Progress 12/16/10 to 12/15/15
Outputs
Progress Report Objectives (from AD-416): 1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety. 2) Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host. Evaluate the effects of antimicrobial alternatives (dietary fermentation products) on swine intestinal health, enteric microbiomes, and foodborne pathogens such as Salmonella, under normal and heat stress conditions. 3) Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety). Develop and evaluate alternatives to antibiotics, emphasizing immunological /vaccine strategies, for impacting the host immunome and reducing the risk of Salmonella transmission to humans. 4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes. 5) Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys). Approach (from AD-416): Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using Campylobacter. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii (M. elsdenii) will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Various technologies will be used to assess the effects of alternatives to antibiotics (e.g. fermentation products) on swine performance and intestinal health. Technologies include intestinal microbiome analyses by QPCR-, phylotype census-, culture-based methods; metagenomic analyses of animal host gene expression (esp. immunome); and traditional immunological techniques. A variable heat stress model for swine will be developed and used to test diurnal heat cycle impacts on animal health and on colonization/shedding of foodborne pathogens such as Salmonella, as well as amelioration of the impacts by antibiotic alternatives. Immunotherapeutic measures (vaccine) will be examined for their impact on the swine immunome and Salmonella shedding. Other areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant Escherichia coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth. This is the final report for project 5030-31320-003-00D terminated in December 2015 and replaced with 5030-31320-004-00D. Substantial results were realized over the five years of the project and some projects are continuing in the new 5030-31320-004-00D project. Under Objective 1, progress was made on identifying potential probiotic bacteria that inhibit the colonization of foodborne pathogens. The native swine gut bacterium Mitsuokella jalaludinii was isolated and shown to inhibit the growth of the foodborne pathogen Salmonella enterica serovar Typhimurium. Culturing a potential antagonist of the foodborne pathogen Campylobacter jejunii (C. jejunii) proved difficult. Many organisms related to potential C. jejuni antagonists were isolated (nearly 200 isolates), but culture efforts and quantitative polymerase chain reaction (qPCR) assays were negative for putative antagonists in the turkeys screened. Significant impacts were made under Objective 2, �Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host�. ARS researchers at Ames, Iowa, defined the side- effects of in-feed antibiotics (carbadox and a combination [sulfamethazine/penicillin/ chlortetracycline]) on the swine gut bacterial community. The use of microbial ecology analyses, such as metagenomics and 16S ribosomal ribonucleic acid (rRNA) amplicon sequencing, were applied to study the changes in the swine gut microbiome during and following antibiotic treatment. Discoveries include that 1) swine microbial communities harbor diverse antibiotic resistance genes regardless of antibiotic treatment, 2) the combination antibiotic co- selects for resistance genes that do not confer resistance to the administered antibiotics, 3) the combination antibiotic leads to an increase in Escherichia coli populations in the gut bacterial community, and 4) carbadox induces bacteriophage-mediated horizontal gene transfer in both the gut microbiome and in cultures of Salmonella enterica serovar Typhimurium. These discoveries are important to scientists, stakeholders, and policy makers who are interested in how antibiotic administration affects resistance gene diversity, horizontal gene transfer, and bacterial diversity. In support of Objective 3, functional metagenomic libraries were constructed from swine fecal deoxyribonucleic acid (DNA) containing over two gigabases of genetic material from the swine gut. Antibiotic resistance genes in these libraries were shown to be present in tandem with other antibiotic resistance genes and mobile elements, informing how antibiotic resistance genes come to be so prevalent in gut microbial communities. Experiments addressing Objective 4 revealed that modulating an established gut microbiota is difficult. Antibiotic-sensitive strains of one bacterium (Megasphaera elsdenii) were administered to piglets, but the presence of these sensitive strains did not exclude eventual colonization by antibiotic resistant strains of the same bacteria. Overall, these data indicate that administration of antibiotic-sensitive strains of bacteria found in the intestinal microbiome is unlikely to be a method to combat colonization with strains that have antibiotic resistance. Objective 5 led to the development of a vaccine to control the human foodborne pathogen Campylobacter jejuni (C. jejuni) in poultry. The vaccine was constructed by putting a gene from C. jejuni into non- virulent strain of Salmonella enterica so that upon oral inoculation into a turkey, the C. jejuni protein would be expressed and the animal would generate an immune response against the protein. Investigations of the immunogenicity and protection of this vaccine in turkeys are ongoing and were incorporated into the new project. Accomplishments 01 Fermentation products as feed additives mitigate some but not all effects of heat stress in pigs. Pigs that experience heat stress show decreased performance, leading to increased production costs and losses to farmers. The exact mechanisms of how heat stress decreases pig performance are unknown. ARS researchers at Ames, Iowa, studied the effects of heat stress on performance, gut structure, and immune functions in post-weaning pigs, and evaluated if a fermented feed additive could help relieve some of the undesired effects of heat stress. The data showed that heat stress damaged gut structure in one part of the small intestine, the jejunum, but not in other gut locations. The pigs that were fed the fermented feed additives had less gut damage, suggesting that the feed additives protected against this kind of damage during heat stress. Additionally, the data did not show any effects of heat stress on immune function by any of the endpoints we measured, suggesting that the decrease in pig performance caused by heat stress is unlikely related to the immune system. The results of this study are useful to scientists, stakeholders, and farmers who are interested in how heat stress affects pig production and what practical solutions could be implemented.
