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

Location: Food Safety and Enteric Pathogens Research

2020 Annual Report

Objectives
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.

Approach
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 antibiotic for disease treatment in swine and is available in different formulations, including injectable and in-feed. The in-feed route is not only easier to administer at the herd level, we showed that 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 to provide data on the impact of in-feed versus injected antibiotics in swine, a trial using therapeutic oxytetracycline either orally (in-feed) or intramuscularly administered to groups of swine was previously performed. Analysis on microbial communities, oxytetracyline levels in different sites, and abundance of antibiotic resistance genes was previously completed. The final analysis on plasmid-enriched samples revealed multiple genetic contexts for the resistance genes and that the majority of antibiotic resistance genes were encoded on small plasmids. Bacitracin methylene disalicylate (BMD) is an antibiotic cocktail commonly used for growth promotion, feed efficiency, and disease prevention and treatment in poultry. BMD is used therapeutically, and subtherapeutic use is permitted under the FDA veterinary feed directive. In support of Objective 1, Subobjective 1A, we analyzed data on microbiota composition and functions generated from a previously conducted animal BMD animal trial. Results indicate BMD impacted turkey intestinal microbiota structure and function (antimicrobial resistance gene content and metabolome). The impact was immediate and had a lasting effect, as many members of the bacterial communities were eliminated, and overall diversity of bacteria in the gut was reduced. Metagenomic sequence data revealed many antimicrobial resistance genes enriched within the microbiota of birds fed the high dose of BMD by the end of the study. 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 further support of Subobjective 1A, a study was completed to assess changes in pig intestinal epithelial T cells, which are important for maintaining a strong intestinal barrier, while pigs were fed a non-amended or antibiotic amended diet beginning at weaning. Tissues were collected from the small and large intestine of pigs at various days post-weaning to assess the abundance and functional phenotype of different T cell populations by flow cytometry. While there were shifts in abundance of different types of T cells across different gut compartments, dietary subtherapeutic antibiotic administration did not impact T cell population abundance or phenotype. Instead, age of the animal and intestinal location seemed to be the major factors associated with population shifts. In addition, evaluation of individual intestinal cells isolated from healthy pigs was completed to deeper characterize modulation of immune status by microbiota and foodborne pathogens. The use of non-antibiotic feed additives to limit antimicrobial usage and improve food safety is a top priority for producers and public health agencies, but research is needed to define the mechanism of how various additives limit colonization of 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 were completed using prebiotics, as opposed to probiotics, as an approach to improve resistance to colonization by foodborne organisms. Results from a series of in vivo studies indicated dietary beta-glucan modulated intestinal microbial populations and markers of intestinal barrier function. Pigs fed a diet supplemented 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. Changes in immune gene expression in the intestine were minimal, but markers of barrier integrity were increased (MUC2 for mucus, tight junction genes). Using a series of in vitro assays, beta-glucan did modulate function of monocytes. Monocytes are important because they migrate into tissues, including the intestine, and trigger immune responses leading to clearance of bacteria, including Salmonella. Monocytes collected from pigs fed a beta-glucan diet were altered compared to pigs on non-amended diet. Currently, there are no commercially available Campylobacter interventions available for pre-harvest reduction in turkeys, and an understanding of the immune response elicited during colonization may provide insights into potential interventions. In support of Objective 2, Subobjective 2A, “Characterize the host response to Campylobacter spp. colonization” turkey immune gene expression in the intestinal tract was assessed by real-time RT-PCR. Tissue samples were collected during the acute (days 3 and 7 post-inoculation) and persistent phases (day 21) of colonization by C. jejuni. Analysis of tissue gene expression was completed, and pro-inflammatory, host-defense peptide and innate marker genes were significantly up-regulated in cecal tonsil early after inoculation (3 days post-inoculation), and to a lesser extent in the cecum at 7 days post-inoculation. By day 21 post-inoculation, very few of the acutely up- or down-regulated genes in cecum or cecal tonsil were differentially expressed compared to non-infected birds, despite continued colonization. These data suggest that turkeys become immunologically tolerant to Campylobacter after colonization. In a separate study, total transcriptomic analysis (RNA-Seq) was used to determine global gene expression in the cecal tonsil of turkeys acutely inoculated (day 2 post-inoculation) with C. coli. Results of this animal study identified differentially expressed genes and pathways. These data have identified gene targets in the turkey cecal tonsil which may be important to consider when developing future Campylobacter mitigation strategies (e.g., vaccines and immunomodulators). In support of Objective 2, Subobjective 2b, “Test whether microbiota-derived short-chain fatty acids (e.g., butyrate and propionate) are involved in development of Treg cells in turkeys,” an animal experiment was performed with day of hatch turkey poults fed a diet with or without an encapsulated butyrate supplement for 49 days. Tregs may change the immune status in poults and allow the poult immune system to respond to C. jejuni and eliminate it from the intestinal tract. Poults were challenged with C. jejuni at the end of the study. Data from this study demonstrated that butyrate supplementation significantly reduced cecal C. jejuni colonization. Intestinal tissues were harvested for gene expression and immunohistochemical detection of immune cells. Analysis of gene expression and immunohistochemistry is ongoing, but early data suggests that butyrate supplementation in the diet may by a useful intervention to reduce Campylobacter colonization in turkeys. In support of Objective 3, Subobjective 3B, “Develop and test novel mucosal vaccines for efficacy against Campylobacter spp.,” progress was made to optimize conditions to express recombinant Campylobacter proteins in Escherichia coli. For three of the five recombinant Campylobacter proteins to be expressed, optimized conditions (e.g., growth temperature, growth medium, length of incubation, IPTG-induction conditions, method to solubilize inclusion body protein and isolation of His-tagged protein from bacterial lysates) were identified. Analysis of serum immunoreactivity towards each of these isolated recombinant proteins is ongoing, and serum from C. jejuni-colonized and -naïve animals is being used to probe for immune recognition of C. jejuni proteins. Additional progress was made to test the efficacy of a recombinant attenuated Salmonella vaccine expressing C. jejuni antigen CjaA in turkeys. Analysis is ongoing to verify expression of the vaccine antigen in vivo.

