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Molecular ecology of Listeria spp., Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin producing E. coli in northern Colorado wilderness areas




Ahlstrom, Christina, author
Nightingale, Kendra Kerr, 1977-, advisor
Goodridge, Lawrence, committee member
Stromberger, Mary, committee member

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Foodborne disease is a substantial concern in the United States and receives a great deal of attention from the government, industry, and the media. Government initiatives have alleviated some of the burden; however, without improved knowledge of the molecular epidemiology of the pathogens in a variety of environments, a comprehensive understanding of foodborne disease will remain out of reach. Listeria monocytogenes, Salmonella, Escherichia coli O157:H7 and non-O157 Shiga toxin producing E. coli (STEC) play a prominent role in the incidence of bacterial foodborne illness in the United States. Molecular subtyping methods are used extensively in foodborne disease surveillance, yet there is a knowledge gap regarding the presence, transmission and molecular ecology of these pathogens in non-food associated environments. We collected foodborne pathogen isolates from pristine wilderness environments to obtain subtyping data that may aid in the interpretation of clinical and food isolates particularly during outbreak investigations. Furthermore, the identification of subtypes present in different environments but not commonly linked to human disease may provide key information regarding the evolution of virulence in these organisms. To achieve these goals, five wilderness locations in Colorado were selected to represent pristine locations and three areas (approximately 100m2) within each location were designated; each area was sampled once during the spring, summer, and fall seasons in 2009 and 2010. A total of 450 soil, 450 water, 90 drag swab (surface soil) and 276 fecal samples were collected. Five soil samples and five water samples from each area were composited and all samples were microbiologically analyzed to detect Listeria spp. (i.e., L. monocytogenes and other Listeria spp.), Salmonella, E. coli O157:H7, and non-O157 STEC. After non-selective pre-enrichment, samples were divided and microbiologically analyzed to detect each target organism using modified versions of the United States Food and Drug Administration Bacteriological Analytical Manual. Up to four presumptive colonies for each target organism from each sample were confirmed by PCR to detect gene fragments specific to each respective organism. Overall, three samples tested positive for L. monocytogenes, including two fecal samples and one water sample. Nineteen samples contained Listeria spp. other than L. monocytogenes, 14 of which were determined to be Listeria welshimeri by sigB sequencing. The remaining five Listeria spp. were presumptively identified as Listeria rocourtiae sp. nov. by 16s rDNA sequencing; however, these isolates demonstrated notably different biochemical properties than L. rocourtiae. Salmonella was found in two samples, including one water and one fecal sample, and five non-O157 STEC were found in one fecal, one sediment, and three water samples. E. coli O157:H7 was not detected in the natural environments in Northern Colorado surveyed in this study. A molecular serotyping PCR assay revealed two L. monocytogenes isolates belonging to the 1/2 b serogroup and two isolates belonging to the 1/2a serogroup, with a single fecal sample containing two different L. monocytogenes serogroups (1/2a and 1/2b). Pulsed field gel electrophoresis typing results indicated three unique DNA fingerprints among the L. monocytogenes isolates. Two unique strains were isolated from a single fecal sample; with one strain isolated from two different fecal samples collected from the same area. All five non-O157 STEC- and both Salmonella -positive samples had unique PFGE fingerprints. All subtyping data has been deposited in PathogenTracker (, a publicly available WWW database. In conclusion, results from this study demonstrate a rare presence of foodborne pathogens in pristine environments and the utility of molecular subtyping from distinct environments. Further characterization of the foodborne pathogen isolates obtained from non-food associated environments in this study will expand our knowledge on the molecular ecology of foodborne pathogens in nature.


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foodborne pathogen
food safety


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