Impacts
(N/A)
Publications
|
Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety. 2) Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host. Evaluate the effects of antimicrobial alternatives (dietary fermentation products) on swine intestinal health, enteric microbiomes, and foodborne pathogens such as Salmonella, under normal and heat stress conditions. 3) Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety). Develop and evaluate alternatives to antibiotics, emphasizing immunological /vaccine strategies, for impacting the host immunome and reducing the risk of Salmonella transmission to humans. 4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes. 5) Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys). Approach (from AD-416): Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using Campylobacter. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii (M. elsdenii) will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Various technologies will be used to assess the effects of alternatives to antibiotics (e.g. fermentation products) on swine performance and intestinal health. Technologies include intestinal microbiome analyses by QPCR-, phylotype census-, culture-based methods; metagenomic analyses of animal host gene expression (esp. immunome); and traditional immunological techniques. A variable heat stress model for swine will be developed and used to test diurnal heat cycle impacts on animal health and on colonization/shedding of foodborne pathogens such as Salmonella, as well as amelioration of the impacts by antibiotic alternatives. Immunotherapeutic measures (vaccine) will be examined for their impact on the swine immunome and Salmonella shedding. Other areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant Escherichia coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth. Bacteria of animal gut microbial communities perform functions that are essential for host health. One group of bacteria of particular importance are those that produce butyrate. Butyrate is a short-chain fatty acid that provides energy and promotes the health of cells in the colon. Following the research objective to characterize intestinal ecological niches of importance to animal health and food safety, ARS researchers in Ames, Iowa, had previously identified several new bacteria from the swine gut that produced butyrate. It was then of interest to develop a method to detect butyrate-producing bacteria under different conditions, such as antibiotic administration or Salmonella challenge. To address objective 1, a new tool was developed this year to detect the presence and abundance of this function in swine gut bacterial communities. Preliminary findings include that the butyrate-producing bacterial community in pigs shedding high amounts of Salmonella in their feces is different than the butyrate- producing bacterial community of pigs that shed low amounts of Salmonella, and this difference is apparent before the pigs were inoculated with Salmonella. These results suggest a possible relationship between gut- associated butyrate-producing bacteria and the potential to shed Salmonella. Campylobacter is one of the most problematic foodborne pathogens of poultry worldwide. Aligned with the objective to evaluate vaccine strategies for Campylobacter, ARS researchers in Ames, Iowa continue to make progress toward the development of a Campylobacter vaccine in turkeys. To address objective 5, a recombinant strain of non-virulent Salmonella enterica was created that produces a surface-exposed antigen of Campylobacter jejuni. Turkeys were orally vaccinated with this strain, followed by challenge with Campylobacter jejuni. Preliminary results suggest that the vaccine reduced cecal colonization and may be effective in pre-harvest reduction of Campylobacter in turkeys, but further refinements are necessary. The widespread distribution of antibiotic-resistance genes among human and animal pathogens has caused an increase in the demand for antibiotic stewardship. However, many aspects of antibiotic-resistance gene ecology remain poorly understood, including the routes of resistance gene transmission among bacteria in the environment. One environment that is often overlooked but is of primary importance is the gut bacterial communities (i.e., normal commensals) of agricultural animals. Toward the objective of evaluating antibiotic effects on antibiotic resistance genes, previous research by ARS researchers in Ames, Iowa has described the antibiotic resistance gene reservoir of the swine bacterial community. This research was continued to address objective 4 and to evaluate the functional and genetic capacities of these resistance genes. Preliminary results suggest that the resistance-gene reservoir in swine is commonly associated with multiple resistance genes and with elements that promote the spread of resistance genes among bacteria. The importance of these results is that they show that swine gut bacteria harbor a reservoir of resistance genes that can readily mobilize to other bacteria.