Accomplishments
1. Informing judicious antibiotic use practices in pigs. Antibiotic resistance is a global concern and calls for improved antibiotic stewardship in human and animal medicine continue. Antibiotics remain an important tool for limiting disease in swine and are commonly delivered by oral administration though injectable formulations are available. While the in-feed route is easier than injected formulations to administer at the herd level, the in-feed formulation may have more impact on intestinal bacterial populations, including those harboring antibiotic resistance genes. ARS researchers in Ames, Iowa, performed a study to compare the impact of in-feed versus injected therapeutic oxytetracycline on levels of oxytetracylcine in blood and feces, intestinal bacteria, and antimicrobial resistance gene abundance. The in-feed route caused a greater shift in gut bacterial communities and increased the abundance of some antibiotic resistance genes when compared to injected route. Specifically, two genes that confer resistance to tetracycline-family antibiotics increased in abundance in the guts of pigs fed oxytetracycline compared to 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 and there were multiple genetic contexts for the resistance genes. This research shows that in-feed oxytetracycline administration has a greater impact on the gut microbiota than intramuscular administration and provides evidence that injection instead of oral antibiotic administration may mitigate the selection pressure for antibiotic resistance, overall, informing judicious use practices.

2. Defined important shifts in pig gut immune cells following weaning. Immune cells located in the lining of the intestinal tract are some of the earliest immune cells to populate the intestine, making them important in shaping early-life intestinal health with impacts on long-term overall health. Early in life, pigs undergo intestinal distress from weaning when they are separated from their mothers, undergo a diet change, and are transferred into a new environment. When weaning occurs, the intestinal immune system is not yet fully developed, and the characteristics of immune cells in the intestinal lining following weaning were undefined. ARS researchers in Ames, Iowa, showed immune cells in the intestinal lining were similar between intestinal locations in younger, more recently weaned pigs but became more different between intestinal locations as pigs continued to age and the immune system became more fully developed. Collectively, these data indicate immune cell populations in the intestinal lining continue to develop after weaning, and early life events may affect how these cells develop. This is important because immune cells in the intestinal lining can impact intestinal health and resistance to foodborne pathogen colonization. Producers may use this data to consider how intervention strategies implemented early in life impact pig intestinal health, overall health, and/or market performance.

