Survival Kinetics of Two Genotypes of Vibrio vulnificus in Oysters(Crassostrea virginica) Stored at Two Different Temperatures

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1 Survival Kinetics of Two Genotypes of Vibrio vulnificus in Oysters(Crassostrea virginica) Stored at Two Different Temperatures Lopez- Joven Carmen 1,2,3, Roque Ana 1 *,. Oliver James D 2 1 IRTA Research Aquaculture Centre, 43540, Sant Carles de la Ràpita, Tarragona, Spain. 2 Dept. Biology, University of North Carolina at Charlotte, 28223, Charlotte, NC, USA. 3 Department of Animal Pathology. Veterinary Faculty, University of Zaragoza, 50013, Zaragoza, Spain. * Corresponding author: Ana.Roque@irta.es; fax: In the United States, Vibrio vulnificus is responsible for 95% of all seafoodborn deaths, having a fatality rate of >50%. This pathogen has two distinct genotypes, the C-genotype isolated from human clinical cases and an E- genotype from environmental sources. To investigate if post-harvest temperature has a significant effect on genotype levels, their survival in oysters (Crassostrea virginica) kept at 22ºC (RT) or 4ºC was examined. Oysters (n=25) were exposed to genetically marked strains of the two genotypes of Vibrio vulnificus, E-type of environmental origin and C-type of clinical origin into separate tanks at ca CFU per millilitre of tank water, along with phytoplankton. After overnight incubation, oysters were removed and placed at either RT or 4ºC. At this time (0h), five oysters exposed to either V. vulnificus strain were removed to determine the levels of C- and E-genotype uptake. Individual oysters (n=5) were subsequently sampled at 3h, 8h, 24h and 48h (RT oysters), or at 8h, 24h, and 3 and 7 days (4ºC oysters) for each V. vulnificus strain. Experiments were performed twice. After an initial increase (ca. 1.5 logs) of the C-genotype in oysters maintained at RT, cells of this genotype returned to their initial levels, whereas those in oysters maintained at 4ºC decreased nearly 2 logs without an initial increase. No significant differences were detected for cells of the E-genotype in oysters held at room temperature, and this genotype decreased ca. 1.0 log at both RT and 4ºC. These results suggest that the E-genotype may be more resistant to low temperature than are C-genotype cells, and that cold temperatures may result in the relatively higher numbers of the E- in oysters compared to C-genotype. Keywords: Survival, Vibrio vulnificus, Oysters, shellfish-borne diseases, epidemiology 1

2 Introduction Vibrio vulnificus is responsible for 95% of all seafood-borne fatalities in the United States (Oliver and Kaper 2007). Recent studies have shown that V. vulnificus has two major genotypes, which could account for differences in virulence. Nilsson et al. (2003) found that the strains fall into two different groups, each harbouring a 17-nucleotide difference within the 16S rrna gene. Likewise, the gene (vvha) encoding the V. vulnificus hemolysin has sequence variations that can also be used to divide this species into the same two groups. These variations were named E type, which correlated with strains isolated from environmental samples, and C type, which correlated with strains isolated from clinical cases (Rosche et al. 2005). Warner and Oliver (2008) subsequently found that, while the levels of C and E genotypes of V. vulnificus in estuarine waters were approximately equal and were equally affected by water temperature, the levels in oysters harvested from within those waters were greatly skewed toward the E genotype. Several studies have reported that endogenous V. vulnificus can multiply in post-harvest shellfish (Cook and Ruple 1989; Cook 1994). The present research was undertaken to investigate the effect of time and temperature on the multiplication of the two genotypes of V. vulnificus in oysters during post-harvest storage at two different temperatures, a factor which might explain the predominance of the E- genotype in these samples. 1. Materials and methods 1.1. Maintenance of shellfish Oysters (Crassostrea virginica) from the North Carolina (USA) coast were obtained from a local seafood distributor and placed in 55-gal (ca. 208 L) holding tanks with artificial seawater (ASW; Instant Ocean, Aquarium Systems, Mentor, OH) at 20 ppt salinity and 23ºC. They were fed phytoplankton Bacterial strains and growth conditions. Two strains of V. vulnificus were used for the present study, one a C-genotype and the other an E-genotype both genetically marked (Rosche et al. 2005). Both strains were inoculated into heart infusion (HI, Difco Laboratories, Detroit, MI, USA) broth containing 3 µl ml -1 kanamycin and 100 µl ml -1 10% arabinose, and grown with shaking at 22 C incubating overnight for oyster uptake studies Experimental design Sampling of five individuals was performed before inoculation of the genetically marked bacterial strains. For uptake studies, two groups of oysters (n=25) were used. The genetically marked strains of the two genotypes were added to oysters at ca CFU per millilitre of tank water, along with phytoplankton. Oysters were allowed to filter the algal-bacterial mixture overnight. After this period, oysters were removed and placed in containers without water, and maintained at either room temperature or 4ºC. Upon removal from tanks, 5 oysters which had been exposed to the E-strain and 5 oysters which had been exposed to the C-strain were sampled (0h). Oysters were weighed and individually homogenized in 3 ml of sterile ASW. Decimal dilutions were made in 2

