Analysis of bacterial communities in the eastern oyster (Crassostrea virginica) with emphasis on Vibrio vulnificus dynamics under refrigeration

Abstract

In this study we determined the effect of refrigeration on the total bacterial, total Vibrio spp., and V. vulnificus populations present within the eastern oyster (Crassostrea virginica), and the effects of cold shock (35°C to 4°C) on the complete V. vulnificus transcriptome. Oysters from two different locations, the Auburn University Shellfish Laboratory, Dauphin Island, AL, and a commercial processor, were compared during two weeks under refrigeration conditions. During the course of the experiment, total aerobic bacteria counts increased by two logs. Ribosomal Intergenic Sequence Analysis (RISA) and Denaturing Gradient Gel Electrophoresis (DGGE) were used to determine changes within the total population, while DGGE was used to evaluate changes of the Vibrio spp. population over the two week period. RISA-derived data showed that the microbial communities at Day 1 were clearly different from both Day 7 and 14 samples. Within the Day 1 cluster, samples were subdivided based on location. On Day 7 and 14, samples could also be subdivided by date and origin. Vibrio-specific DGGE also allowed for the clustering of samples by location on Day 1, which also clustered away from Days 7 and 14. Bands corresponding to both V. parahaemolyticus and V. vulnificus decreased in prevalence, while those corresponding to V. mimicus increased during the two weeks of refrigeration, particularly on Day 7. Interestingly, V. vulnificus counts determined by colony dot-blot hybridization remained unchanged throughout the experiment. Vibrio vulnificus isolates were recovered from oysters and genotyped as 16S type A, B or AB by restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (ALFP) analyses. 16S type B isolates (typically associated with clinical cases) comprised 53% of the isolates. An important note was the identification of 16S type B-specific AFLP bands that differentiated type B from type A and AB. A DNA microarray of V. vulnificus showed that 165 genes out of 4,488 altered their expression profiles by more than twofold. The highest induction observed occurred in two of the five categorized cold shock genes, cspA and cspB, which showed a complementary and persistent expression pattern during cold shock suggesting a homologous role. Other genes showing a significant fold increase included ribosomal genes, protein folding regulators, and membrane genes.