Oysters are suspension feeders that filter phytoplankton detritus and bacteria from the water environment. Potential human pathogens are ingested when oysters feed. Regulations require that each state test rearing waters at least six times per year for the presence of coliforms. Unfortunately, there is no FDA requirement for states to test waters for human pathogens such as Salmonella, since it is assumed that the presence or absence of monitored coliforms reflects the status of enteric pathogens. However, data indicates a complete lack of correlation between the presence of coliforms and Salmonella in market oysters. Therefore, hundreds of thousands of coastal acres currently designated as shellfish rearing waters may contain human pathogens that remain undetected due to established FDA regulations. <P> Salmonella Newport is an emerging pathogen and is currently ranked by CDC as the third most frequently reported Salmonella serovar from human sources. Along with the rise in prevalence, multi-drug resistance has emerged in S. Newport from 1% of isolates tested in 1998 to 21% of isolates tested in 2003, with the most common genotype being S. Newport-MDR-AmpC, with MDR-AmpC referring to resistance to chloramphenicol, streptomycin, sulfamethosazole, tetracycline, amoxicillin-clavulanic acid, ampicillin, cefoxitin, ceftiofur, and cepthalothin. In our published studies, we detected Salmonella in oysters from 12 of 36 bays nationwide. S. Newport was the predominant serovar (82%) and one genotype was dominant at an overall prevalence rate of 98%. Submission of this S. Newport PFGE pattern to PulseNet resulted in a match to pattern number JJPX01.0014, a pattern representing 3.1% of all S. Newport genotypes currently in the national database. Additionally, this was the first MDR-AmpC pattern identified by PulseNet. This pattern has been persistent in the database since 1999, and has been linked to Salmonella gastroenteritis outbreaks in 39 states and Canada. Lastly, cattle were previously identified as a source, suggesting that the S. Newport JJPX01.0014 genotype presence in oysters is due to contamination by agricultural runoff into oyster rearing waters. Therefore, we hypothesize that certain genes used in the colonization of oysters by S. Newport genotype JJPX01.0014 are either not expressed, or are absent from, other S. Newport genotypes routinely found in agricultural run-offs. <P>
The objectives of this study are: <UL> <LI> Comparative genomic hybridization (genomotyping) will be used to identify genes present in S. Newport genotype JJPX01.0014 but absent in S. Newport genotypes that are less fit in colonizing and surviving in oysters <LI> Transposon site hybridization (TraSH) analysis will be used to determine genes directly involved in the colonization and survival within oysters. </uL> Data from these assays will provide considerable information on the interaction of S. Newport with oysters and identify genes that are essential for these unique host-parasite interactions. Mutants will be generated to test the role of selected genes and a diagnostic assay capable of detecting S. Newport JJPX01.0014 will be developed for use in environmental samples.
NON-TECHNICAL SUMMARY: Salmonella Newport is ranked by CDC as the third most frequently reported Salmonella serovar. The impact of this increased incidence is exponential, due to S. Newport emerging as a major multi-drug (MDR) resistant pathogen. In our previous studies, we detected S. Newport in oysters from 12 of 36 bays nationwide, with prevalence levels as high as 77.8% in oyster meat. Submission of the major (98%) S. Newport PFGE pattern to PulseNet/CDC resulted in a match to pattern JJPX01.0014, a MDR pattern recovered from clinical cases of salmonellosis from 39 states and Canada. Previously identified sources indicate cattle, therefore suggesting significant agricultural runoff into oyster rearing waters. We hypothesize that there are genes expressed by S. Newport genotype JJPX01.0014 that are not expressed, or absent, in other S. Newport genotypes routinely found in agricultural run-offs. We will conduct survival studies with JJPX01.0014 and 10 other bovine S. Newport strains. Then, two profiling methods using DNA microarray technology will be used to permit fitness genes to be identified. An assessment of genome content of JJPX01.0014 with other bovine strains will identify genes present in JJPX01.0014, but not in other strains. Transposon site hybridization (TraSH) will detect genes directly involved in survival in market oysters. Genes unique to JJPX01.0014 will be used to develop a nested PCR assay for detection of the agent in environmental waters. Data from these assays will allow a greater understanding of the environmental persistence of S. Newport JJPX01.0014. <P>
APPROACH: Since cattle has previously been identified as a source, suggesting that the S. Newport JJPX01.0014 genotype presence in oysters is due to contamination by agricultural runoff into oyster rearing waters, we hypothesize that certain genes used in the colonization of oysters by S. Newport genotype JJPX01.0014 are either not expressed, or are absent from, other S. Newport genotypes routinely found in agricultural run-offs. We will test the hypothesis by conducting the following experiments. 1. Conduct in vivo survival studies and competitive inhibition experiments with S. Newport JJPX01.0014 and other bovine derived S. Newport genotypes to identify genetic and phenotypic differences (i.e. JJPX01.0014 growth/transmission advantages). The JJPX01.0014 genotype is the major S. Newport isolate found in market oysters but numerous S. Newport genotypes (~20) are shed by dairy cows and dispersed into oyster-growing waters. We will compare the survival rate of individual S. Newport strains in oysters in laboratory aquaria, and survival properties of S. Newport JJPX01.0014 in the presence of other S. Newport strains to identify JJPX01.0014 colonization/survival advantages. 2. Identify S. Newport JJPX01.0014 genes required for colonization of and survival in oysters. The purpose of this aim will be two-fold. First, we will use genomotyping to identify genes present in S. Newport genotype JJPX01.0014 but absent in S. Newport genotypes that are less fit in colonizing and surviving in oysters. Then, we will use TraSH to directly identify JJPX01.0014 genes required for fitness in the oyster, including those genes shared with other S. Newport and those genes unique to the JJPX01.0014 genotype. These two assays will allow us to identify genes essential to survival. 3. Produce S. Newport JJPX01.0014 isogenic mutants and test mutants for survival in oysters. Genes identified in the above assays as putative virulence factors will be mutated via allelic exchange and the isogenic mutants examined for their ability to colonize and survive in market oysters. These results will then be used to develop a diagnostic assay to detect S. Newport JJPX01.0014 in oyster growing waters. 4. Develop a diagnostic assay to detect S. Newport JJPX01.0014 in environmental samples. Genes unique to S. Newport JJPX01.0014 will be used to develop a nested PCR assay to detect S. Newport JJPX01.0014 in oyster growing waters. This assay will be useful to state regulatory officials attempting to ensure that waters used for oyster production are S. Newport JJPX01.0014-free. It will also be useful for both regulators and oyster farmers for source tracking and cleanup of contaminated growing waters to reduce or eliminate the presence of S. Newport JJPX01.0014 in oysters.