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The Centers for Disease Control and Prevention estimate that 76 million illnesses are caused by foodborne pathogens annually in United States. To address this problem, many strategies are currently being used to reduce pathogen carriage by food animals by promoting the growth of beneficial bacteria in the intestine. Commensal intestinal bacteria, formulated as probiotics, competitive exclusion formulations, or direct-fed microbials, have been widely used world-wide in food animals as a means of reducing Salmonella. However, their promise has not been realized in the U.S. because technology was not available that provided accurate compositional and mechanistic information necessary for FDA approval. <P>
In order to address these issues and to develop more effective products we must elucidate the compositional and functional interactions of the gastrointestinal microbial community in order to understand the effect of these communities on colonization by enteropathogens. The burgeoning field of molecular ecology has provided new methodology and a new conceptual view of the intricate interrelations that shape microbial communities. <P>
Our long-term goal is to understand how the intestinal microbial community affects health and production. The central hypothesis of this work is that communication, competition and resource partitioning are responsible for modifying the capacity of Salmonella to establish a stable population within the intestine of poultry. Very little is known about microbial competition, predation, or metabolism within the intestinal environment. <P>
Successful completion of the experiments proposed in this application will not only contribute significantly to producing Salmonella-free poultry, but will also contribute fundamentally to the understanding of how commensal bacteria regulate the outcome of intestinal microbial interactions. <P>
To this end, we propose the following Aims: <Ol> <LI> Perform quantitative compositional and functional metagenomic analysis of the broiler intestinal microbial community. The hypothesis of this Aim is that exclusive communities differ from Salmonella-permissive communities in their composition, metabolic activity, bacteriocin production and quorum sensing systems in the chicken intestinal ecosystem. <LI> Determine the in vivo transcriptional response of Salmonella to the presence of permissive or exclusive intestinal microbial communities. The hypothesis of this Aim is that exclusive communities may modify the expression of specific metabolic pathways or virulence genes of Salmonella reducing its ability to establish a stable population within the intestine.</OL> This information will allow us to determine the colonization, competition and invasion behavior of Salmonella in the presence of a complex intestinal microbial community. Only when we understand the mechanisms by which exclusive communities confer resistance to pathogen colonization can we effectively design strategies to prevent colonization of food animals by enteropathogens.
NON-TECHNICAL SUMMARY: Salmonellosis is one of the most significant foodborne diseases in the United States, with an estimated 800,000 to 4 million human infections occuring each year, and represents an annual loss of approximately $4 billion from the U.S. economy. Poultry and eggs are considered major contributors to disease burden associated with Salmonella and serve as a significant reservoir of Salmonella in the human food chain. The US Department of Agriculture reported an upward trend in Salmonella contamination of poultry from 2002 through 2005, with about 16% of broiler chicken samples testing positive in 2005. As a consequence, the poultry industry is facing the challenge of reducing carcass contamination as the government strives to achieve a 50% reduction in human illnesses by 2010. To address this problem, many strategies are currently being used to reduce pathogen carriage by food animals by promoting the growth of beneficial bacteria in the intestine. Beneficial intestinal bacteria, formulated as probiotics, competitive exclusion formulations, or direct-fed microbials, have been widely used world-wide in food animals as a means of reducing Salmonella. However, their promise has not been realized in the U.S. because technology was not available that provided accurate compositional and mechanistic information necessary for FDA approval. In order to address these issues and to develop more effective products, a better understanding of the composition and functional interactions of the gastrointestinal microbial community is needed so that we understand the effect of these communities on colonization by enteropathogens.
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APPROACH: In Aim 1 we will perform quantitative compositional and functional metagenomic analysis of the broiler intestinal microbial community in order to reveal how exclusive communities differ from Salmonella-permissive communities. Comparative metagenomic analysis will be performed on sequence datasets provided by pyrosequencing. The composition of the communities will be determined by evaluating the distribution of 16S rDNA genes; gene expression profiles will be determined using northern blots. Salmonella load among the different communities will be determined using quantitative qRT-PCR. Comparative metagenomic tools, including phylotype analysis and KEGG subsystems-based annotation, will be used to examine the distribution of metabolic, quorum sensing, bacteriophage and bacteriocin-related loci within the community DNA and RNA in order to reveal the ecology of community interactions. In Aim 2 we will determine the in vivo transcriptional response of Salmonella to the presence of permissive or exclusive intestinal microbial communities in order to reveal the mechanisms of colonization of the broiler intestinal tract. The hypothesis of this Aim is that exclusive communities may modify the expression of specific metabolic pathways or virulence genes of Salmonella reducing its ability to establish a stable population within the intestine. The effect of different communities on gene expression of Salmonella will be monitored with a combination of quantitative qRT-PCR to detect the expression of known loci and genome-wide microarray hybridization to detect novel regulatory interactions.