Impacts
(N/A)
Publications
- Allen, H.K., An, R., Handelsman, J., Moe, L. 2015. A response regulator from a soil metagenome enhances resistance to the beta-lactam antibiotic carbenicillin in Escherichia coli. PLoS One. 10(3):e0120094. DOI: 10.1371/ journal.pone.0120094.
- Allen, H.K. 2014. Antibiotic resistance gene discovery in food-producing animals. Current Opinion in Microbiology. 19:25-29.
|
Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety. 2) Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host. Evaluate the effects of antimicrobial alternatives (dietary fermentation products) on swine intestinal health, enteric microbiomes, and foodborne pathogens such as Salmonella, under normal and heat stress conditions. 3) Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety). Develop and evaluate alternatives to antibiotics, emphasizing immunological /vaccine strategies, for impacting the host immunome and reducing the risk of Salmonella transmission to humans. 4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes. 5) Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys). Approach (from AD-416): Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using Campylobacter. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii (M. elsdenii) will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Various technologies will be used to assess the effects of alternatives to antibiotics (e.g. fermentation products) on swine performance and intestinal health. Technologies include intestinal microbiome analyses by QPCR-, phylotype census-, culture-based methods; metagenomic analyses of animal host gene expression (esp. immunome); and traditional immunological techniques. A variable heat stress model for swine will be developed and used to test diurnal heat cycle impacts on animal health and on colonization/shedding of foodborne pathogens such as Salmonella, as well as amelioration of the impacts by antibiotic alternatives. Immunotherapeutic measures (vaccine) will be examined for their impact on the swine immunome and Salmonella shedding. Other areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant Escherichia coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth. Foodborne pathogens of humans (Campylobacter, Escherichia coli (E. coli), Salmonella) are non-pathogenic members of the intestinal bacterial communities of farm animals. These pathogens can be transmitted through the food chain to humans. Consistent with the research objective to determine the effects of environmental influences on food safety and animal health, an experimental model has been developed to assess the impacts of heat stress on swine performance, physiology, immunology, and especially the swine intestinal microbiome which includes foodborne pathogens. The heat stress model employed a diurnal rhythm approach (high- low temperature cycles every 24 hours) to mimic farm conditions. Preliminary findings indicate heat stress impacts E. coli populations and generates an (energy-draining) immunological response of stressed swine to gastrointestinal bacteria. The immediate and specific value of the heat stress model will be to evaluate and identify strategies to abrogate the impact of heat stress, for example through the use of dietary additives. This model will also be useful for evaluating heat stress impacts on other animal species and for predicting and preparing United States animal agriculture for the possibility that global warming will be a component of climate change. Essential contributors to animal health and food safety are the 100 trillion bacteria colonizing the intestinal tract. Following the research objective to determine the effects of antimicrobials on farm animal microbiomes, the impact of the commonly used performance enhancing antibiotics ASP250 (combination of aureomycin, sulfamethazine, penicillin) and carbadox on bacteria colonizers and on the microbial gene content of the swine intestinal tract were determined. The development of a vaccine to control the human foodborne pathogen Campylobacter in poultry advanced through the creation of a recombinant strain of non-virulent Salmonella enterica X9241 carrying the CjaA gene of Campylobacter jejuni (C. jejuni). This gene encodes the surface- exposed, outer membrane protein CjaA, an antigen of C. jejuni. Demonstrating the immunogenicity of this strain in turkeys for Campylobacter will now proceed. Accomplishments 01 