3 phosphate buffered saline (PBS) and inoculated onto HI agar to determine total heterotrophic bacterial counts, onto CPC+ agar (Warner and Oliver 2007) to determine total V. vulnificus levels and onto Tn agar or pgtr agar to enumerate V. vulnificus CVD713 or V. vulnificus Env1, respectively. Following 24h incubation at room temperature, colonies were counted. Five individual oyster samples were subsequently taken at 3h, 8h, 24h and 48h (for oysters maintained at room temperature) or at 0h, 8h, 24h, and 3 and 7 days (for oysters maintained at 4ºC) for both genotypes. Experiment was performed twice Statistical analysis Significant differences over time among the three media used for each strain, and between the two temperatures at which the oysters were stored at, were investigated with a one way-anova with post-hoc Student Newman-Keuls Multiple Comparison Test (GraphPad Prism v 4.00 for Windows, GraphPad Software, San Diego, CA, USA). Analyses of the duration of bacterial survival was performed employing univariate analysis of variance with analysis of covariance (ANCOVA) (SPSS Statistics v17.0, SPSS Inc. software, Chicago, IL, USA). 2. Results Sampling of five individuals was performed before inoculation of the marked bacterial strains, at which time the total heterotrophic bacterial load was ca CFU g -1. Immediately after inoculation of the genetically marked strains, the total bacteria number at both temperatures on HI was ca CFU g -1, and ca CFU g -1 on CPC+ and pgtr or Tn Agar. The counts on HI were consistently higher than those recorded on the other media, for all samples and treatments, without significant differences over the time course of study. The total heterotrophic counts increased less than 1 log over the time of study at room temperature for both strains, whereas they were decreasing 1 log at 4ºC for both strains. Total V. vulnificus levels remained constant at room temperature and increased less than 1 log at 4ºC over the time for E-genotype whereas they increased 1 log at room temperature and decreased slightly at 4ºC over the time for C-genotype. Data is presented in figure 1, shows briefly evolution of the C-genotype and E-genotype in their specific media. After an initial increase (ca. 1.5 logs) of the C-genotype in oysters maintained at RT, cells of this genotype returned to their initial levels, whereas those in oysters maintained at 4ºC decreased nearly 2 logs (168 hours) without an initial increase. No significant differences were detected for cells of the E-genotype in oysters held at room temperature, and this genotype decreased ca. 1.0 log at both RT and 4ºC. Comparing the two genotypes (on their specific media) differences were not found at room temperature, however differences were detected at 4ºC where E-types counts were consistently higher (p<0.001). 3

4 6.0 Room Temperature Strain ºC Strain log CFU / g 2.0 log CFU / g Time Time Fig. 1. Variations in V. vulnificus C- and E-genotype levels per ml of oyster homogenate when held at room temperature. (ANCOVA, F 1, 96 = 1.08, P = 0.30). Variations in V. vulnificus C- and E- genotype levels per ml of oyster homogenate when held at 4 o C. (ANCOVA, F 1, 96 = 6.15, P = 0.02). Values obtained from inoculations onto specific media agar plates. 3. Discussion The present study assessed the effect of post-harvest temperature (22ºC or 4ºC) on the C- genotype and E-genotype strains of V. vulnificus densities in C. virginica. Sampling before inoculation showed a load of total heterotrophic bacteria on the order of 10 4 CFU g - 1 which is consistent with previous environmental studies (Sokolova et al. 2005; Wright et al. 1996). At time 0h, the CFU on HI counts at both temperatures and for both strains were around 10 5 CFU g -1. This count increased less than 1 log over the course of study, which is in agreement with a previous study by Hood et al. (1983). Cook and Ruple (1989) observed that total bacterial CFU g -1 in oyster meat increased four-fold from harvest to the processing plant. Counts on HI were higher than those recorded on the other media, which would be expected in that such a general medium should provide better growth for different types of cells than would any specific medium (Elliott et al. 1995; Warner and Oliver 2007). CPC+ provides a good recovery of cells from oyster and water samples without enrichment, and does not provide a selective advantage to either genotype (Warner and Oliver 2007). There were no significant differences between the counts on CPC+ and Tn agar, nor between CPC+ and pgtr at the two temperatures studied. This indicates that the oysters were virtually free of V. vulnificus before starting the experiment. In the present study, a decrease of 1 log for the E-genotype strain and 2 logs for the C- genotype strain were estimated when oysters were held at 4 C. Significant differences were detected in the development of the bacterial load for each strain over time, suggesting that the survival of these strains is compromised at 4 C. However, the E- genotype showed a moderate decline over time at 4 C, whereas the C-genotype showed a rapid decrease at this temperature (Fig. 1). In general, the results of the present study seemed to be in agreement with a previous study by Hood and collaborators (1983) who observed the effects of storage on loads of 4

