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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

2017 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
Understanding the effects of antibiotics on both the host and bacteria in the gastrointestinal tract is warranted due to the importance of antibiotics in turkey and swine production for treating and preventing disease, and historically for improving growth. In support of Objective 1a, data were analyzed from a study in which turkeys were fed diets formulated with two different concentrations, a high dose and a low dose, of the commonly used antibiotic formulation bacitracin methylene disalicylate (BMD) to evaluate the effects of antibiotic-induced changes on turkey gene expression and bacteria in the intestine, known as the microbiome. Preliminary results suggest that BMD modulates the bacteria present in the intestine. Overall, both concentrations of BMD had immediate and lasting impacts on the structure of the microbiota, reducing the types of bacteria in the BMD-treated turkeys through the end of the study. Another method undertaken to analyze the bacterial communities in the samples was metagenomics, which is the study of the collective genomes of all the bacteria in the sample. Metagenomic sequence data revealed many antibiotic resistance genes enriched within the microbiota of birds fed the high dose of BMD by the end of the study. The resistance genes potentially confer resistance to beta-lactam, aminoglycoside, tetracycline, vancomycin, and macrolide antibiotics, suggesting that BMD co-selects for multiple antibiotic resistance genes. Additional analysis will explore functional shifts within the microbiota of birds receiving low dose and high dose BMD. In addition to the intestinal microbiota, the effect of in-feed BMD on the turkey’s intestinal health was assessed by sequencing messenger RNA (mRNA) isolated from turkey intestinal tissue (cecum). Analysis of the genes expressed is ongoing, but the data suggest that feeding BMD to turkeys significantly alters intestinal gene expression for nutrient acquisition and innate immunity. Direct antibiotic exposure of bacteria to antibiotics can be a selective pressure for antibiotic resistance. Antibiotics are commonly administered to animals in-feed because of the ease of use of administration, but in-feed may also be a mechanism in which bacteria and antibiotics are in contact and result in selection for antibiotic resistance genes. Thus, one mechanism to limit the selective pressure of antibiotics on intestinal bacteria but still provide a judicious alternative for adequate animal treatment is to administer the antibiotic differently. Thus, in support of Objective 1a, a study evaluating differential effects of route of antibiotic administration on swine microbiota, antibiotic resistance genes, and intestinal immune status was conducted. Pigs were administered the same antibiotic, oxytetracycline, either orally (in-feed) or injected (intramuscular). Plasma, nasal washes, and intestinal samples (gut contents and tissues) were collected at various times after administration. The results indicate that orally administered oxytetracycline significantly disrupted the gut microbiota by four days after initiating treatment, whereas the injected antibiotic did not disrupt the microbiota by that time. By seven days following the oxytetracycline injection, the gut microbiota was disrupted, but not to the extent of the microbiota of the pigs that were administered oxytetracycline in-feed. Injected antibiotic led to higher plasma concentrations of oxytetracycline than in-feed administration, whereas in-feed administration led to higher nasal fluid concentrations than injection. Plasmid-borne antibiotic resistance genes are also being evaluated from this study, with the preliminary results showing many transferrable antibiotic resistance genes in the swine gut. To better inform judicious use practices, a follow-up study was performed to evaluate the efficacy of direct oral versus in-feed antibiotic administration on treating bacterial pneumonia associated with Pasteurella multocida. Data generated from this study will inform producers and veterinarians on management practices that limit the impact of antibiotic administration on antibiotic resistance, while still providing disease treatment. Alternatives to in-feed antibiotics are urgently needed as the United States livestock and poultry industries move to use fewer antibiotics in animal production. A study was completed with partners at Iowa State University to evaluate the effects of the non-antibiotic dietary feed additives inulin, resistant starch, or encapsulated butyrate for nursery-age pigs on their immune system and microbiome. A group fed a non-amended diet or diet with typical levels of chlortetracycline was included as controls. This work supports Objective 1b, “Test the efficacy of novel probiotics as non-antibiotic feed additives to improve gut health”, where we have focused on prebiotics, as opposed to probiotics, as an approach to improve gut health due to utility of use by producers. Results indicate that inclusion of resistant starch in the diet of nursery pigs significantly improved growth performance when compared to non-amended diet. In addition, the data indicate resistant starch enriches beneficial bacterial communities in the gut, and these beneficial bacteria produce butyrate, a small molecule known to promote gut health. These data, along with ongoing studies, indicate non-antibiotic alternatives can be used to enhance performance in pigs. In further support of Objective 1b, a series of studies were completed to evaluate the impact of beta-glucan on the swine immune response and intestinal microbiota. Beta-glucan is a component of yeast cell wall, a known modulator of immunity, and is a proposed alternative to antibiotics. Groups of pigs were fed a non-amended diet, or diet with beta-glucan, and samples were collected to evaluate fecal microbial diversity and host immune response to secondary stimulation in the laboratory. Preliminary results suggest that dietary beta-glucan can modulate intestinal health, and analyses are on-going. In addition to the animal study, the impact of beta-glucan on the immune cells (monocytes and macrophages) has been completed and results indicate that the beta-glucan zymosan significantly alters how macrophages respond to immunostimulatory bacterial components. Specifically, the macrophage response is reduced in comparison to cells that were not exposed to zymosan, suggesting that zymosan helps the immune system to not over respond to bacteria, which results in inflammation. These results provide a potential mechanism for zymosan modulation of pig immune responses, and additional analysis is ongoing to evaluate beta-glucan’s potential use as a dietary anti-inflammatory. Low level inflammation may contribute to limiting growth of pigs, and non-antibiotic methods to limit inflammation are sought by producers. Vaccination of food-producing animals is a proposed pre-harvest control strategy to lessen the introduction of Campylobacter jejuni (C. jejuni) into the human food chain by reducing levels in the animal. Turkeys naturally carry C. jejuni in their intestinal tract and are a potential source of human infection when a carcass becomes contaminated during processing. To identify vaccine targets, C. jejuni was cultured in different media conditions and differentially expressed genes were identified. Six genes were selected and cloned into a bacterial expression system to enable purification of the gene products. Preliminary data suggest that the cloning was successful and laboratory experiments are now being performed to purify each protein. These results will help define candidate C. jejuni proteins to be used as vaccine antigens for limiting C. jejuni in turkeys. This work is being done in support of Objective 3B: Develop and test novel mucosal vaccines for efficacy against Campylobacter challenge. The resistance gene mcr1 in bacteria confers resistance to colistin, which is an antibiotic of last resort to treat certain human infections. Recently, two Escherichia coli (E. coli) strains of swine origin that harbor the mcr1 gene were discovered in the United States. We obtained the strains to determine how the mcr1 gene arose in E. coli strains from swine. In addition, other mcr1-containing E. coli strains from agricultural animals were submitted for genome sequencing to compare the genetic mechanisms for transferring the mcr1 gene. Preliminary analyses showed that the isolates from United States swine carry the mcr1 gene on a different mobile element than the isolates from Chinese poultry. Additionally, most of the mcr1-containing isolates carry additional mobile elements and antibiotic resistance genes, suggesting that using diverse antibiotics, not just colistin, could select for further dissemination of this gene into other bacteria. These results are important for informing the movement of the mcr1 gene among bacteria in the environment and identifying ways to prevent its transmission.

