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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

2019 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
Oxytetracycline is an important therapeutic option for disease treatment in swine and is available in different formulations, including injectable and in-feed. While the in-feed route is easier to administer at the herd level, the in-feed approach may have more impact on intestinal bacterial populations, including those harboring antibiotic resistance genes. In support of Objective 1, Subobjective 1A “Determine the effects of industry-relevant antibiotics on the swine microbiota” and provide data on the impact of in-feed versus injected antibiotics in swine, therapeutic oxytetracycline was either orally (in-feed) or intramuscularly administered to groups of swine. The in-feed route of antibiotic administration had a greater impact on the gut bacterial communities and the abundance of some antibiotic resistance genes than did the intramuscular route. Two genes that confer resistance to tetracycline-family antibiotics increased in abundance in the guts of pigs fed oxytetracycline compared to bacteria in intestine of pigs in non-medicated and injected groups. In addition, increased abundance of a gene that confers resistance to an antibiotic not administered (aminoglycoside-family antibiotics) was detected in the in-feed group. Concentration of oxytetracycline was greatest in feces of pigs given in-feed oxytetracycline, compared to injected group, whereas levels were highest in plasma of the injected group. Final data analysis on plasmid sequences from specific bacterial populations is being completed. This research shows that that in-feed antibiotic administration has a greater impact on the gut microbiota and antibiotic gene resistance levels than intramuscular administration. The use of non-antibiotic feed additives to control disease, improve animal health, and limit antimicrobial usage is a top priority for producers, but research is needed to define the mechanism of how various additives improve health and limit colonization with gut pathogens and foodborne organisms. In support of Objective 1, Subobjective 1B, “Test the efficacy of novel probiotics as non-antibiotic feed additives to improve gut health” studies are ongoing using prebiotics and immunomodulators, as opposed to probiotics, as an approach to improve gut health and identify associated mechanisms. The compound beta-glucan can modulate pig immune cell function in vitro, and beta-glucan is a non-antibiotic feed additive believed to modulate immune status in vivo. Results from a series of in vivo studies suggest that dietary beta-glucan modulates intestinal microbial populations, and markers of intestinal barrier function. Subsequently, a study was completed to test whether in-feed beta-glucan would alter pig immune status and subsequently reduce Salmonella shedding following experimental challenge of nursery-aged pigs. Pigs fed a diet with beta-glucan prior to and during Salmonella challenge shed less Salmonella in their feces over time compared to pigs fed the standard, non-amended diet. Data analysis is ongoing to further characterize intestinal immune status of pigs fed dietary beta-glucan and identify the mechanism of limiting Salmonella shedding. In further support of Subobjective 1b to test the efficacy of non-antibiotic feed additives to improve gut health, analysis is ongoing to identify mechanisms by which various non-antibiotic feed additives alter intestinal immune and barrier status. Intestinal samples collected from pigs fed diets with dietary fibers including resistant corn starch and potato starch, and immune-stimulating beta-glucan, therapeutic levels of zinc and copper, or antibiotics were assessed for changes in mucin gene expression and mucus type. In addition, changes in immune cell phenotype and abundance in intestinal samples is being assessed by flow cytometry and immunohistochemistry. Mucus plays an important role in intestinal barrier function, and dietary potato starch increased the number of cells expressing mucin-2, an important protein component of mucus. Dietary antibiotic and metals appear to alter the population of immune cells in the intestine, which may be a factor in altering metabolism. Data analysis is ongoing to further characterize immune cell population changes in the intestine of nursery pigs that grow better when administered in-feed antimicrobial diets. In addition, changes in intestinal immune cell metabolic capacity are ongoing. The data generated from the evaluation will inform nutritionists and producers as to how the various feed additives impact the gut health of nursery-aged swine. Currently, there are no commercially available Campylobacter intervention strategies available for pre-harvest reduction in turkeys, and an understanding of the immune response elicited during colonization may provide insight on intervention methods. In support of Objective 2, Subobjective 2A “Characterize the host response to Campylobacter spp. colonization” the turkey immune gene expression in intestinal tract was assessed by real-time PCR. Tissue samples were collected at the acute (days 3 and 7) and later phase (day 21) of colonization. Targets under evaluation include host-defense peptides and innate immune markers, at multiple intestinal sites. Preliminary data suggests little change in pro-inflammatory gene expression in the cecum early after inoculation (day 3), but expression of pro-inflammatory, host-defense peptide and innate markers were localized to the cecal tonsil. By day 7, pro-inflammatory and host defense peptide gene expression in the cecal tonsil was undetectable. By day 21 post-inoculation, gene expression was not different between non-inoculated and inoculated birds, despite continued colonization, indicating the turkeys were tolerant to Campylobacter. Analysis of gene expression is ongoing, but these data suggest that intervention strategies may need to focus on modulating the immune response in the cecal tonsil to reverse immune tolerance and reduce Campylobacter colonization. Some human Campylobacter outbreaks have been linked to ingesting food products containing chicken liver, which may be due to spreading from the intestinal tract to the liver. In further support of Objective 2 to explore extra-intestinal reservoirs of Campylobacter, turkeys were experimentally inoculated with Campylobacter jejuni (C. jejuni) and the amount of C. jejuni in the intestine and liver was determined. The intestinal samples were positive for C. jejuni. C. jejuni was recovered in some, but not all, of the liver samples of C. jejuni inoculated turkeys. These data have identified liver as a reservoir of Campylobacter, an important consideration for processing and consumption of turkey liver in human and companion animal products. Microbial succession is the process of development and change in intestinal microbiota over time and has long-term implications for animal health. In commercially-reared birds, microbiota succession begins at hatch, with bacteria from the hen passed to the eggshell, potentially colonizing chicks as they emerge from the egg. In support of Objective 3, Subobjective 3A, samples were analyzed from a series of animal trials comparing microbial succession of chicks hatched in isolators in the presence of only eggshell associated microbes, environmental bacteria, or chicks hatched under conventional conditions. Microbiota DNA sequence data indicate cecal bacterial communities were different between the three treatment groups, and the conventional birds most resembled chicks hatched in the presence of the egg microbiota, suggesting the eggshell is an important microbial source for microbiota development in chicks. Analysis is ongoing to identify the bacterial members uniquely acquired from the eggshell versus the environment. The data will identify the developmental periods chicks are vulnerable to colonization with foodborne organisms, as well as identify opportunities best suited for microbiota modulation strategies. In support of Objective 3, Subobjective 3B “Develop and test novel mucosal vaccines for efficacy against Campylobacter spp.” isolation of recombinant-expressed Campylobacter proteins identified through a unique screen is ongoing. To date, 13 putative Campylobacter antigens have been cloned into prokaryote expression vectors and five have been selected for expression in Escherichia coli. The recombinant proteins will be useful in understanding immune response to C. jejuni colonization and utility of C. jejuni proteins in assays to identify colonized animals. Also, the proteins may serve as novel Campylobacter vaccine antigen.

