Theses and Dissertations


Title: In vitro and in vivo cold shock response in Vibrio vulnificus

Name: Limthammahisorn, Suttinee

Degree: PhD

Chair: Yolanda Brady

Resides: FAA Library

University: Auburn

Location: Auburn, Alabama

Date: 2007

Pages: 167

Keywords: oyster, disease, vibrio, bacteria, cold shock, pathogen

Abstract:

Vibrio vulnificus is a serious food-borne pathogen associated with the consumption of raw oysters. The Interstate Shellfish Sanitation Conference (ISSC) recommends refrigeration of Gulf Coast oysters after harvesting to minimize pathogen risks. Cooling temperature control of harvested oysters could potentially induce cold shock response in V. vulnificus. In order to determine the potential for cold shock mediated survival of V. vulnificus, my research investigated in vitro and in vivo cold shock response in V. vulnificus regarding its growth and the molecular mechanisms underlying this phenomenon. The effect of cold shock on V. vulnificus cultures was investigated under ISSC guidelines by the drop plate method. V. vulnificus cultures were incubated at 35, 25, 20, and 15°C for 10 h, 12 h, 14 h, and 36 h, respectively, and then abruptly shifted to 7.2°C and 4°C for 168 h. The mean counts of V. vulnificus after 7 day refrigeration at 7.2°C was significantly greater (P<0.05) than at 4°C in all treatments. Cold adaptation response enhanced cell culturability and was observed when cells were adapted to 15°C prior to cold shock at 7.2°C. When V. vulnificus was artificially inoculated into oysters, its growth during cold shock was investigated using colony hybridization. After oysters were kept at 35°C and 25°C for 10 h and 12 h, respectively, and abruptly shifted to 4°C, cells exhibited a moderate decline in culturability. Oysters kept at 15°C prior to a 4°C shift for 168h, showed a slight decline in V. vulnificus cell numbers. These findings suggested that incubation of V. vulnificus at 15°C could induce cold adaptation and should be avoided during post-harvest conditions. Four cold shock genes (csps) encoding small cold shock proteins in V. vulnificus were cloned. Analysis of in vitro and in vivo cold shock gene expression was displayed by reverse transcriptase polymerase chain reaction (RT-PCR). All csps tested in this study can not be considered cold shock inducible genes under in vitro condition, while some csps showed weak induction after cold shock under in vivo experiments. Besides, the oxidative related genes (oxyR and katG) were induced during in vitro study, while these transcripts were not detected during in vivo experiment. The transcript levels of 16SrRNA and rpoS were constant throughout the study. Overall, these results demonstrated that gene expression of V. vulnificus was different between in vitro and in vivo conditions during cold shock.

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