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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

2018 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:
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 gut pathogens. The in-feed prebiotic compound resistant starch (as raw potato starch) modulates the microbiota and stimulates a community of gut bacteria to produce butyrate, which is a molecule known to improve gut health. Additional data suggest that pigs with more butyrate-producing bacteria in their gut shed less of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Therefore, a study was completed to test whether in-feed raw potato starch would reduce Salmonella shedding following experimental challenge of nursery-aged pigs. This work supports Objective 1b, “Test the efficacy of novel probiotics as non-antibiotic feed additives to improve gut health” through the use of prebiotics, as opposed to probiotics, as an approach to improve gut health due to utility of use by producers. Four weeks of in-feed raw potato starch modulated the gut microbiota to be different from that of the pigs fed standard nursery diet without raw potato starch. In addition, pigs fed a diet with raw potato starch prior to and during S. Typhimurium challenge shed less S. Typhimurium in their feces over time compared to pigs fed the standard diet. Data analysis is ongoing to characterize the intestinal immune response and changes in bacterial populations to better understand the mechanism of limiting S. Typhimurium shedding. Collectively, these data indicate that dietary raw potato starch can serve as a prebiotic in nursery-aged swine to modulate the gut in a manner that may reduce shedding of the foodborne pathogen S. Typhimurium. In further support of Objective 1b to identify non-antibiotic alternatives that modulate pig health, a study was conducted with ARS collaborators in Ames, Iowa, to evaluate the impact of various non-antibiotic feed additives on the swine gut microbiota and immune status. Dietary fibers including resistant corn starch and beet pulp, phytogens, immune-stimulating beta-glucan, medium-chain and short-chain fatty acids, zinc and copper, or antibiotics were fed to separate groups of nursery-aged pigs. Fecal and blood samples were collected through the trial, and intestinal samples were collected from pigs that were necropsied after four weeks on respective diets, which is standard for nursery studies. Nucleic acid has been extracted from fecal samples for analysis of microbiota, and from intestinal samples for analysis of intestinal cell gene expression. Preliminarily, dietary beta-glucan modulated immune responses of peripheral blood cells, and altered intestinal gene expression. The data generated from this study will inform nutritionists and producers as to how the various feed additives impact the gut health of nursery-aged swine. Many non-pathogenic bacteria that normally colonize the intestine of animals, referred to as commensals, may possess beneficial traits. Microbiota modulation with the use of probiotics is one proposed strategy for improving animal health, and thus, it is important to identify beneficial traits possessed by commensal bacteria to use this approach. In support of Objective 2b, a novel butyrate-producing bacterium was isolated from the intestinal tract of a healthy chicken and subsequently characterized. The strain belongs to the Megasphaera genus and has been detected in both chickens and turkeys. Whole-genome sequencing of the isolate identified genomic segments that are divergent from other Megasphaera species, thus the species in poultry are divergent from Megasphaera species found in mammals. The isolate was confirmed to produce butyrate, a fermentation product with beneficial properties that is produced by some gut bacteria. Ongoing analyses may identify adaptations by Megasphaera to the poultry gut and provide information to enhance beneficial functions for testing as a probiotic to improve poultry health. Microbial succession, which is the process of development and change in intestinal bacteria populations over time, starts at birth and has long-term implications for animal health. In commercially-reared birds, microbiota succession begins at hatch, with bacteria from the hen that passed to the egg potentially colonizing chicks as they emerge from the egg. In support of Objective 3a, gnotobiotic (germ-free) isolators were used to evaluate gut microbiota colonization in newly hatched chicks in the absence of environmental bacteria. The adapted gnotobiotic system provided a mechanism to assess the contribution of only egg-shell bacteria from the hen to the “normal” microbiota development of chicks. Cecal samples were collected at various time points post-hatch from chicks hatched in isolators devoid of environmental bacteria (other than the egg itself) and conventionally raised chicks (presence of environmental bacteria) for comparison of intestinal tract microbiota. Analysis of bacterial membership is ongoing, but nucleic acid sequence data indicate commensal bacteria prominent in older birds were present on the shell at hatch, suggesting that many of the bacteria in poultry intestines are obtained from the egg rather than from the environment. These data are useful for identifying developmental periods when animals are vulnerable for pathogen colonization (including the egg) as well as opportunities best suited for microbiota modulation strategies. Reagent availability, particularly antibodies that label immune cells and cytokines, is critical for evaluating changes to immune status and animal health. Currently, there are very few turkey-specific antibodies available that allow for evaluation of the turkey immune response. To address Objective 2a, monoclonal antibodies were generated against turkey immune proteins CD3 epsilon chain (CD3e), interleukin (IL)-6, IL-8, IL-10, IL-13, IL17F, IL-22, and IgA heavy chain. Ten hybridoma clones were generated for each turkey protein, and secreted antibodies were evaluated for immunoreactivity against turkey proteins using western blotting and immunohistochemistry. The ten anti-CD3e clones were also analyzed using flow cytometry. Clones secreting antibody reactive against turkey IL-6, IL-8, IL-10, IL-13, IL-17F, IL-22 and IgA heavy chain were identified. None of the cloned antibodies reacted with turkey CD3e by flow cytometry. The developed reagents will be used to characterize the immune response following Campylobacter colonization in the intestinal tract, as well as to understand immune-modulating properties of various in-feed, non-antibiotic additives.