3. Eggshell and environmental bacteria colonize the intestinal tract of birds. Growing birds experience developmental changes, including shifts in their intestinal bacteria that have long-term implications for poultry health. Identification of the sources of bacteria that colonize the chicken can inform producers and veterinarians on intervention strategies to enhance beneficial bacterial populations. ARS researchers in Ames, Iowa, determined that both eggshell and environmental bacteria contributed to the total microbial community in the gut. The bacteria that colonized the gut produced byproducts, or metabolites, known to enhance intestinal health by altering immune status and strength of the intestinal barrier. Bacteria on the eggshell colonized birds immediately after hatch and were especially dominant early in life in the upper intestinal tract. However, bacteria from the environment, such as the bedding and water, continued to colonize birds as they aged. Ensuring beneficial bacteria have the opportunity to colonize poultry is essential for maintaining gut health and food safety in animal production. 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.

4. Turkey liver may be a source of Campylobacter jejuni food product contamination. C. jejuni remains the main bacterial foodborne pathogen in humans. Ingestion of contaminated poultry products is the most common route by which humans are infected, and foods prepared with Campylobacter contaminated chicken liver were identified as a source of human disease. Reducing the amount of Campylobacter in turkey products entering the human food supply may decrease the prevalence of human infection. ARS researchers in Ames, Iowa, investigated if C. jejuni disseminated to liver tissue of turkeys following intestinal colonization. Turkeys were intestinally colonized with C. jejuni, and liver was cultured for C. jejuni presence. Turkey liver was identified as a reservoir of C. jejuni after intestinal colonization. Our results provide vital information to scientists and consumers regarding a food safety risk when turkey liver is prepared for human or animal consumption.

