Skip to main content
ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Research Project #430363

Research Project: Intestinal Microbial Ecology and Metagenomic Strategies to Reduce Antibiotic Resistance and Foodborne Pathogens

Location: Food Safety and Enteric Pathogens Research

2021 Annual Report

1. Characterize the microbiome of swine and turkeys and investigate the effects of antibiotics and non-antibiotic feed additives on the expression and transmission of virulence, fitness or antimicrobial resistance genes in intestinal microbial populations. a. Determine the effects of industry-relevant antibiotics on the swine and turkey gut microbiotas and host gut tissues. b. Test the efficacy of novel probiotics as non-antibiotic feed additives to improve gut health. 2. Assess the interaction of the intestinal immune system and commensal bacteria in swine and turkeys to determine how the microbiota or foodborne pathogens affect tissue innate immunity and acquired immunity, and evaluate non-antibiotic feed additives as an effective strategy to control colonization by foodborne pathogens. a. Characterize the host response to Campylobacter spp. colonization and subsequent changes in intestinal microbiota. b. Test whether microbiota-derived short-chain fatty acids (e.g., butyrate and proprionate) are involved in development of Treg cells in turkeys. 3. Evaluate environmental and host influences on gut bacterial ecological niches and foodborne pathogen control strategies, including vaccines, on phenotypic and genotypic characteristics of foodborne pathogens. a. Identify microbes that initially colonize turkey poults following hatching and evaluate how host development interacts with microbiota succession through the 14-week growth cycle. b. Develop and test novel mucosal vaccines for efficacy against Campylobacter spp. challenged turkeys.

The research addresses food safety at the first link in the food production chain, namely the food-producing animals on the farm. The research investigates the bacterial communities and the animal’s immune response in the intestinal tract, as well as the interactions between them that lead to health and food safety. Experiments are planned to: 1) examine the environmental, microbial, and immunological factors affecting Campylobacter colonization of turkeys by challenging gnotobiotic and conventional turkey poults with Campylobacter after a different dietary amendments and examining the resulting immune response and Campylobacter colonization; 2) investigate collateral effects of therapeutic antimicrobials on animal intestinal bacterial populations by administering antibiotics to young pigs or turkey poults and monitoring their microbiota and immune response over time, and gut tissues at necropsy; 3) define the bacterial and immunological events during initial colonization of the intestinal tract in newly-born piglets and turkeys by monitoring the bacterial colonization of the gut and the immune responses that ensue; 4) examine novel, antibiotic-free intervention strategies to improve animal health and to reduce foodborne pathogen carriage in animals by developing a vaccine against Campylobacter and by administering novel prophylactic treatments to pigs to prevent Salmonella Typhimurium colonization. This basic research will supply knowledge and tools in support of applied research to control foodborne pathogens.

Progress Report
Substantial results were realized over the five years of the project. This is the final report for this project which terminated in December 2020. See the report for the bridge project, 5030-31320-005-00D, “Intestinal Microbial Ecology and Metagenomic Strategies to Reduce Antibiotic Resistance and Foodborne Pathogens” for additional information. Some projects are continuing in the two new Unit projects 5030-32000-225-00D, “Intestinal Microbial Ecology and Non-Antibiotic Strategies to Limit Shiga Toxin-Producing Escherichia coli (STEC) and Antimicrobial Resistance Transmission in Food Animals" which started in March 2021 and 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 started in August 2021. Under Objective 1, progress was made in characterizing shifts in microbial membership in both turkeys and pigs under different treatments, including antibiotics and non-antibiotic feed additives. Specifically, the impact of bacitracin methylene disalicylate (BMD), a common antibiotic administered to turkeys, on turkey intestinal microbiota structure and function (metabolome) was thoroughly characterized. Bacteria associated with metabolic function were identified and may serve as a probiotic for improving turkey performance without antibiotics. Metagenomic sequencing of cecal bacteria revealed antimicrobial resistance genes enriched in the groups fed the high dose of BMD. The antimicrobial resistance genes potentially conferred resistance to beta-lactam, aminoglycoside, tetracycline, vancomycin, and macrolide antibiotics, suggesting that BMD co-selected for genes that confer resistance to compounds other than bacitracin. In addition, under Objective 1, substantial progress was made to understand the impact of route of antibiotic administration on microbiota and antibiotic resistance genes in the feces. Intestinal bacterial community shifts were greatest in pigs administered oral (in-feed) antibiotic when compared to injected administration, and abundance of antimicrobial resistance genes in fecal bacteria were increased in the in-feed group as well. Antimicrobial resistance genes in commensal organisms may transfer to other commensals, including foodborne pathogens, especially during times of major shifts in bacterial populations or under disturbing conditions (eg, antibiotic exposure). Thus, the research indicates injected antibiotic may still provide beneficial effects against disease and limit the disturbances on the intestinal microbiota. In addition, non-antibiotic feed additives including resistant potato starch and beta-glucan were shown to have beneficial effect on pig intestinal microbiota by increasing abundance of beneficial members. Furthermore, resistant potato starch led to decreased intestinal inflammation, which is associated with reduced colonization by the foodborne pathogen Salmonella. Collectively, methods to minimize negative impacts on piglet intestinal microbiota were identified. Many novel organisms from the turkey and swine microbiota were cultured, characterized, and made available to researchers through deposition in public culture repositories. The characterization also informs future microbiota experiments by assigning genus to specific 16S rRNA sequences in the database used for identifying specific taxa in 16S rRNA amplicon datasets. Under Objective 2, substantial progress was made in understanding the pig intestinal immune system through weaning and under different dietary conditions. As noted above, resistant potato starch improved intestinal immune status. In addition, beta-glucan was shown to alter pig innate immune cells. Specifically, cells from beta-glucan fed pigs produced different amounts of inflammatory cytokines than cells from pigs on the non-amended diets, indicating beta-glucan can modulate the immune response of some immune cells. The modulation of monocytes is important, because they play a role in limiting intestinal disturbances upon infection. Upon Salmonella challenge, both resistant potato starch and beta-glucan limited the amount of Salmonella shed by pigs. Substantial progress was made under Objective 2 in turkeys as well, with challenge models for both Campylobacter jejuni and Campylobacter coli developed. C. jejuni was detected in the liver of inoculated poults, indicating dissemination of the foodborne pathogen outside the intestinal tract. The turkey intestinal immune response following C. coli challenge was elevated, but relatively quickly returned to normal despite the continued presence of C. coli. Preliminary trials performed in turkeys suggest dietary butyrate may limit C. jejuni colonization. Butryate is a short-chain fatty acid produced by commensal organisms, and increasing levels in the turkey ceca may be a method to reduce colonization. In support of Objective 3, some progress was made, but somewhat limited due the lack of required reagents and change in personnel. As noted above, the short-chain fatty acid butyrate was able to limit colonization of turkeys with C. jejuni, though it was unclear if it was associated with an increase in intestinal T-regulatory cells in the turkey. A major advance under Objective 3 was the development of procedures and protocols to hatch poults and chicks in gnotobiotic isolators to better understand microbiota source and succession. Thus, differences in the source of microbiota could be identified (egg versus environment), and it was noted that the eggshell is a significant source of microbes that first colonize chicks. However, environmental bacteria also colonize the bird. Bacteria play important roles in the maturation of the poultry gut, and modulation of the microbes on the egg or in the environment could enhance poultry production and limit foodborne pathogen colonization.