All intestinal compartments are not equal. Do intestinal microbes play a key role in food safety and swine health? Using phylotype analyses (molecular identification of bacteria) and metagenomic techniques (identification of genes by sequencing), ARS researchers in Ames, Iowa, with European collaborators, detected microbiome differences among intestinal compartments. Microbial populations and gene contents at the end of the small intestine (ileum) were appreciably different from those in large intestine compartments (cecum and colon). The ileum microbes were dominated by bacteria known as Firmicutes (gram-positive bacteria), and two bacterial genera, Turicibacter and Anaerobacter. Unlike large intestine counterparts, bacteria-associated genes in the ileum contained a minimum of genes for degrading plant material, and, surprisingly, a large number of bacterial virus genes. The end of the ileum is an important region for development and proper functioning of swine intestinal immune system. These results have uncovered the unique microbial composition of the swine ileum, thereby enabling specific therapies for improving gut health and food safety to focus on those microbes in that important intestinal compartment. 02 Antibiotic collateral effects. There is a need for antibiotic alternatives in swine production. Carbadox is a widely used antibiotic in the United States swine production due to its dual ability to enhance swine performance and, at higher concentrations, to prevent swine intestinal diseases caused by Escherichia coli (E. coli) and Brachyspira hyodysenteriae. ARS researchers in Ames Iowa examined the results of carbadox feeding (at performance enhancing levels) on fecal bacteria from the swine intestinal tract. Carbadox had an immediate impact on both the bacterial community structure and bacterial membership in the swine intestine. Just after carbadox feeding, there were relative increases in bacteria known as Prevotella, Roseburia, Faecalibacterium. These bacterial genera are potential partners in the production of the short chain fatty acids (propionate, butyrate) and directly benefit growth and intestinal health of their animal hosts. These findings are significant because they offer a possible explanation for the performance enhancing ability of carbadox. The results also suggest that measurements of the Prevotella-Roseburia- Faecalibacterium consortium might usefully be applied to identify non- antibiotic alternatives for enhancing swine health and growth. 03 Carbodox collateral effects. What are the effects of subinhibitory concentrations of antibiotics? Carbadox is a widely used antibiotic in United States swine production for enhancing animal performance and controlling intestinal disease. ARS researchers in Ames, Iowa found that carbadox, at subinhibitory concentrations, induces in vitro production of bacterial viruses (prophages) by the foodborne pathogen Salmonella enterica Typhimurium LT2. These viruses were found to transfer genes for virulence and antibiotic resistance among strains of Salmonella in laboratory cultures. These results are significant because they suggest that carbadox use may be a factor driving Salmonella evolution on the farm. These in vitro results underscore the need for studies to examine the influence of carbadox on the spread of antibiotic resistance genes in vivo, that is, in the intestinal tract.
Impacts
(N/A)
Publications
- Looft, T.P., Allen, H.K., Casey, T., Alt, D.P., Stanton, T.B. 2014. Carbadox has both temporary and lasting effects on the swine gut microbiota. Frontiers in Microbiology. DOI: 10.3389/fmicb.2014.00276.
- Bearson, B.L., Allen, H.K., Brunelle, B.W., Lee, I.S., Casjens, S.R., Stanton, T.B. 2014. The agricultural antibiotic carbadox induces phage- mediated gene transfer in Salmonella. Frontiers in Microbiology. 5:1-8.
- Allen, H.K., Trachsel, J., Looft, T.P., Casey, T. 2014. Finding alternatives to antibiotics. Annals of the New York Academy of Sciences. Available:
- Allen, H.K., Stanton, T.B. 2014. Altered egos: antibiotic effects on food animal microbiomes. Annual Review of Microbiology. 68:297-315.
- Looft, T.P., Allen, H.K., Cantarel, B., Levine, U.Y., Bayles, D.O., Alt, D. P., Henrissat, B., Stanton, T.B. 2014. Bacteria, phages, and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations. Journal of the International Society for Microbial Ecology. 8(8) :1566-1576. DOI: 10.1038/ismej.2014.12.
