Characterization of the response of Vibrio vulnificus to sublethal stresses during oyster handling and processing

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Title: Characterization of the response of Vibrio vulnificus to sublethal stresses during oyster handling and processing
Author: Drake, Stephenie Lynn
Advisors: Dr. Lee-Ann Jaykus, Committee Chair
Abstract: Vibrio vulnificus, a naturally occurring marine bacterium, causes severe disease in at-risk individuals consuming contaminated raw shellfish. The organism can be difficult to discriminate from natural microflora present in the product, complicating the evaluation of process control efficacy. The purpose of this study was to construct a strain of V. vulnificus expressing green fluorescent protein (Vv-GFP-K) which could be readily distinguished from background flora. Once constructed, the objectives were to compare the physiological characteristics of Vv-GFP-K to the wild-type parent (Vv-WT); to use Vv-GFP-K to evaluate survival of the bacterium under various environmental stresses relevant to food processing; and to assess the effect of sodium pyruvate media supplementation on recovery efficiency, with particular reference to sublethally injured cells. V. vulnificus strain ATCC 27562 was engineered to express GFP and kanamycin resistance using methods of conjugation. Comparisons were made between Vv-GFP-K and Vv-WT with respect to growth characteristics, heat tolerance (45 degree C), freeze/thaw tolerance (-20 degree and -80 degree C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage (5 degree C), cold adaptation (15 degree C) and starvation. Recoveries were evaluated using non-selective [tryptic soy agar-2%NaCl, (TSAN2)] medium with and without sodium pyruvate supplementation. To represent the food matrix, seeding studies were done with either shellstock or shucked oysters and the survival of Vv-GFP-K in the food matrix was evaluated after exposure to cold and acid stress. Specifically, cooling regimens designated (i) rapid cooling (iced); (ii) conventional cooling (5 degree C); and (iii) mild abusive cooling (temperature dropped to 5 degree C over 8 hr) were evaluated. Acetic and citric acids at pH values ranging between 3.5 to 5.0 were evaluated in the acid studies. In most cases, Vv-GFP-K was comparable to Vv-WT with respect to growth, survival, thermal inactivation, and freeze thaw survival. There were differences between Vv-WT and Vv-GFP-K with respect to acid tolerance, although these differences disappeared with sodium pyruvate supplementation of media. In broth studies dealing with organic acids, Vv-GFP-K was rapidly inactivated with acetic acid. Similar, but not as dramatic results were seen for citric acid. As pH values declined, the positive impact of pyruvate supplementation on cell recovery disappeared. In the refrigeration studies done in the matrix, there were no apparent differences in Vv-GFP-K levels for all three treatments within the first few days of storage. In all cases, levels dropped 1 log10 after 8 days refrigerated storage. By the end of the study (21 d), Vv-GFP-K levels were nondetectable for both iced and conventionally cooled product, however mild abusively cooled oysters still had levels approximating 103 CFU/oyster. Vv-GFP-K levels remained stable for up to 24 hrs within the oyster meat under acidic conditions at various pH values. The oyster meat provided a protective environment that prevented inactivation of Vv-GFP-K. Similarities between Vv-GFP-K and Vv-Wt with respect to growth and survival suggest that it may be an appropriate surrogate for evaluating processing stress tolerance. Sodium pyruvate supplementation of media may aid in the recovery of V. vulnificus cells sublethally injured by exposure to food processing-related stresses, although the efficacy of pyruvate supplementation was highly dependent upon the specific stress. Cooling alone cannot be relied upon to eliminate V. vulnificus. Furthermore, specific cooling methods or organic acids appear to make little difference in the survival of V. vulnificus during extended refrigerated storage of whole (shellstock or shucked) oysters.
Date: 2004-08-15
Degree: MS
Discipline: Food Science

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