Amy Christie

Document Type

Thesis - University Access Only

Award Date

2007

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Enteric bacteria such as E.coli 0157:H7 and Salmonella are widely associated with cattle, and as food-associated pathogens. Food products are required to be free of these pathogens but despite various measures taken during processing, food-associated outbreaks still occur. The ecology of these bacteria is inadequately understood and several analyses point to the role of soil as a primary reservoir. Salmonella is believed to maintain stable dividing populations in soils, E. coli is not. The aim of this work was to ascertain whether E. coli O 157 :H7 is able to grow and survive in soil organic matter, and then to study its physiology by proteomic analysis. Another aim was to decipher how E. coli O l 57 :H7 and Salmonella enterica subsp. enterica ser. Typhimurium (S. Typhimurium) are able to grow using soil organic matter of various soil types. Liquid extracts of various soils, Soil Extractable Soluble Organic Matter (SESOM) were prepared by extraction with wann MOPS buffer and filtered to sterility to avoid chemical modification by heat. E.coli 0157:H7 strain 933D (stxir) and S. Typhimurium were incubated while shaking at 30°C in SESOM, using Luria-Bertani broth (LB) as the control, and growth was determined by increase in optical density. Survival was determined by increase in optical density and plate counts. BacLight stained cells were viewed by fluorescence = microscopy to determine survival. Biomass was cultured at midexponential phase in LB and SESOM and on day 3 for cultures grown in LB, 1/40 strength LB, and SESOM and was subjected to 2D electrophoresis (pH 4-7). Gels were stained using silver and Colloidal Coomassie staining, and analyzed using PD Quest software. Unique protein spots were identified by MALDI TOF MS of tryptic digests for both mid-exponentially-grown and 3 day populations. Both E.coli O157:H7 and S. Typhirnuriurn were able to grow exponentially in the various SESOM indicating that, contrary to current thinking, soils can support the growth of enteric pathogens, and therefore are a possible source of contamination of agricultural products. Proteorni analyses were performed on mid-exponential and 3 day cultures. Ca. 560 spots could be visualized on mid-exponential 2D gels. Mid-exponential SESOM-grown populations had I 96 over-accumulated (>2-fold) spots, while mid-exponential LB-grown populations had 144 over-accumulated spots. Both mid-exponential populations had at least 36 unique protein spots absent in the other, indicating a substantial physiological switch in ability to grow on soil organic matter. Stationary phase SESOM-grown populations remained culturable for 18 days while neither LB, 1/40 strength LB nor glucose minimal medium supported extended longevity. Viability could be confirmed by BacLight. Results also indicated a unique phenotype of populations growing on soil organic matter.

Library of Congress Subject Headings

Escherichia coli O157:H7

Soil microbiology

Soils

Format

application/pdf

Number of Pages

96

Publisher

South Dakota State University

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