5 natural bacterial flora in C. virginica stored as shellstock at 2, 8, 20, and 35 C for seven days, during which time mean levels of total bacteria increased. Both genotypes are found in approximately equal proportions in estuarine waters, but not inside the oysters from these waters, where the E-genotype is found in much higher proportion (Warner and Oliver, 2008). Although the mechanisms by which the E- genotype is found at higher levels than genotype-c in oysters is not yet understood, the results presented here suggest that the E-genotype of V. vulnificus survives better than the C-genotype within oysters held at 4ºC. Therefore, it seems the E-genotype has a selective advantage at reduced temperature, implying that treatment with cold might be a tool for controlling food borne infections caused by this pathogen. Acknowledgments INIA projects RTA and RTA , and an award ( ) from the US Dept. Agriculture. CLJ has an INIA PhD scholarship and was supported by INIA to spend three months in JDO s laboratory at the University of North Carolina at Charlotte to undertake experimental work. We thank Brett Froelich for his assistance in collecting oysters and his suggestions during the experiment, Carles Alcaraz for his help and advice with the statistical analysis and Melissa Jones and Carmen Amaro for helpful discussions during the preparation of the manuscript. References Cook, D. W., Ruple. A. D., 1989, Indicator bacteria and Vibrionaceae multiplication in post-harvest shellstock oysters. J. Food Prot. 52, Cook, D. W., 1994, Effect of time and temperature on multiplication of Vibrio vulnificus in postharvest gulf coast shellstock oysters. Appl. Environ. Microbiol. 60, Elliott, Kaysner, Jackson and Tamplin., 1995, In FDA bacteriological analytical manual, 8 th ed. AOAC International, Gaithersburg, Md. Hood, M. A., Ness G., Rodrick G., Blake N., 1983, Effect of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis. Appl. Environ. Microbiol. 45, Nilsson, W. B., Paranjype R. N., DePaola A., Strom M., 2003, Sequence polymorphism of the 16S rrna gene of Vibrio vulnificus is a possible indicator of strain virulence. J. Clin. Microbiol. 41, Oliver, J.D. and Kaper J., Vibrio species. In: Doyle M.P., Beuchat L.R. (Eds.) Food Microbiology: Fundamentals and Frontiers, 3 nd ed. Amer. Soc. Microbiol., Washington, D.C. pp Rosche, T. M., Yano Y., Oliver J. D., 2005, A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlates with clinical or environmental isolation. Microbiol. Immunol. 4, Sokolova, I. M., Leamy L., Harrison M., Oliver J. D., 2005, Intrapopulational variation in Vibrio vulnificus levels in Crassostrea virginica (Gmelin 1971) is associated with the host size but not with disease status or developmental stability. J. Shellfish Res. 24, Warner E., Oliver J. D., 2007, Refined medium for direct isolation of Vibrio vulnificus from oyster tissue and seawater. Appl. Environ. Microbiol. 73, Warner, E., Oliver, J. D., 2008, Population Structures of two genotypes of Vibrio vulnificus in Oysters (Crassostrea virginica) and Seawater. Appl. Environ. Microbiol. 74, Wright, A. C., Hill R. T., Johnson J. A., Roghman M.-C., Cowell R. R., J. Glenn Morris, 1996, Distribution of Vibrio vulnificus in the Chesapeake Bay. Appl. Environ. Microbiol. 62,