1. The in-feed antibiotic Bacitracin methylene disalicylate (BMD) impacts turkey intestinal microbiota structure and function (metabolome). Concern for antibiotic resistance and restrictions on agriculture antibiotic use has heighted the need to identify the mechanism of action of in-feed antibiotics. BMD is an in-feed antibiotic with label use for feed efficiency (low dose) and therapeutic (high dose) applications in poultry production. ARS researchers in Ames, Iowa, identified immediate and lasting changes to the microbiome of turkeys after feeding low or high dose BMD. BMD reduced the number of bacterial members immediately after treatment, a trend that lasted after the antibiotic was removed from the diet. BMD induced functional shifts among hundreds of metabolites, many within bacterial populations likely related to the feed efficiency applications of BMD. Several bacterial members of the microbiota were associated with the increased presence of beneficial metabolites with in the turkey intestine. Bacteria associated with metabolic functional shifts in turkeys offer promising targets for non-antibiotic methods to enhance poultry gut health.

2. Non-antibiotic, in-feed product alters gut microbiota and immune status in nursery pigs. The use of non-antibiotic feed additives to control disease, improve animal health, and limit antimicrobial usage is a top priority for producers, but research is needed to define the mechanism of how various additives improve intestinal health. Nursery age pigs were fed a diet with raw potato starch (or non-amended diet) and within four weeks of in-feed raw potato starch the gut microbiota exhibited shifts associated with gut health. Intestinal concentrations of the beneficial short-chain fatty acid butyrate were increased, and markers of intestinal mucosal defense were detected by ARS researchers in Ames, Iowa, in pigs fed resistant starch. Collectively, dietary raw potato starch is a commercially available product that can serve as a prebiotic in nursery-aged swine to modulate the gut in a manner to promote intestinal integrity.

3. Campylobacter jejuni (C. jejuni) responds to animal mucus in a source dependent manner for environment specific adaptation. C. jejuni is the leading cause of bacterial foodborne illness in the US and typically occurs after ingesting C. jejuni contaminated poultry products, though other food products can also be a source. C. jejuni does not make poultry sick and understanding how C. jejuni colonizes the intestine of poultry without causing disease may help identify strategies to eliminate C. jejuni in food animals. Mucus lines the intestinal tract where C. jejuni grows, and mucus can serve as an energy source for the bacteria. ARS researchers in Ames, Iowa, determined C. jejuni responds differently when grown on chicken or turkey mucus, compared to cow, pig, or sheep mucus. Changes were associated with factors essential for survival in the poultry gut. Binding of C. jejuni to intestinal cells was also altered by the source of mucus, suggesting that mucus may be an environmental cue for C. jejuni growth and attachment to intestinal epithelial cells. The interaction between mucus and C. jejuni may be disrupted to modulate C. jejuni colonization in poultry food animals.

