A comparison of strategies for the detection and recovery of Vibrio vulnificus from marine samples of the western Mediterranean coast

We hare compared the effectiveness of culture-based methods and a DNA-based method for the detection, of Vibrio vulnificus from seawater and three types of shellfish collected from the coastal waters of Valencia, Spain. For culture-based methods we used two selective media, thiosulphate-citrate-bile salts sucrose (TCBS), and cellobiose-polymyxin B-colistin (CPC) agars with and without previous enhancement in alkaline-saline-peptone-water (APWS). Presumptive colonies were confirmed as V. vulnificus by the polymerase chain reaction (PCR) using previously described 23S rRNA V. vulficus-specific sequences as primers (Dvu 9V and Dru 45R). Direct detection was accomplished by a nested-PCR procedure developed for environmental samples, with the above mentioned primers for the second amplificaton. Of 32 seawater samples, only one yielded positive results by direct detection by PCR, whereas five were positive by culture methods. Of the 32 bivalve samples, two were positive by PCR and five by culture methods. From a total of 675 presumptive colonies selected on the two media, only 48 (20 from seawater and 28 from bivalves) were confirmed as V. vulnificus by PCR. Forty-six v. vulnificus isolates were obtained after enrichment and only two after direct inoculation on CPC. Except for one sampling, positive results by direct detection did not correlate with confirmed strains obtained from culture media. API 20E profiles were recorded for all isolates previously identified as V. vulnificus, revealing that around 20% of the strains were sucrose-positive. Fur our samples, the best strategy consisted in the combination of culture based methods (3 h enrichment in APWS at 40 degrees C followed by CPC at the same temperature) and DNA-based procedures (specific PCR amplification of the presumptive colonies with primers Dvu 9V and Dvu 45R) which allowed the detection and accurate identification of V. vulnificus in less than 48h. This is the first report on the detection of cells of V. vulnificus naturally present in seawater and edible shellfish in the Spanish Mediterranean coast.