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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

2016 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. 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 improving growth. In support of Objective 1a, a study was completed in which turkeys were fed diets formulated with two different doses of the commonly used antibiotic bacitracin methylene disalicylate (BMD) to evaluate the effects of antibiotic-induced disturbances on turkey intestinal gene expression and the microbiome. Deoxyribonucleic acid (DNA) has been extracted and the 16S ribosomal ribonucleic acid (rRNA) gene has been sequenced for microbiome analysis and preliminary results suggest that BMD modulates intestinal bacterial membership and function. Methods to isolate high quality ribonucleic acid (RNA) from turkey intestinal tissues have been optimized and isolation from experimental samples for analysis of turkey gene expression is ongoing. Campylobacter jejuni (C. jejuni) isolates with appropriate antibiotic resistance markers, for ease of isolation and enumeration from intestinal samples, have been generated and will be used in future studies of BMD treatment with C. jejuni inoculation. Further analysis will identify common effects on the microbiome and transcriptome due to antibiotic treatment, and whether antibiotic resistance increases with antibiotic administration. In further support of Objective 1a, a study was completed in swine evaluating the differential effect of route of antibiotic administration on swine microbiota, antibiotic resistance genes, and intestinal immune status. Pigs were administered the same antibiotic oxytetracycline, either orally (in-feed) or injected. Intestinal samples (contents and tissues) were collected over time and will be used to determine changes in microbiota, antibiotic resistance genes, and host immunity relative to time of administration and route. Data generated from this study will inform producers and veterinarians on management practices that limit the impact of antibiotic administration on antibiotic resistance. 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 to evaluate the effects of the non-antibiotic feed additive resistant starch in the diet of nursery-age pigs on the immune system and microbiome. This work supports Objective 1b, “Test the efficacy of novel probiotics as non-antibiotic feed additives to improve gut health”, and although resistant starch is a prebiotic, it is an approach to improve gut health. Pigs were fed either a non-amended diet or diet with resistant starch, and data indicate favorable changes to immune cell populations in the large intestine that are beneficial to intestinal health, with no negative impact on growth of the pigs. Preliminary data also suggest that the butyrate-producing bacteria in the swine gut were altered by the resistant starch. A repeat study with collaborators at Iowa State University is ongoing, and includes extended measurement of various production parameters. Collectively, these data will be used to inform producers on ability of resistant starch to enhance intestinal health of pigs. To address Objective 3a, a germ-free turkey model is being developed to study interactions between the gut microbiota and the host immune system. The first step in establishing a germ-free system is to develop tools for removing the native bacteria from the egg’s surface without compromising the developing embryo in the egg. Different antiseptic and disinfection methods to sterilize eggs were tested for embryotoxicity of turkey eggs. Preliminary results suggest that immersion of turkey eggs for longer than 10 minutes in a single compound caused increased embryotoxicity. Embryotoxicity was avoided by allowing the eggs to sit for 5 minutes between two disinfection steps. These results suggest that a combination of two antiseptic baths separated by an air incubation step will provide the best disinfection of surface microbes from turkey eggs so that germ free turkeys can be generated for fulfillment of research objectives. The resistance gene mcr1 in bacteria confers resistance to colistin, which is an antibiotic of last resort to treat certain human infections, was recently discovered in bacteria in China. Just this year in the United States, the mcr1 gene was identified in bacteria isolated from a human patient. An ARS collaborator at Athens, Georgia, screened thousands of isolates from the National Antimicrobial Resistance Monitoring System (NARMS) and identified two Escherichia coli (E. coli) strains of swine origin that harbor the mcr1 gene. The strains were then sent to ARS researchers at Ames, Iowa, to determine how the mcr1 gene arose in these E. coli strains from swine. The deoxyribonucleic acid (DNA) sequence surrounding the mcr1 gene is being characterized to provide information that will be useful in understanding how the mcr1 gene was incorporated into the E. coli genome. In addition, laboratory experiments are being conducted to determine if this gene readily transfers to other bacteria from swine. These results will be important for informing the movement of the mcr1 gene among bacteria in the environment and identifying ways to prevent its transmission.