4. Accomplishments
1. Defined collateral effects of the in-feed antibiotic, carbadox, on the total gut bacterial community. Carbadox is an antibiotic not used in humans but widely used in U.S. pig farms. It is important to study possible side-effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Carbadox can induce phages in swine gut bacteria and Salmonella enterica serovar Typhimurium, a foodborne pathogen. Phages are viruses that infect bacteria and are important because they kill bacteria and they transfer genetic material between cells. ARS researchers in Ames, Iowa, fed carbadox to nursery-aged swine to characterize its impact on the swine gut microbiota, particularly phages. Two days after the initiation of carbadox in the feed, gut bacteria in the carbadox-fed pigs expressed different genes than the gut bacteria in the non-medicated pigs. The differences indicated that the gut bacteria in the carbadox-fed pigs were not multiplying or metabolizing carbohydrates as they normally would, and that phages were being induced in the gut microbiota. In addition, phage genetic material encoded antibiotic resistance genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This research highlights the collateral effects of antibiotics and demonstrates the need for scientists, policy-makers, regulators, and farmers to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered.

2. Identified contemporary isolates of Campylobacter jejuni (C. jejuni) able to colonize the intestinal tract of commercial turkeys. Campylobacter jejuni is the main bacterial foodborne disease in humans, and ingesting contaminated poultry products is the most common route by which humans are exposed. Reducing the amount of Campylobacter in turkeys prior to slaughter is a mechanism to reduce the amount of Campylobacter in retail poultry products and lower disease transmission to humans. To test intervention strategies to limit Campylobacter in turkeys, a research model using contemporary strains of C. jejuni in turkeys was developed by ARS researchers in Ames, Iowa, and different Campylobacter-selective media for Campylobacter enumeration from intestinal samples were evaluated in collaboration with ARS researchers in Athens, Georgia. Contemporary and common lab isolates of C. jejuni colonized the intestinal tract of turkeys, with the cecum as a preferred site of colonization. In addition, experimental Campylobacter-selective media with sulfamethoxazole and commercial Campylobacter agar were equally reliable to enumerate the number of Campylobacter in turkey intestinal samples. The establishment of the animal model is critical for testing pre-harvest strategies to reduce the amount of Campylobacter in turkeys, which will promote a safe food supply.

3. Characterized a novel beneficial bacterium from the swine gut. Modulating the gut microbial community, or microbiota, via the administration of novel probiotics is one potential way to improve animal health. ARS researchers in Ames, Iowa, isolated bacteria from the swine gut and discovered a bacterium that was different from all other known bacterial species. Characterization of the bacterium, named Butyricococcus porcorum, by comparison to its closest known relatives indicate it is a coccus-shaped anaerobe with an extracellular capsule that grows on simple sugars but not complex carbohydrates, and it produces butyrate. Although the genome sequence specifies that the isolate encodes sporulation genes, sporulation could not be induced in the laboratory. The inability to sporulate limits use as a probiotic because it is a strict anaerobic bacterium, meaning it is non-viable upon exposure to oxygen. The strain was deposited in international microbial repositories, which can be accessed by researchers globally. The research and isolate are useful to scientists who study the gut microbiota because this is the first characterization of a bacterium that had previously only been detected by nucleic acid sequencing alone. Bacterial characterization studies such as these are needed in order to know how the various members of the gut microbiota function, and characterizing bacteria in culture enables subsequent inferences from studies that examine nucleic acids alone.


Review Publications
Choi, J., Rieke, E.L., Moorman, T.B., Soupir, M.L., Allen, H.K., Smith, S., Howe, A. 2018. Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance. FEMS Microbiology Ecology. 94(4). https://doi.org/10.1093/femsec/fiy006.

Sylte, M.J., Inbody, M.H., Johnson, T., Looft, T.P., Line, J.E. 2018. Evaluation of different Campylobacter jejuni isolates to colonize the intestinal tract of commercial turkey poults and selective media for enumeration. Poultry Science. 97(5):1689-1698. https://doi.org/10.3382/ps/pex384.

Shippy, D.C., Bearson, B.L., Holman, D.B., Brunelle, B.W., Allen, H.K., Bearson, S.M. 2018. Porcine response to a multidrug-resistant Salmonella enterica serovar I 4,[5],12:i:- outbreak isolate. Foodborne Pathogens and Disease. 15(5):253-261. https://doi.org/10.1089/fpd.2017.2378.

Johnson, T.A., Looft, T.P., Severin, A.J., Bayles, D.O., Nasko, D.J., Wommack, E., Howe, A., Allen, H.K. 2017. The in-feed antibiotic carbadox induces phage gene transcription in the swine gut microbiome. mBio. 8(4):e00709-17. https://doi.org/10.1128/mBio.00709-17.

Trachsel, J., Humphrey, S.B., Allen, H.K. 2018. Butyricicoccus porcorum sp. nov. a butyrate-producing bacterium from swine intestinal tract. International Journal of Systematic and Evolutionary Microbiology. 68:1737-1742. https://doi.org/10.1099/ijsem.0.002738.

Lindblom, S., Gabler, N., Dilger, R., Olson, Z.F., Loving, C.L., Kerr, B.J. 2018. Influence of feeding thermally peroxidized soybean oil on oxidative status in growing pigs. Journal of Animal Science. 96:545-557. https://doi.org/10.1093/jas/sky005.

Latham, E.A., Pinchak, W.E., Trachsel, J., Allen, H.K., Callaway, T.R., Nisbet, D.J., Anderson, R.C. 2018. Isolation, characterization and strain selection of a Paenibacillus species for use as a probiotic to aid in ruminal methane mitigation, nitrate/nitrite detoxification and food safety. Bioresource Technology. 263:358-364. https://doi.org/10.1016/j.biortech.2018.04.116.