella vaccines against turkey-associated Salmonella enterica serovar Reading was evaluated. Both vaccines were cross-protective (i.e., immunity induced by the vaccine Salmonella serotype was protective against another Salmonella serotype), reducing intestinal colonization and systemic transmission of Salmonella Reading. These vaccination data provide valuable information to producers on mitigation strategies to reduce Salmonella in their food animal commodities.

1. Prevalent genetic features of an emerging foodborne Salmonella serotype identified using publicly available database. Of the >2600 Salmonella serovars, Salmonella enterica serovar I 4,[5],12:i:- (serovar I 4,[5],12:i:-) has emerged as one of the most common causes of human human foodborne illness and the most frequent multidrug-resistant (MDR) Salmonella serovar in the United States. To assess global distribution and identify common genetic features associated with the emergent Salmonella serotype, ARS researchers in Ames, Iowa, analyzed 13,612 sequences available in the NCBI Pathogen Detection database. Of particular interest were genes that confer resistance to antimicrobial agents such as antibiotics and heavy metals, and the researchers determined that 71% of the Salmonella sequences contained a DNA cassette with metal tolerance genes (copper, arsenic, silver) while 55% contained a genetic module with mercury tolerance genes as well as multiple antibiotic resistance genes (ampicillin, streptomycin, sulfisoxazole, and tetracycline). Furthermore, host-associations of serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database suggested that swine-associated strain sequences were the most frequent food-animal source globally, though it’s unclear if this is due to sampling bias or an increased association with swine. This study illustrates how the sizable databases of whole genome sequences generated through genomic surveillance efforts can be utilized by regulatory agencies and research scientists to identify prevalence of key genetic features in emerging foodborne pathogens for prediction of Salmonella outbreaks or development of new technologies.

2. In-feed additive promotes beneficial intestinal bacteria and reduces Salmonella fecal shedding in pigs. Swine producers desire in-feed, non-antibiotic products to enhance pig production and control infectious diseases. ARS researchers in Ames, Iowa, evaluated a commercially available in-feed prebiotic compound, resistant potato starch (RPS), and discovered that, compared to a non-amended diet, RPS-fed pigs had an enrichment of health-associated gut microorganisms, increased concentrations of health-associated short chain fatty acids (SCFAs) in their intestinal tract, and lower amounts of Salmonella fecal shedding. However, not all of the RPS-fed pigs exhibited the same response. Specifically, RPS-fed pigs with higher abundances of specific bacterial taxa had greater concentrations of certain SCFAs and shed less Salmonella. The data suggest that RPS may be an effective option for swine producers to enhance gut health and limit Salmonella colonization and shedding in swine, provided the appropriate bacterial communities that can utilize RPS as a substrate are present in the gut.

3. Horizontal transfer of antimicrobial resistance genes occurs between Campylobacter species isolated from turkeys and swine. Campylobacter are commonly found in the intestinal tract of poultry, but rarely cause disease in birds. In humans, undercooked contaminated poultry is the main source of Campylobacter infections. In recent years, there has been a rise in antibiotic-resistant Campylobacter, which can severely limit treatment options. Identification of factors contributing to antibiotic resistance spread in Campylobacter, especially in the natural gut environment, will inform the development of control strategies along the food supply chain. ARS researchers in Ames, Iowa, detected the natural transmission of antibiotic resistance genes between Campylobacter species and strains. This was evaluated in vitro through co-culture experiments and in vivo with dual-strain inoculation of turkeys, followed by whole genome sequencing of parental and newly emerged strains. Multiple events of antibiotic resistance gene transfer from one strain to another were detected under laboratory conditions. Four pairs of resistant and nonresistant Campylobacter strains, that demonstrated in vitro gene exchange, were administered to turkeys orally, however, only one resistance transfer was detected. Transfers of genes conferring resistance to multiple classes of antibiotics were detected. This study highlights the potential for antimicrobial resistance transfer across Campylobacter species and strains originating from turkeys or swine, which may have implications for farms hosting both species in close proximity. These data inform producers on control strategies to minimize transfer of antimicrobial resistance by minimizing or eliminating cross-contamination between swine and turkey production systems.

