1a. Objectives (from AD-416):
This study will evaluate the survival and proliferation of virulent and avirulent strains of V. vulnificus and V. parahaemolyticus in marine waters and in oysters (Crassostrea virginica) in the presence and absence of algae. It will compare the role of single versus continuous algae addition on V. parahaemolyticus and V. vulnificus survival and proliferation. Mechanisms of potential vibrio suppression by oyster-associated factors released into seawater will be evaluated. This research may lead to recommendations for vibrio monitoring and for the continued sale of raw shellfish in the marketplace.
1b. Approach (from AD-416):
Research will be conducted in three distinct areas. The first will entail an evaluation of the growth of streptomycin-resistant virulent and avirulent strains of V. parahaemolyticus and V. vulnificus in sterile, natural seawater after the addition of either a single dose of algae or during continuous algae supplementation to determine whether algae affects vibrio blooms in seawater. Vibrio levels will be monitored in the seawater using our (ARS) quantitative pour-plate method on Luria-Bertani media supplemented with streptomycin at the following time intervals: 0, 1, 24, 48, and 72 h, with colonies enumerated after 24 h. The second area of research will involve combined oyster and seawater studies, where the uptake and persistence of V. parahaemolyticus and V. vulnificus strains will be determined in freshly acquired oysters after the oysters are acclimated to the water conditions. Water will be spiked with the appropriate vibrio strains and oysters will be collected at 24, 48, and 72 h to determine vibrio levels. Seawater will also be evaluated for specific vibrio levels at 0, 1, 24, 48, and 72 h. Samples will be assayed by our pour-plate method with streptomycin in the media. The final area of research will be to evaluate whether shellfish give off a substance which is vibriocidal. Oysters will be maintained in a tank of seawater for 24-48 h, seawater will be collected, and vibrios will be introduced to the water. The same source seawater (without added oysters) will be inoculated with each of the vibrios to serve as controls. Water will be collected and assayed by the pour-plate method at 0, 24, and 48 h to determine possible suppression of vibrio outgrowth by materials (proteins, including possible enzymes, or other substances) secreted by the oysters. All experiments will be performed three times and each time in triplicate for each vibrio strain tested.
3. Progress Report:
The research was performed principally by a UD employee stationed at the ARS Laboratory at Dover, Delaware, under the technical guidance of ARS scientists. Vibrio bacteria, particularly Vibrio parahaemolyticus and Vibrio vulnificus are major causes of shellfish-associated illness and deaths in the US. This study demonstrated the inability of various strains of streptomycin-resistant V. vulnificus and V. parahaemolyticus (Vp), including a pandemic Vp O3:K6 strain, to survive or proliferate in natural seawater or to persist in Eastern oysters. This inability to persist in seawater was greater when seawater contained high levels of algae. Some factor associated with the seawater and possibly present in the oysters caused a reduction in Vibrio levels. To begin to identify the cause of Vibrio decline in seawater (with or without the presence of oysters), we determined if the inhibitory factor was heat sensitive. In autoclaved seawater, levels of freshly added Vp O3:K6 increased 1000-fold within 72 h; however, in non-heated natural seawater, the levels of VP decreased 47-fold in 72 h. In a search to identify the factor(s) responsible for the beneficial reductions in vibrios, we identified a variety of Vibrio predatory bacteria (VPB) which used vibrios as prey. When these organisms are secreted from shellfish or are present in seawater, they have a vibriocidal effect. Thirty VPB were isolated and subsequently characterized by scanning electron microscopy. They resembled Bdellovibrio-and-like organisms (BALOs), such as Bdellovibrio bacteriovorus and Bacteriovorax stolpii. Vibrio and VPB counts were monitored in natural seawater containing oysters after the addition of Vp O3:K6. Vibrios diminished 1000-fold to non-detectable levels while VPB increased 1000-fold within 48 h. VPB levels were also monitored in natural seawater from four sites in Delaware and from seawater obtained from the Gulf Coast and Hawaii. VPB were present at levels as high as 300/100 ml of seawater. Thus, these studies suggest that VPB may be a major regulator of vibrios in seawater and shellfish and offer a new paradigm on the importance of VPB in the regulation of vibrios in the marine environment. Since BALOs rely on flagellar motility to find their prey, we hypothesize that high algae levels may have physically interfered with the bacteria’s ability to locate its prey. A manuscript on this work has been submitted for publication. Further work to quantify and characterize these predatory bacteria is underway and will continue through the remainder of this study. Characterization will include DNA sequencing and host specificity studies to determine if VPB isolates are also infectious toward other pathogens (E. coli, Salmonella and Shigella species).