<p>Aim 1: To characterize allelic variation of known and predicted intestinal colonization factors from 1,000well documented S. enterica isolates from our Salmonella Reference Center collection of~ 50,000 strains, using a recently developed targeted MPS strategy. Correlations will beevaluated between (a) strains carrying defined combinations of allelic variants and (b)strain-specific phenotypes/metadata (host species, environment, disease signs orantimicrobial resistance profiles) to determine if specific sets of colonization factor alleles areassociated with bacterial pathotype or antibiotype. </p>
<p>Aim 2: To determine if cause-effect relationship exist between specific allelic variant(s) andstrain specific colonization phenotypes using in vitro binding assays and intestinalcolonization studies in animals. </p>
<p>Aim 3: To determine if cause-effect relationship exist between allele specific adhesin andantimicrobial resistance phenotypes using in vitro and in vivo studies that detect theacquisition of antimicrobialresistance genes by horizontal gene transfer.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Salmonella is a foodborne pathogen that causes substantial livestock morbidity and mortality. Salmonellosis, when notself-limiting, requires antimicrobial therapy, particularly for neonates or systemic infections. Assuch, the increasing prevalence of multidrug resistant Salmonella raises substantial concernsregarding the efficacy of current therapy. Infection and intestinal colonization by Salmonellainvolves the participation of colonization factors that mediate bacterial binding and invasion ofthe intestines. Some Salmonella colonization factors have slight differences in their DNA and protein sequences that is likely responsiblefor host specificity. Recent data suggest thatcolonization factors promote the transfer of antimicrobial resistance genes byincreasing the local density of Salmonella in colonized intestines. For this project, a
recently developed novelsequencing strategy, together with laboratory and food animal studies will be used to determine whether specific Salmonella colonization factors withtheir specific sequences determine host species specificity and facilitate the transfer of antibiotic resistance genes.The result of theseinvestigations will guide future development of novel inhibitors to reduce both intestinalcolonization by Salmonella and the expansion of antibiotic-resistant Salmonella.
<p>APPROACH:<br/> 1)We shall use our Targeted massive parallel sequencing (MPS) technique. Briefly, PCR primer pairs will be prepared from the list of targeted genes colonization factor genes. MPSwill be combined with a barcoding system to identify the Salmonella strain of origin for eachsample and with target enrichment to sequence only the DNA of interest. 454pyrosequencing with the Roche/454 GS FLX sequencer using Titanium chemistry will be performed.We shall utilize the recently developed Fluidigmmicrofluidic chipthat allows for 48 DNA template samples to be amplified separately with 48 differentat the primer pairs in a total of 2,304 independent PCR, each in its own nanoliter chamber. In addition to uniformly amplifying DNA and avoiding allelic bias by PCRamplification in separate compartments, this technique is also advantageous in that amplicons aresimultaneously
barcoded by introducing two different sets of primer pairs in each chamber, onethat is region specific with a small constant tag, and another one that hybridizes to this tag andcarries barcodes and 454 sequencing tags. Thus, all the PCR products are collected and parallelsequenced together, with barcode signatures identifying the strain provenance for each successfulsequence read.The quality of the sequencing data will be checked with various software programs. The prevalence of allelic variants among each set of targetgenes will be compared by the Fisher's exact test with Bonferroni correction for multiplecomparisons, Random Forest and/or multivariate logistic (or other) regression methods. Biases will be minimized whenever possible by ensuring similar sized independentgroups of isolates. 2) Genetic engineering to construct recombinant bacteria and Salmonella with switched
colonization factor alleles.Bacterial binding studies with new cell lines (porcine, bovine, human, avian). Standard methods, determination of adherant CFUs. Oral administration of bacteria to chicken and/or calves, using published procedures and determining fecalshedding and organ colonization (endpoint). 3) The effect of colonization factors on the efficiencey of horizontal transfer of antibiotic resistance genes will be tested in vitro and in vivo, using standard techniques.The effect of high cell density onconjugation efficiency will be determined in liquid cultures and on filters. We will determine if bacterial binding to specific cellsinfluences conjugation efficiency. In vivo conjugation will be tested by checking for Salmonella transconjugants in chicken and/or calves infected orally with bacteria contaning conjugative plasmids that havean antibiotic resistance gene. Efforts: The
results will be published in peer-reviewed scientific journals, posted on genome data web site(s) and presented at scientific meetings. Evaluation: Finding new colonization factor alleles and new associations with specific hosts or with the presence of antibiotic resistance genes. Finding biological relevance of these association through in vitro or/and in vivo studies. Acceptance of the studies and data by peer-reviewers, resulting in scientific publications.