Location: Food Safety and Enteric Pathogens Research
2021 Annual Report
Objectives
Our objectives target three factors that influence Salmonella colonization, pathogenesis, and persistence. These factors include virulence mechanisms of Salmonella, the tactical response from the host, and interactions with the microbiota residing within the host. Our systematic approach integrates these research areas into three complementary objectives:
Objective 1: Investigate the impact of antibiotic usage on influencing Salmonella virulence mechanisms and enhancing antibiotic resistance.
Objective 2: Develop novel non-antibiotic intervention strategies such as beneficial microbes and vaccines to limit Salmonella colonization, persistence and shedding.
Objective 3: Evaluate immune networks and identify porcine genes for their relationship with the host microbiota to reduce Salmonella colonization, persistence, and shedding.
Approach
The common goal of each research objective is to identify targets for the development of novel antibiotic alternatives to reduce both Salmonella transmission through the food chain and antibiotic usage on the farm. To accomplish these objectives, experiments are planned to examine molecular mechanisms in Salmonella that are influenced by antimicrobial resistance and host colonization, elucidate porcine genetic pathways associated with decreased Salmonella colonization, and investigate interactions between Salmonella and host microbiota that affect Salmonella colonization and persistence. We plan to: 1) identify antibiotics that enhance virulence properties in multidrug-resistant (MDR) Salmonella, as well as those antibiotics that have no effect on virulence; this useful information will aide producers and veterinarians when determining antibiotic therapy for the treatment of infectious diseases; 2) genome sequence and transcriptionally analyze MDR Salmonella isolates that phenotypically respond to antibiotic exposure; 3) measure the effect chlortetracycline treatment has on limiting or exacerbating Salmonella shedding and altering the microbiota in swine; 4) evaluate a cross-protective Salmonella vaccine in turkeys for reduction of Salmonella colonization and transmission; 5) systematically characterize changes in the porcine immune response and gastrointestinal microbiota during Salmonella colonization; 6) assess biotherapeutic treatments as alternatives to antibiotics for treatment of swine colonized with Salmonella.
Progress Report
Progress reported below is continued from the expired project 5030-32000-113-00D “Analysis of Virulence and Antibiotic Resistance Mechanisms of Salmonella and Development of Intervention Strategies” and incorporates the objectives from the soon to be new project, 5030-32000-227-00D, “Analysis of Genetic Factors that Increase Foodborne Pathogen Fitness, Virulence, and Antimicrobial Resistance Gene Transfer, to Identify Interventions against Salmonella and Campylobacter in Food Animals” which was recently certified by peer review panel.
Food animal production systems can benefit from non-antibiotic interventions that control bacterial pathogens and support animal health. Resistant potato starch (RPS) has been shown to promote health-associated microbial communities in the intestinal tract of food animals and maintaining a healthy gut can limit bacterial pathogens such as Salmonella. In support of Objective 3b, a study was performed to determine if RPS affects Salmonella colonization in pigs. Pigs were fed a diet with and without RPS and inoculated with multidrug-resistant Salmonella serovar I 4,[5],12:i:-. In the RPS-fed group of pigs, reduced Salmonella shedding was associated with specific microbial communities as well as increased levels of short chain fatty acids (SCFAs) which can have beneficial impacts on host tissues. These data suggest that specific bacterial food webs may play a role in the RPS-associated reduction in Salmonella shedding in swine and that bacterial metabolism associated with SCFA production may be important for this effect. Current research is evaluating the bacteria enriched in the intestinal microbiota of the RPS-fed pigs to determine if RPS supplementation could be an effective means to reduce MDR Salmonella serovar I 4,[5],12:i:- in swine.