4. The novel intestinal bacteria Turicibacter bilis, has novel roles in the poultry intestinal tract. Livestock and poultry production in the U.S. is responsible for feeding a growing global population. Understanding how microbial communities in the intestinal tracts of food animals impact health, production, and food safety is critical to supporting the industry and food supply. Members of the Turicibacter genus have been detected in the small intestine of many animal species using culture-independent methods, and they are associated with altered fat metabolism in their host. ARS researchers in Ames, Iowa, isolated novel Turicibacter strains from the eggshell of two separate white leghorn chicken flocks and the ileum of a healthy pig. The novel strains were determined to be a new species and designated Turicibacter bilis. The T. bilis genome is divergent from close bacterial relatives and it has specialized genes for breaking down host bile acids. T. bilis grows better in in the presence of avian bile, reflecting its adaptations to the small intestine environment. The characterization of T. bilis provides the first biochemical and genomic information on the species and adds new data to what is known about the group of bacteria related to T. bilis. T. bilis may impact poultry and swine growth by modifying compounds produced by the animal (bile) in the intestinal tract and subsequently impact digestion. Studies like this fill gaps in researchers’ understanding of the microbiota membership in the intestinal tracts of livestock species. Bacterial functions that impact host digestion need to be identified to determine if modulation of these activities can improve animal nutrition and resilience to colonization by foodborne pathogens.

5. Antimicrobial peptide kills multidrug-resistant Salmonella. Non-antibiotic interventions against Salmonella for use in food animal production and processing are highly desirable, especially with ~20% of tested Salmonella being resistant to one or more antibiotics. In search of non-antibiotic therapeutics, ARS researchers in Ames, Iowa, evaluated four bovine antimicrobial peptides (NK-lysins) for their ability to kill Salmonella in vitro. One of the antimicrobial peptides, NK2A, caused bacterial membrane permeability, resulting in leakage of the cell’s contents and bacterial death. Industry, academic and government researchers could utilize this innovative, non-antibiotic strategy to aid efforts by food animal producers and processors to control the spread of MDR Salmonella infections in animal and human populations.

Review Publications
Trachsel, J.M., Bearson, B.L., Brunelle, B.W., Bearson, S.M. 2022. Relationship and distribution of Salmonella enterica serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database. Biomed Central (BMC) Genomics. 23(1). Article 268.
Trachsel, J.M., Bearson, B.L., Kerr, B.J., Shippy, D.C., Byrne, K.A., Loving, C.L., Bearson, S.M. 2022. Short chain fatty acids and bacterial taxa associated with reduced Salmonella enterica serovar I 4,[5],12:i:- Shedding in swine fed a diet supplemented with resistant potato starch. Microbiology Spectrum. 10(3). Article e0220221.
Bearson, S.M. 2021. Salmonella in swine: Prevalence, multidrug resistance, and vaccination strategies. Annual Review of Animal Biosciences. 10:373-393.
Dassanayake, R.P., Atkinson, B.M., Mulls, A.S., Falkenberg, S.M., Nicholson, E.M., Casas, E., Narasimhan, B., Bearson, S.M. 2021. Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates. Nature Scientific Reports. 11(1). Article 19276.
Johnson, J., Zwirzitz, B., Oladeinde, A.A., Milfort, M.C., Looft, T.P., Chai, L., Zock, G.S., Sommers, M., Tunim, S., Aggrey, S.E. 2021. Succession patterns of the bacterial community in poultry litter after bird removal and sodium bisulfate application. Journal of Environmental Quality. 50(4):923-933.
Oladeinde, A.A., Abdo, Z., Press, M.O., Cook, K.L., Cox Jr, N.A., Zwirtzitz, B., Woyda, R., Lakin, S.M., Thomas Iv, J.C., Looft, T.P., Cosby, D.E., Hinton Jr, A., Guard, J.Y., Line, J.E., Rothrock Jr, M.J., Berrang, M.E., Herrington, K., Zock, G.S., Plumblee Lawrence, J.R., Cudnik, D., House, S.L., Ingram, K.D., Lariscy, L., Wagner, R., Aggrey, S.E., Chai, L., Ritz, C. 2021. Horizontal gene transfer is the main driver of antimicrobial resistance in broiler chicks infected with Salmonella enterica Serovar Heidelberg. mSystems. 6(4):e00729-21.
Maki, J., Looft, T.P. 2022. Turicibacter bilis sp. nov., a novel bacterium isolated from the chicken eggshell and swine ileum. International Journal of Systematic and Evolutionary Microbiology. 72(1). Article e0.005153.
Guernier-Cambert, V., Trachsel, J.M., Maki, J.J., Qi, J., Sylte, M.J., Hanafy, Z., Kathariou, S., Looft, T.P. 2021. Natural horizontal gene transfer of antimicrobial resistance genes in Campylobacter spp. from turkeys and swine. Frontiers in Microbiology. 12(1). Article e732969.
Oladeinde, A.A., Abdo, Z., Zwirzitz, B., Woyda, R., Lakin, S.M., Press, M.O., Cook, K.L., Cox Jr, N.A., Thomas, J.C., Looft, T.P., Rothrock Jr, M.J., Zock, G.S., Plumblee Lawrence, J.R., Cudnik, D., Ritz, C. 2022. Litter commensal bacteria can limit the horizontal gene transfer of antimicrobial resistance to Salmonella in chickens. Applied and Environmental Microbiology.88(9).