|
Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): 1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety. 2) Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host. 3) Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety). 4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes. 5) Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys). Approach (from AD-416): Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using C. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Metagenomic, culture, and PCR methods will be used to assess the effects of dietary antibiotics (ASP250, carbadox, and other antibiotics) on swine and turkey microbiomes. Specific areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant E. coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth. The diversity of bacterial viruses in the swine intestinal tract was analyzed. In support of Objective 4 we applied statistics to mathematically discount the rare observations that are common to these types of data, and published a description of an accessible program (CatchAll version 3.0) that implements the statistical procedures. CatchAll was used to analyze data from swine bacterial viruses, showing higher estimations of phage diversity than previously realized. Viruses of bacteria are important drivers of bacterial evolution and of ecosystem functions, so discovering that the swine gut harbors thousands of different phages is important for understanding the ecology of bacteria, their viruses, and how they change under different conditions. Further investigations into these viruses might affect our strategies to modulate gut bacteria and improve animal health and food safety. Bacteria likely to be keystone members of the intestinal microbial communities of healthy swine were isolated in pure culture and characterized. In support of Objective 1 eleven isolates were found to produce butyrate, an important nutrient for swine intestinal tissue health. Six were strains of Megasphaera elsdenii. Ten to twelve diverse bacterial species (e.g. Cloacibacillus porcorum) intimately associated with the swine intestinal tissues (notably ileum) were also isolated. Molecular assays are being developed for these bacterial subpopulations and will be used along with immunological and metagenomic tools to measure the impacts of heat stress and dietary additives on the swine. The research will facilitate the validation, discovery, and development of alternatives to antibiotics for improving swine health and reducing foodborne pathogens. This research and our previous research on dietary antibiotic effects on swine gut microbes led to a cooperative research agreement and invitations to publish three journal articles. Accomplishments 01 Developed software for measuring viral diversity in the intestinal tract. ARS researchers in Ames, Iowa, together with collaborators at Cornell University and the University of Idaho modified and developed a computer program, CatchAll, to estimate the total number of bacterial viral (phage) species in Deoxyribonucleic acid (DNA) samples (metagenome) made from feces and other environmental samples. No previously published programs were adequate. The researchers will apply CatchAll to new swine fecal datasets to learn how the numbers of different gut bacteriophages change over time and with antibiotic treatment. This program and its proven usefulness will benefit virologists, bacteriologists, ecologists, and environmental scientists studying viruses of bacteria and how they interact with their hosts and the environment.
Impacts
(N/A)
Publications
- Stanton, T.B. 2013. A call for antibiotic alternatives research. Trends in Microbiology. 21(3):111-113.
- Levine, U.Y., Looft, T.P., Allen, H.K., Stanton, T.B. 2013. Butyrate- producing bacteria, including mucin degraders, from the swine intestinal tract. Applied and Environmental Microbiology. 79(12):3879-3881. Available:
- Allen, H.K., Bunge, J., Foster, J.A., Bayles, D.O., Stanton, T.B. 2013. Estimation of viral richness from shotgun metagenomes using a frequency count approach. BMC Microbiome. 1(5). Available:
- Looft, T.P., Levine, U.Y., Stanton, T.B. 2012. Cloacibacillus porcorum sp. nov., a mucin-degrading bacterium from the swine intestinal tract and emended description of the genus Cloacibacillus. International Journal of Systematic and Evolutionary Microbiology. 63:1960-1966.
- Looft, T.P., Allen, H.K. 2012. Collateral effects of antibiotics on mammalian gut microbiomes. Gut Microbes. 3(5):463-467.
- Allen, H.K., Levine, U.Y., Looft, T.P., Bandrick, M.M., Casey, T. 2013. Treatment, promotion, commotion: Antibiotic alternatives in food-producing animals. Trends in Microbiology. 21(3):114-119.
