Isolation, enumeration and antibiotic profiling of Vibrio vulnificus and V. parahaemolyticus from coastal Virginia

Vibrio vulnificus and V. parahaemolyticus are gram-negative, halophilic bacteria that reside in estuarine waters and are associated with infections in humans. These bacteria can cause gastroenteritis through their presence in raw fish and shellfish consumed by humans. V. vulnificus can also produce wound infections leading to severe septicemia, and in some cases, death if not treated promptly. With increasing incidence of infections due to these two organisms, research efforts have focused on potential reservoirs and environmental conditions that can increase human exposure to, and infection, with these species of bacteria. This study was conducted in order to examine the role of Gracilaria, a non-native invasive algal species, as a potential reservoir of V. parahaemolyticus and V. vulnificus. Water and sediment samples were collected weekly in triplicate from mud flats of coastal Virginia for a six week period from early July to mid-August. Plots were set up as two treatments — undisturbed and Gracilaria mats removed. Samples were taken for three weeks prior to algal removal to establish baseline data. Subsequently, algae were removed from half of the plots, and samples were collected for another three weeks. All samples were processed and analyzed by means of dilution, vacuum filtration, and plating on differential media in order to accurately determine the abundance of Vibrio spp. on the coastal flats. DNA was extracted, from “presumptive” Vibrio spp., and amplified by Polymerase Chain Reaction (PCR) using primers specific for the vvhA gene (V. vulnificus) and the tdh gene (V. parahaemolyticus) to confirm identification of isolates. A resistance profile was developed for confirmed isolates using a 12 antibiotic panel. The removal of G. vermiculophylla from the intertidal mudflats did not have a significant impact on the concentration of V. parahaemolyticus or V. vulnificus in either water or sediment. All V. vulnificus isolates (n=181) tested in this study were resistant in some aspect, intermediate or resistant, to all 12 antibiotics tested. Additionally, 68.51% of V. vulnificus and 98.25% of V. parahaemolyticus tested were resistant in some aspect to multiple antibiotics. Future studies should aim at sampling over a longer period and including more sampling areas so that the association between the Vibrio species with the algae can be better understood and also give a more in depth picture of the development of antibiotic resistance among the same