1. Identified specific bacterial species common to swine worldwide. The gut bacterial community, or microbiota, is an essential contributor to animal health. In agricultural animals such as swine, knowledge of what this microbiota looks like in health and disease is important so that humans can use feed additives or probiotics to change the microbiota towards health and away from disease. ARS researchers in Ames, Iowa, undertook a meta-analysis of all publically available swine microbiota data in an effort to gain insight into commonalities and differences among swine gut microbiota datasets. The results showed that each discrete study yielded a significantly different microbiota, suggesting that technical variation between labs and experiments may mask true biological differences. Gut location (i.e., small versus large intestine) was the second most important variable relating to the microbiota, reflecting the different functions of the microbiota in each location. Despite overall differences, some bacterial genera were detected in over 90 percent of gastrointestinal samples; representing bacteria that are well adapted to the swine gut and may serve as markers of a typical swine gut microbiota. These findings will help scientists and producers understand which bacteria can be expected in the swine gut in order to benchmark future studies and to inform potential interventions to engineer a healthy microbiota.

2. Developed protocol to sterilize turkey eggs and hatch germ-free poults. Raising animals in the absence of bacteria is an important way to study specific interactions between the gut bacteria (microbiota) and the development of the host immune system. ARS researchers in Ames, Iowa, established a germ-free system for raising turkeys by evaluating the sterilization of eggs prior to hatching. This is important because the eggshell is the source for a hatchling’s gut microbiota, so the shell must be sterilized to ultimately raise a germ-free animal. Disinfectants and antiseptics were evaluated for killing bacteria present on the egg’s surface without compromising the developing turkey embryo in the egg. Following the developed sterilization protocol, turkey poults were hatched that were free of detectable bacteria, as determined by microbiological culture and molecular testing. This method will be useful to scientists interested in studying the interactions between the gut microbiota and the host immune system in a germ-free poultry system, as well as researchers interested in methods to sterilize eggs.

Review Publications
Kumar, S., Bass, B., Bandrick, M., Loving, C.L., Brockmeier, S., Looft, T.P., Trachsel, J., Madson, D.M., Thomas, M., Casey, T., Frank, J.W., Stanton, T., Allen, H.K. 2017. Fermentation products as feed additives mitigate some ill-effects of heat stress in pigs. Journal of Animal Science. 95:279-290. doi: 10.2527/jas.2016.0662.
Castaneda Correa, A., Trachsel, J., Allen, H.K., Corral-Luna, A., Gutierrez-Banuelos, H., Ochoa-Garcia, P.A., Ruiz-Barrera, O., Hume, M.E., Callaway, T.R., Harvey, R.B., Beier, R.C., Anderson, R.C., Nisbet, D.J. 2017. Effect of sole or combined administration of nitrate and 3-nitro-1-propionic acid on fermentation and Salmonella survivability in alfalfa-fed rumen cultures in vitro. Bioresource Technology. 229:69-77. doi: 10.1016/j.biortech.2017.01.012.
Holman, D.B., Brunelle, B.W., Trachsel, J., Allen, H.K. 2017. Meta-analysis to define a core microbiota in the swine gut. mSystems. 2(3):e00004-17. doi: 10.1128/mSystems.00004-17.
Sylte, M.J., Chandra, L.C., Looft, T.P. 2017. Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults. Poultry Science. 96(7):2412-2420. doi: 10.3382/ps/pex022.
Olson, Z.F., Sandbulte, M.R., Kunzler Souza, C., Perez, D.R., Vincent, A.L., Loving, C.L. 2017. Factors affecting induction of peripheral IFN-gamma recall response to influenza A virus vaccination in pigs. Veterinary Immunology and Immunopathology. 185:57-65. doi: 10.1016/j.vetimm.2017.01.009.