Browsing by Author "Drake, Stephenie Lynn"

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    Characterization of the response of Vibrio vulnificus to sublethal stresses during oyster handling and processing
    (2004-08-15) Drake, Stephenie Lynn; Dr. Lee-Ann Jaykus, Committee Chair
    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.
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    THE ECOLOGY VIBRIO VULNIFICUS AND VIBRIO PARAHAEMOLYTICUS FROM OYSTER HARVEST SITES IN THE GULF OF MEXICO
    (2008-12-02) Drake, Stephenie Lynn; Dr. Lee-Ann Jaykus, Committee Chair
    The Vibrionaceae are environmentally ubiquitous to estuarine waters. Two species in particular, V. vulnificus and V. parahaemolyticus, are important human pathogens that are transmitted by the consumption of contaminated molluscan shellfish. Molluscan shellfish harvesting guidelines stipulate refrigeration within a defined time based on harvest season. Nonetheless, even the more stringent summer guidelines allow for extended storage of oysters at ambient temperatures, providing an opportunity for bacterial proliferation. Recent risk assessments have been based on limited data sets; accordingly, the purpose of this study was to address some of these data gaps in the V. vulnificus and V. parahaemolyticus risk assessments. The objectives of this study were to (i) quantify the levels of total estuarine bacteria, total Vibrio spp., and specific levels of non-pathogenic and pathogenic V. vulnificus and V. parahaemolyticus over the harvest period; (ii) determine if length of harvest time affects the levels of V. vulnificus; and (iii) determine if survival rates under environmental stress conditions differ by strain for both V. vulnificus and V. parahaemolyticus. Oyster and water samples were harvested seasonally from 3 U.S. Gulf Coast sites over 2 years. Environmental parameters were monitored during harvesting. Both surface and bottom water samples (1 L) were taken at the beginning of harvesting and at the end of harvesting. Oyster samples (15 specimens for each time point) were taken at 0, 2.5, 5.0, 7.5, and 10 hrs intervals after being held at ambient temperature during harvesting. Samples were processed for fecal coliforms, Salmonella, total Vibrio spp., aerobic plate count, and total estuarine bacteria using the appropriate media. For enumeration of total V. parahaemolyticus, pathogenic V. parahaemolyticus, and V. vulnificus was done using colony lift hybridization (tlh, tdh+ and/or trh+, and vvhA gene targets, respectively). MPN methods were also used to obtain estimates of pathogenic V. parahaemolyticus (tdh+ and/or trh+) counts. Representative V. parahaemolyticus and V. vulnificus isolates were subjected to phenotyping; V. vulnificus isolates were also subjected to genotyping. Different statistical analysis including the application of generalized linear mixed models (GLMMs), Pearson’s correlations, and analysis of variance (ANOVA) were used to establish relationships where appropriate. The first manuscript describes the field study which examined the distribution and variation in the levels of V. parahaemolyticus and V. vulnificus (both total and pathogenic strains) in shellfish and their overlay waters, and established the effect of seasonal and environmental/ecological factors on these distributions. The best estimate of growth for V. parahaemolyticus (tlh) in oysters based on water temperature was 0.054 log10 per degree C, while the best estimate for growth for V. vulnificus (vvhA) in oysters based on water temperature was 0.068 log10 per degree C. Estimated relationships between tlh and vvhA growth rates and air temperature were consistent with the risk assessments. No statistical relationship could be established between pathogenic V. parahaemolyticus (tdh+ and/or trh+) growth rates and air temperatures. In the second study, we specifically looked at the effect of extended boat deck storage of commercially harvested oysters held at ambient air temperature on the levels of V. vulnificus. During summer, increases in V. vulnificus were as high as 1.4 log10 (CFU/g) after 10 h storage at ambient air temperatures; for spring/fall oysters, a 1.0 log10 increase was observed; and in winter, increases were <0.5 log10. Statistically significant (p<0.05) correlations between ambient air temperature and increases in V. vulnificus counts were noted. Summer V. vulnificus isolates contained a higher percentage (52%) of genotype B strains, which are more often associated with clinical illnesses which was statistically significant. Spring/fall isolates were more often genotype A (80%), associated with environmental origin. In the third study, we examined the relationship of phenotypic characteristics (D-mannitol fermentation) and V. vulnificus genotypes. For a total of 469 V. vulnificus isolates, there was a high degree of concordance between 16SrRNA type and vcg variant type (92-94%) and between either genotyping method and mannitol fermentation (84-91%). Fermentation of D-mannitol may be an appropriate initial screen prior to the application of more labor intensive V. vulnificus genotyping methods. In the fourth study, strains of V. parahaemolyticus and V. vulnificus with different virulence genes/markers were evaluated for their survival under physiological stress conditions. Although there were no statistically significant differences in the recovery of V. parahaemolyticus by strain type under cold stress, there were statistically significant differences in D-values when comparing different V. parahaemolyticus strains subjected to starvation. In some instances, culture media and conditions statistically impacted V. parahaemolyticus (starvation) and V. vulnificus (cold) recovery, extending D-values by 1-3 days when media with pyruvate supplementation and aerobic incubation were used. For V. vulnificus, there were no statistically significant differences in recovery when comparing genotypes under conditions of both starvation and cold stress. In the final study, we looked an alternative method for monitoring oyster temperature during refrigerated cool-down. No statistically significant differences in oyster cooling rates were observed when comparing thermocouple versus button data logger data, irrespective of location in the commercial sacks (top, middle, and bottom) or temperature monitor location (internal vs. external). Taken together, this research provides information which can be used to fill key data gaps in the current risk assessments for V. vulnificus and V. parahaemolyticus. These data can be used in future iterations of the risk assessments to help better predict risk and inform policy as risk managers seek to reduce the disease burden associated with these important foodborne pathogens.