|
Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): 1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety. 2) Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host. 3) Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety). 4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes. 5) Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys). Approach (from AD-416): Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using C. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Metagenomic, culture, and PCR methods will be used to assess the effects of dietary antibiotics (ASP250, carbadox, and other antibiotics) on swine and turkey microbiomes. Specific areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant E. coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth. A) Research on the impact of antibiotics on intestinal microbiomes revealed detectable changes in the bacteriophages (viruses that infect bacteria) due to in-feed antibiotics. By studying a gene that is required for phages to get into a bacterial genome (called an integrase), the results suggested that phage production was stimulated by the in-feed antibiotics carbadox and aureomycin, sulfamethazine, penicillin 250 (ASP250). Antibiotic resistance genes were detected in the phage deoxyribonucleic acid (DNA), although there was no statistically significant difference in the abundance of resistance genes between the medicated and non-medicated animals. Phages are important drivers of bacterial evolution in an ecosystem because they transfer genes between bacteria. The result that in-feed antibiotics increased phage numbers is an indication that antibiotic treatment is providing an evolutionary stimulus to gut microbial communities. B) Megasphaera elsdenii (M. elsdenii) is a common, non-pathogenic bacterium that inhabits the intestinal tracts of animals and humans. It provides nutritional and health benefits to its animal hosts. The genomes (DNAs) of two M. elsdenii parent strains (strain 14-14 and strain 24-50C) and three offspring strains produced from the matings of the parent strains were extensively sequenced and analyzed. The genome sequence comparisons showed M. elsdenii strain 14-14 efficiently transferred resistance to the antibiotics tetracycline, ampicillin, and tylosin to strain 24-50C. There were, however, two different genetic mechanisms for the transfer. Understanding both bacterial reservoirs of antibiotic resistance and the genetic mechanisms for the spread and the evolution of resistance provide tools for rationally assessing the effects of agricultural antibiotic use. This research was recently cited in the Food and Drug Administration�s final Guidance for Industry Number 209 entitled, �The judicious use of medically important antimicrobial drugs in food-producing animals�. This citation demonstrates the importance of this research for regulatory agencies. Accomplishments 01 Functional metagenomics assay for alternatives to antibiotics. ARS researchers in Ames, Iowa developed and successfully tested a high throughput, robotic-based assay to identify �toxic� genes whose products either inhibit Escherichia coli (E. coli) growth or disrupt E. coli bacteria. This �functional metagenomic� assay screens all deoxyribonucle acids (DNAs) in subfractions of environmental samples in order to find genes toxic for foodborne pathogens, such as E. coli. The researchers pl to add to their collection of inhibitory genes while starting to identif the cloned toxic genes. This research and products benefit animal producers, animal health and food safety industries, commodity groups, a pharmaceutical industries seeking alternatives to existing antibiotics.
Impacts
(N/A)
Publications
- Allen, H.K., Looft, T.P., Bayles, D.O., Humphrey, S.B., Levine, U.Y., Alt, D.P., Stanton, T.B. 2011. Antibiotics in feed induce prophages in swine fecal microbiomes. mBio [serial online]. 2(6). Available:
- Bunge, J., Bohning, D., Allen, H.K., Foster, J. 2012. Estimating population diversity with unreliable low frequency counts. Pacific Symposium on Biocomputing (PSB) [serial online]. 17:203-212. Available:
- Anderson, R.C., Krueger, N.A., Genovese, K.J., Stanton, T.B., MacKinnon, K. M., Harvey, R.B., Edrington, T.S., Callaway, T.R., Nisbet, D.J. 2012. Effect of thymol or diphenyliodonium chloride on performance, gut fermentation characteristics, and Campylobacter colonization in growing swine. Journal of Food Protection. 75:758-761.
- Levine, U.Y., Bearson, S.M., Stanton, T.B. 2012. Mitsuokella jalaludinii inhibits growth of Salmonella enterica serovar Typhimurium. Veterinary Microbiology. 159(1-2):115-122.
- Stanton, T.B., Humphrey, S.B., Stoffregen, W.C. 2011. Chlortetracycline - resistant intestinal bacteria in organically-raised and feral swine. Applied and Environmental Microbiology. 77(20):7167-7170.
- Bunge, J., Woodard, L., Bohning, D., Foster, J.A., Connolly, S., Allen, H. K. 2012. Estimating population diversity with CatchAll. Bioinformatics. 28(7):1045-1047.
- Looft, T.P., Johnson, T., Allen, H.K., Bayles, D.O., Alt, D.P., Cole, J., Hashsham, S., Stedtfeld, R., Stedtfeld, T., Chai, B., Tiedje, J., Stanton, T.B. 2012. In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences. 109(5):1691-1696.
- Stanton, T.B., Humphrey, S.B. 2011. Persistence of antibiotic resistance: evaluation of a probiotic approach using antibiotic-sensitive M. elsdenii strains to prevent colonization of swine by antibiotic-resistant strains. Applied and Environmental Microbiology. 77(20):7158-7166.