Review Publications
Atkinson, B.M., Bearson, B.L., Loving, C.L., Zimmerman, J.J., Kich, J.D., Bearson, S.M. 2019. Detection of Salmonella-specific antibody in swine oral fluids. BMC Porcine Health Management. 5(29). https://doi.org/10.1186/s40813-019-0136-7.
Maki, J.J., Klima, C.L., Sylte, M.J., Looft, T.P. 2019. The microbial pecking order: utilization of intestinal microbiota for poultry health. Microorganisms. 7(10):376. https://doi.org/10.3390/microorganisms7100376.
Oladeinde, A.A., Cook, K.L., Lakin, S., Abdo, Z., Looft, T.P., Herrington, K., Zock, G.S., Plumblee Lawrence, J.R. 2019. Horizontal gene transfer and acquired antibiotic resistance in S. Heidelberg following in vitro incubation in broiler ceca. Applied and Environmental Microbiology. 85(22):e01903-19. https://doi.org/10.1128/AEM.01903-19.
Mou, K.T., Allen, H.K., Alt, D.P., Trachsel, J.M., Hau, S.J., Coetzee, J.F., Holman, D.B., Kellner, S.G., Loving, C.L., Brockmeier, S. 2019. Shifts in the swine nasal microbiota of swine in response to different dosing regimens of oxytetracycline administration. Veterinary Microbiology. 237(1084020). https://doi.org/10.1016/j.vetmic.2019.108386.
Mir, R., Schaut, R.G., Looft, T.P., Allen, H.K., Sharma, V.K., Kudva, I.T. 2020. Recto-anal junction (RAJ) fecal microbiomes of cattle experimentally challenged with Escherichia coli O157:H7. Frontiers in Microbiology. 11:693. https://doi.org/10.3389/fmicb.2020.00693.
Bucher, M., Zwirzitz, B., Oladeinde, A.A., Cook, K.L., Plymel, C., Zock, G., Aggrey, S., Ritz, C., Looft, T.P., Lipp, E., Agga, G.E., Sistani, K.R. 2020. Reused poultry litter microbiome with competitive exclusion potential against Salmonella Heidelberg. Journal of Environmental Quality. 49(4):869-881. https://doi.org/10.1002/jeq2.20081.
Mir, R.A., Schaut, R.A., Allen, H.K., Looft, T.P., Loving, C.L., Kudva, I.T., Sharma, V.K. 2019. Cattle intestinal microbiota shifts following Escherichia coli O157:H7 vaccination and colonization. PLoS One. 14(12):e0226099. https://doi.org/10.1371/journal.pone.0226099.
Sylte, M.J., Shippy, D.C., Bearson, B.L., Bearson, S.M. 2020. Detection of Campylobacter jejuni liver dissemination in experimentally colonized turkey poults. Poultry Science. 99(8):4028-4033. https://doi.org/10.1016/j.psj.2020.03.042.
Nielsen, D.W., Maki, J.J., Looft, T.P., Ricker, N., Sylte, M.J. 2020. Complete genome sequence of Campylobacter jejuni strain NADC 20827 isolated from commercial turkeys. Microbiology Resource Announcements. 9(1):e01403-19. https://doi.org/10.1128/MRA.01403-19.
Maki, J.J., Nielsen, D.W., Looft, T.P. 2020. Complete genome sequence and annotation for romboutsia sp. strain CE17. Microbiology Resource Announcements. 9(23):e00382-20. https://doi.org/10.1128/MRA.00382-20.
Maki, J., Nielsen, D.W., Looft, T.P. 2020. Complete genome sequence and annotation for turicibacter sanguinis MOL361T (DSM 14220). Microbiology Resource Announcements. 9(25):e00475-20. https://doi.org/10.1128/MRA.00475-20.
Ricker, N., Trachsel, J.M., Colgan, P., Jones, J., Choi, J., Lee, J., Coetzee, J., Howe, A., Brockmeier, S., Loving, C.L., Allen, H.K. 2020. Toward antibiotic stewardship: Route of antibiotic administration impacts the microbiota and resistance gene diversity in swine feces. Frontiers in Veterinary Science. 7:255. https://doi.org/10.3389/fvets.2020.00255.
Boettcher, A.N., Li, Y., Ahrens, A.P., Kiupel, M., Byrne, K.A., Loving, C.L., Cino-Ozuna, A., Wiarda, J.E., Adur, M., Schultz, B., Swanson, J.J., Snella, E.M., Ho, C., Charley, S.E., Kiefer, Z.E., Cunnick, J.E., Putz, E.J., Giuseppe, Dell A., Jens, J., Sathe, S., Goldman, F., Westin, E.R., Dekkers, J.C.M., Ross, J.W., Tuggle, C.K. 2020. Novel engraftment and T cell differentiation of human hematopoietic cells in Art-I- IL2RG-IY SCID pigs. Frontiers in Immunology. 11:100. https://doi.org/10.3389/fimmu.2020.00100.
Liu, H., Feye, K.M., Nguyen, Y.T., Rakhshandeh, A., Loving, C.L., Dekkers, J.C., Gabler, N.K., Tuggle, C.K. 2019. Acute systemic inflammatory response to lipopolysaccharide stimulation in pigs divergently selected for residual feed intake. BMC Genomics. 20(728). https://doi.org/10.1186/s12864-019-6127-x.
Wiarda, J.E., Trachsel, J.M., Bond, Z.F., Byrne, K.A., Gabler, N.K., Loving, C.L. 2020. Intraepithelial T cells diverge by intestinal location as pigs age. Frontiers in Immunology. 11:1139. https://doi.org/10.3389/fimmu.2020.01139.
Putz, E.J., Putz, A.M., Boettcher, A., Charley, S., Sauer, M., Palmer, M.V., Phillips, R., Hostetter, J.M., Loving, C.L., Cunnick, J.E., Tuggle, C.K. 2019. Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine. PLoS One. 14(10):e0223483. https://doi.org/10.1371/journal.pone.0223483.
Maki, J.J., Bobeck, E.A., Sylte, M.J., Looft, T.P. 2020. Eggshell and environmental bacteria contribute to the intestinal microbiota of growing chickens. Journal of Animal Science and Biotechnology. 11:60. https://doi.org/10.1186/s40104-020-00459-w.
Byrne, K.A., Loving, C.L., McGill, J.L. 2020. Innate immunomodulation in food animals: evidence for trained immunity? Frontiers in Immunology. 11:1099. https://doi.org/10.3389/fimmu.2020.01099.