4. Campylobacter jejuni (C. jejuni) induces minimal inflammation in the turkey cecum following colonization. C. jejuni is the main bacterial foodborne disease in humans and ingesting contaminated poultry products is the most common route by which humans are infected. Understanding how turkeys immunologically respond to C. jejuni may provide insight on methods to limit bird carriage. ARS researchers in Ames, Iowa, developed a research model to study the host-response in the intestinal tract of turkeys to C. jejuni, and improved methods to detect C. jejuni in animal samples. Inoculation with C. jejuni induced inflammation in the intestinal tract of turkeys immediately following colonization, but the response was not sustained and quickly resolved. The identification of differences in host genes expressed following C. jejuni colonization of turkeys is a critical first step to develop Campylobacter intervention strategies that promote a safe food supply.

5. The poultry intestine harbors unique bacteria with beneficial functions. Commensal bacteria are important for animal health because they assist feed digestion, immune system stimulation, displacement of pathogens, and production of beneficial compounds. However, many commensal bacteria in the poultry gut are not studied and their functions are unknown. ARS researchers in Ames, Iowa, isolated, characterized, and sequenced the complete genome of a novel commensal bacterium isolated from the chicken intestine, Megasphaera stantonii (M. stantonii). M. stantonii likely colonizes several poultry species, including chickens and turkeys. Similar species are found in swine and cattle intestine, but this poultry isolate has a highly divergent genome, resulting in the designation of M. stantonii as a novel species. M. stantonii produces butyrate, a short-chained fatty acid that has beneficial effects on the animal intestine and immune system. M. stantonii was deposited into multiple bacterial culture collections to make the strain available globally to researchers investigating poultry health. Identifying important functions in the commensal population and enhancing growth of these beneficial bacteria is a promising way to improve animal health.

Review Publications
Looft, T.P., Cai, G., Choudhury, B., Lai, L.X., Lippolis, J.D., Reinhardt, T.A., Sylte, M.J., Casey, T.A. 2019. Avian intestinal mucus modulates Campylobacter jejuni gene expression in a host-specific manner. Frontiers in Microbiology. 9:3215.
Boettcher, A., Loving, C.L., Cunnick, J., Tuggle, C. 2018. Development of severe combined immunodeficient (SCID) pig models for translational cancer modeling: future insights on how humanized SCID pigs can improve preclinical cancer research. Frontiers in Oncology. 8:559.
Li, Q., Schmitz-Esser, S., Loving, C.L., Gabler, N.K., Gould, S.A., Patience, J.F. 2018. Exogenous carbohydrase added to a starter diet reduced markers of systemic immune activation and decreased Lactobacillus in weaned pigs. Journal of Animal Science. 97(3):1242-1253.
Bearson, S.M., Bearson, B.L., Sylte, M.J., Looft, T.P., Kogut, M.H., Cai, G. 2019. Cross-protective Salmonella vaccine reduces cecal and splenic colonization of multidrug-resistant Salmonella enterica serovar Heidelberg. Vaccine. 37(10):1255-1259.
Bakker, M.G., Looft, T., Alt, D.P., Delate, K., Cambardella, C.A. 2018. Bulk soil bacterial community structure and function respond to long-term organic and conventional agricultural management. Canadian Journal of Microbiology. 64(12):901-914.
Johnson, T., Sylte, M.J., Looft, T.P. 2019. In-feed bacitracin methylene disalicylate modulates the turkey microbiota and metabolome in a dose-dependent manner. Scientific Reports. 9:8212.
Trachsel, J., Briggs, C., Gabler, N.K., Allen, H.K., Loving, C.L. 2019. Dietary resistant potato starch alters intestinal microbial communities and their metabolites and markers of immune regulation and barrier function in swine. Frontiers in Immunology. 10:1381.
Li, Q., Burrough, E.R., Gabler, N.K., Loving, C.L., Sahin, O.A., Gould, S.A., Patience, J.F. 2019. A soluble and highly fermentable dietary fiber with carbohydrases improved gut barrier integrity markers and growth performance in F18 ETEC challenged pigs. Journal of Animal Science. 97(5):2139-2153.
Sylte, M.J., Johnson, T.A., Meyer, E.L., Inbody, M.H., Trachsel, J., Looft, T., Leonardo, S., Wu, Z., Zhang, Q. 2019. Intestinal colonization and acute immune response in commercial turkeys following inoculation with Campylobacter jejuni constructs encoding antibiotic-resistance markers. Veterinary Immunology and Immunopathology. 210:6-14.
Hansen, R.L., Duenas, M.E., Looft, T., Lee, Y.J. 2018. Nanoparticle microarray for high-throughput microbiome metabolomics using matrix-assisted laser desorption ionization mass spectrometry. Analytical and Bioanalytical Chemistry. 411(1):147-156.
Maki, J.J., Looft, T. 2018. Complete genome sequence of Megasphaera stantonii AJH120T, isolated from a chicken cecum. Microbiology Resource Announcements. 7:e01148-18.
Maki, J.J., Looft, T., 2018. Megasphaera stantonii sp. nov., a butyrate-producing bacterium isolated from the cecum of a healthy chicken. International Journal of Systematic and Evolutionary Microbiology. 68:3409-3415.
Hedblom, G.A., Reiland, H.A., Sylte, M.J., Johnson, T.J., Baumler, D.J. 2018. Segmented filamentous bacteria - metabolism meets immunity. Frontiers in Microbiology. 9:1991.