|
Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety. 2) Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host. 3) Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety). 4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes. 5) Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys). Approach (from AD-416) Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using C. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Metagenomic, culture, and PCR methods will be used to assess the effects of dietary antibiotics (ASP250, carbadox, and other antibiotics) on swine and turkey microbiomes. Specific areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant E. coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth. IBC-0260 BSL-Exempt; Recertified 08/03/11; IBC-0303 Recertified 6/11/11; IBC-0312 Recertified 11/15/10; IBC-0331 certified 02/16/11. Swine siblings were fed either a commercial diet containing aureomycin, sulfamethazine, and penicillin (ASP250) or the same diet without antibiotics (control group). After two weeks ASP250 fed swine shed increased Escherichia coli populations in their feces. Bacterial genes important for energy production and metabolism in the swine digestive tract increased in the antibiotic treated group, a finding consistent with the animal performance enhancing properties of the ASP250-diet. The ASP250 diet was found, not surprisingly, to raise the fecal levels of genes encoding resistance for the ASP250 antibiotics. Additionally, increases in resistance genes, not in the ASP250 formulation were detected. One example was a gene for aminoglycoside (streptomycin) resistance. A possible explanation is that the ASP250 diet indirectly enriched for aminoglycoside resistance genes because they are carried on the same genetic transfer elements as the genes for aureomycin-, sulfamethazine-, or penicillin-resistance. To our knowledge, this is the first study combining these multiple molecular and culture based technologies to evaluate �collateral� effects of antibiotics. Investigations of antibiotic feeding effects on intestinal microbiota are important for understanding how growth enhancing antibiotics work and how antibiotic alternatives should behave. The genome sequence of the swine commensal (non-pathogenic) anaerobic bacterium Megasphaera elsdenii (M. elsdenii) strain 14-14 was determined, analyzed, and is being assembled. In addition to the previously detected mosaic (hybrid) gene for tetracycline resistance, there were a large number (six or seven) of other antibiotic-resistance genes identified. A second tetracycline resistance gene was discovered in close proximity to the mosaic gene. The M. elsdenii 14-14 genome contains numerous (greater than 40) genes considered or known to be involved in cell-to-cell transfer of antibiotic resistance genes in other bacteria. Consequently, these results indicate that the 14-14 strain of M. elsdenii, a common non- pathogenic bacterium in the intestinal tracts of animals and humans, is multiple drug resistant and appears well-equipped with mobilization mechanisms for transferring the antibiotic resistance genes to other bacteria. These findings are important for two reasons. Information from this analysis will facilitate research to evaluate M. elsdenii�s roles both in the transfer of antibiotic resistance and in the evolution of antibiotic resistance in the swine intestinal tract. Further, these results underscore the importance of considering both nonpathogenic and pathogenic bacteria in strategies to mitigate against antibiotic resistance in the food chain. Accomplishments 01 Dietary antibiotic effects on the swine intestinal microbiome. ARS researchers at Ames, Iowa in collaboration with scientists at Michigan State University examined effects of feeding certain antibiotics to swin They used a combination of phylotyping (bacterial identification by 16S rRNA gene sequences), metagenomic (sequencing and bioinformatic analysis of bacterial genes), quantitative PCR (qPCR), and culture-based approach to investigate differences between swine fed a commercial diet containin aureomycin, sulfamethazine, and penicillin (ASP250) and siblings fed the same diet without antibiotics (control group). By comparison with contro animals, swine fed ASP250 diets for two weeks: had higher Escherichia co numbers in their feces; had elevated levels of bacterial genes for energ production and metabolism (consistent with the animal performance enhancing properties of the ASP250-diet); and had higher levels of genes encoding resistance both for the ASP250 antibiotics and other antibiotic in feces. Investigations of dietary antibiotic effects on intestinal microbiomes are important for understanding how certain antibiotics enab animals to grow efficiently with less feed consumption and how antibioti alternatives should behave. Additionally, feeding subtherapeutic doses o antibiotics to farm animals will likely have collateral effects which should be considered in cost-benefit analyses of antibiotic use. Information is currently being used in the selection of antibiotics and design of antibiotic alternatives.
Impacts
(N/A)
Publications
- Levine, U.Y., Teal, T.K., Robertson, G.P., Schmidt, T.M. 2011. Agriculture's impact on microbial diversity and associated fluxes of carbon dioxide and methane. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. Available:
- Hamir, A.N., Greenlee, J.J., Stanton, T.B., Smith, J.D., Doucette, S., Kunkle, R.A., Stasko, J.A., Richt, J.A., Kehrli, Jr., M.E. 2011. Experimental inoculation of raccoons (Procyon lotor) with Spiroplasma mirum and transmissible mink encephalopathy (TME). Canadian Journal of Veterinary Research. 75(1):18�24.
|
|