4. Accomplishments
1. Clusters of antibiotic resistance genes enriched together stay together in swine agriculture. Antibiotic-resistant bacteria are a global health concern that causes an estimated $20 billion in health care costs each year. Tracking the source of antibiotic resistance is a complicated issue because antibiotic usage and resistance is widespread. This widespread distribution is due to the location of resistance genes in the deoxyribonucleic acid (DNA) of bacteria on mobile genetic elements, which are portions of DNA that move between bacteria as a method to share their antibiotic resistance genes with their bacterial neighbors. ARS researchers at Ames, Iowa, in collaboration with researchers at Michigan State University, examined the associations between antibiotic resistance genes and mobile genetic elements in fecal or manure samples from Chinese swine farms and United States research swine. Many resistance genes and mobile genetic elements were found enriched together, meaning when one gene increased or decreased in abundance, partner genes increased or decreased in nearly identical fashion. These findings will help veterinarians and producers understand the risks associated with antibiotic selection, and develop best practices for prudent agricultural antibiotic use.

2. Defining the function and diversity of bacterial genes important for butyrate production in the swine gut. The proper balance of commensal gut bacteria is critical for optimum animal health and resistance to disease. In the large intestine, bacteria turn undigested food into various compounds that nourish host tissues, including the compound butyrate. Butyrate has been shown to improve gut and immune health. Currently, the community of butyrate-producing bacteria is not well studied in swine. ARS scientists at Ames, Iowa, have characterized the way in which some swine-associated bacteria produce butyrate and developed tools to investigate this community of bacteria. The results show that the swine gut is home to diverse butyrate-producing bacteria and the potential for butyrate production can be studied by sequencing the gene that encodes butyrate production. This work is important for scientists developing non-antibiotic alternatives for swine because butyrate-producing bacteria are a choice for probiotics and the tools developed from this work will allow for the identification of these bacteria for eventual isolation and development. In particular, butyrate-producing bacteria as a probiotic may serve to decrease carriage of foodborne pathogens in swine.

3. Development of a pipeline for amplifying and analyzing amplicons of the V1-V3 region of the 16S ribosomal ribonucleic acid (rRNA) gene. The technologies used to sequence the most common gene for studying the vast majority of unculturable bacteria, the 16S rRNA gene, are constantly changing and in need of validation. ARS researchers at Ames, Iowa, adapted existing procedures to sequence a particular portion of the 16S rRNA gene on a sequencing instrument that produces millions of sequences in a single run. The procedures were validated by analyzing an artificial bacterial community that was assembled in the laboratory. The results showed that the error rate associated with this procedure can be reduced by removing low-abundant sequences from the analysis, and that removal of these sequences improves the accuracy of bacterial community analyses. These procedures will be used by researchers who are interested in similar regions of the 16S rRNA gene and who are in need of quality-control steps to speed up the analysis of large datasets while maintaining quality of sequence data.

Review Publications
Johnson, T.A., Stedtfeld, R.D., Wang, Q., Cole, J.R., Hashsham, S.A., Looft, T.P., Zhu, Y., Tiedje, J.M. 2016. Antibiotic resistance genes enriched together stay together: Lessons from swine agriculture. mBio. 7(2):e02214-02215. doi: 10.1128/mBio.02214-02215.

Powell, E.J., Cunnick, J.E., Knetter, S.M., Loving, C.L., Waide, E.H., Dekkers, J.C., Tuggle, C. 2016. NK cells are intrinsically functional in pigs with Severe Combined Immunodeficiency (SCID) caused by spontaneous mutations in the Artemis gene. Veterinary Immunology and Immunopathology. 175:1-6. doi: 10.1016/j.vetimm.2016.04.008.

Allen, H.K., Bayles, D.O., Looft, T.P., Trachsel, J., Bass, B., Alt, D.P., Bearson, S.M., Nicholson, T.L., Casey, T. 2016. Pipeline for amplifying and analyzing amplicons of the V1-V3 region of the 16S rRNA gene. BMC Research Notes. 9(380). doi: 10.1186/s13104-016-2172-6.