The spread of Campylobacter from animals to humans through contaminated food is a leading cause of foodborne illness. Antibiotic-resistant Campylobacter is a serious threat to public health, because resistant strains can reduce antibiotic treatment options for campylobacteriosis. Campylobacter is known to naturally pick up DNA from other bacteria (competent), but there is limited understanding of the spread of antimicrobial resistance between Campylobacter in the animal host, particularly turkeys. In support of Objective 2a, a turkey study was designed to evaluate the possible transfer of antimicrobial resistance genes (ARGs) between Campylobacter strains of the same or different species (C. coli or C. jejuni) originating from different host species (swine or turkeys). Birds were inoculated with two Campylobacter strains with different antibiotic resistance patterns, followed by re-isolation from turkey cecal contents to determine if novel resistant patterns emerged. Results from the animal study were compared against laboratory (in vitro) results that were optimized for gene transfer. In vitro, independent horizontal gene transfer events were detected, leading to the acquisition of ARGs in the recipient strain, and one such event was observed in turkeys. This study provides insights into the possible role of animal hosts, wherein ARGs could spread between Campylobacter strains and species, as well as between different animal hosts.
Understanding the risks for ARG acquisition is complicated by potential transfer of genetic material from distantly related bacteria to potential human pathogens such as Salmonella. Key knowledge gaps include understanding which commensal bacteria can transfer ARGs to foodborne pathogens in vivo, what resistance is being transferred, and if there are critical windows in the animal’s lifecycle that transfer occurs. In support of Objective 2a, a study was conducted with day old chicks inoculated with a strain of Salmonella enterica serovar Heidelberg (S. Heidelberg), that was susceptible to several common antibiotics to evaluate if ARGs found within the microbiota could be transferred to S. Heidelberg. S. Heidelberg quickly acquired tetracycline resistance in vivo, and sequence data is being analyzed to identify additional genes that were transferred to S. Heidelberg. These results have informed the design of a follow up study, evaluating the role of eggs in harboring antibiotic resistant commensals that may be responsible for Salmonella’s acquisition of ARGs; however, new procurement regulations (NDAA 889) delayed the procurement of the eggs for this study. It is expected that these data will direct mitigation strategies to reduce the commensal sources of AMR and acquisition by foodborne pathogens.
Bacterial metabolites are important for host intestinal health, including the bacterial fermentation product, butyrate. Butyrate has been shown to improve intestinal health and reduce intestinal pathogen colonization through its antimicrobial properties. Butyrate can be produced by members of the microbiota, or directly fed to animals as a prebiotic to obtain desired outcomes. In support of Objective 3b, a turkey study was conducted in which a commercially available in-feed encapsulated butyrate product was fed to turkeys to evaluate the effect on C. jejuni colonization. In the study, day of hatch poults were fed a butyrate amended or control diet (no butyrate added) for 35 days and then inoculated with C. jejuni. Encapsulated butyrate significantly reduced C. jejuni cecal colonization 14 days post-inoculation compared to the control diet. Additional work is needed to understand the effect of butyrate on the turkey intestinal mucosa. These results have informed the design of follow up studies to compare endogenous and exogenous sources of butyrate in turkeys as a potential intervention for foodborne pathogens.
Accomplishments
1. Salmonella induced a robust immune response in turkeys but did induce clinical disease. Poultry is a common source of Salmonella-associated foodborne illness, and approximately six percent of illnesses are attributed to consumption of improperly prepared or handled turkey products. Evaluating the response of turkeys to Salmonella is critical in developing strategies to minimize colonization and reduce food safety risk. ARS scientists in Ames, Iowa, evaluated bacterial load and blood cell gene expression responses of turkeys challenged with the foodborne pathogen Salmonella enterica serovar Typhimurium. A large increase in the expression of turkey immune genes was detected in blood cells, while Salmonella colonized the gastrointestinal tract and systemically disseminated to the spleen without causing overt clinical signs of disease in the turkeys. These analyses revealed genes and pathways by which turkey blood immune cells responded to the pathogen and can provide potential targets for developing intervention strategies or diagnostic assays to mitigate S. Typhimurium colonization in turkeys, thereby assisting producers in reducing the presence of Salmonella in commercial poultry flocks for improved food safety.
