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We propose a multidisciplinary approach for developing science-based microbial food safety criteria. Current regulations for foodborne pathogens are commonly based on historical taxonomic identifications that may not correlate to the ability of an organism to cause human disease. This project will identify distinct genetic criteria that define the specific human pathogenic potentials of clonal bacterial groups focusing on E. coli O157:H7, Streptococcus agalactiae, and Mycobacterium paratuberculosis.
<OL> <LI> Assemble a collection of animal, human, and environmental (where applicable) bacterial isolates to be characterized by molecular subtyping methods. <LI>Epidemiologically define the relative human pathogenic potentials of different clonal groups of these pathogens. <LI>Explore the functional and genetic basis of differences in the abilities of clonal subgroups to cause human disease using tissue culture and animal models.
NON-TECHNICAL SUMMARY: Foodborne diseases cause an estimated 76 million illnesses and 5000 deaths annually in the US. To protect consumers from these diseases it is important to understand exactly which bacteria can cause human disease. This project examines bacteria from humans and animals with bacterial diseases and from contaminated foods by DNA fingerprinting methods to define which bacterial types cause human foodborne disease.
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APPROACH: Objective 1: Human, food, and animal isolates of E. coli O157:H7, St. agalactiae, and M. paratuberculosis will be collected in collaboration with the NYS Veterinary Diagnostic Laboratory, the Dept. of Argriculture and Markets, and the Dept. of Health. All isolates will be characterized by DNA subtyping methods including Southern blotting, ribotyping and sequencing of selected virulence genes. Objective 2: Bacterial subtype distribution among human, food, and animal isolates will be statistically analyzed to identify specific subgroups linked to human infections. Objective 3: Selected subgroups differing in human and animal host specificities will be used to infect appropriate tissue culture cell lines to determine differences in their pathogenic potentials.
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PROGRESS: 2000/10 TO 2003/09<BR>
Most food safety regulations regarding bacterial foodborne pathogens specify action limits for the presence of specific bacterial species. These classical taxonomic definitions of bacterial species often do not correlate to the ability of a group of bacteria to cause human disease. Rather, somewhat related bacteria that may differ in their abilities to cause human and/or animal disease may be grouped together into the same species. This project is designed to develop a phylogenetic and population genetics framework to allow the specific definition of bacterial clonal groups with human pathogenic potential. Our work specifically focused on Streptococcus agalactiae, which is not only a major cause of mastitis in cattle, but is also responsible for severe invasive disease in adult and neonate humans, as well as asymptomatic infections in women. To probe the population genetics and the interspecies transmission potential of this species, we used molecular subtyping and phenotypic methods to characterize temporally matched bovine milk and clinical human invasive isolates (52 each) collected in New York state over 18 months. EcoRI ribotyping differentiated 17 ribotypes and DNA sequencing of the housekeeping gene sodA and of a 950 bp fragment of hylB, encoding hyaluronidase, differentiated 17 and 7 allelic types, respectively. Human and bovine isolates were not randomly distributed between ribotypes and hylB and sodA allelic types. Combined analysis of all subtyping methods allowed differentiation of 39 clonal groups, 26 groups contained only bovine isolates. Only two clonal groups contained both human and bovine isolates. EcoRI ribotyping also showed a significantly higher genetic diversity among bovine isolates (SID = 0.90 +/- 0.05) as compared to human isolates (0.42 +/- 0.15), providing further evidence that human and bovine isolates represent distinct populations. Eight human but no bovine isolates showed an IS1548 transposon insertion in hylB. Hyaluronidase activities determined for 43 representative isolates showed that human isolates on average showed lower activities than bovine isolates. Isolates with the IS1548 insertion in hylB showed no hyaluronidase activity. In addition, tissue culture invasion assays showed that human and bovine isolated did not differ in their ability to invade human epithelial cells. Our data show that (i) EcoRI ribotyping and hylB and sodA sequencing provide discriminatory subtype analysis of S. agalactiae, (ii) human invasive and bovine S. agalactiae represent distinct populations with limited interspecies transmission, and (iii) hyaluronidase activity is not required for all human infections.
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IMPACT: 2000/10 TO 2003/09<BR>
Most current food safety and public health regulations use classical taxonomic species definitions to identify microorganisms that represent a public health hazard. Bacteria within a given species may vary tremendously in their ability to cause human disease though. This work clearly shows that bacteria within a given species, even if associated with animal and human disease, are not necessarily transmitted between these hosts. A better understanding of bacterial host specificity for zoonotic and foodborne pathogens will contribute to the development of science-based regulations and intervention strategies targeting those bacterial subtypes that have the potential to cause human disease.