Prevalence and antimicrobial resistant patterns of Vibrio parahaemolyticus and Vibrio vulnificus isolated from Pacific oysters (Crassostrea Belcheri) in Thailand

Abstract

V. parahaemolyticus and V. vulnificus are major causes of foodborne diseases from seafood consumption. Both bacteria are significant public healthy problem in many countries. V. parahaemolyticus is cause of human gastroenteritis while V. vulnificus can cause septicemia with high mortality rate. Since, raw pacific oyster (Crassostrea Balcheri) is popular seafood menu. The contamination of these microorganisms on pacific oyster will be risk of foodborne infection. This study covered prevalence, densities and antimicrobial resistance patterns of V. parahaemolyticus and V. vulnificus in the pacific oyster. During April 2007 to March 2008, 360 samples of pacific oysters were collected from 5 different locations of the coast area of Bandon bay, Suratthani province, where the most oysters were cultured in 5 locations; Chaiya, Cha-ngor, Kadae, Tatong and Kadae-gae. Those samples were examined carefully to get the information and the data of the prevalence of two significant species of harmful bacteria, V. parahaemolyticus and V. vulnificus. The occurrence of antimicrobial resistant strains was also studied. The densities of both microorganism were determined by using a modification of the most probable number (MPN) technique described in the FDA-BAM (2004). Agar dilution method used for doxycycline, norfloxacine, ciprofloxacine and cefotaxime susceptibility test (CLSI, 2008). The biochemical method was used for the identification of the isolates. The vvhA gene was applied to confirm V. vulnificus strains by PCR method. Multiplex PCR method was used to identification of V. parahaemolyticus strains, the tlh gene was used to confirm species specific, tdh and trh gene determine the presence of the thermostable direct hemolysin gene and TDH-related hemolysin gene, respectively which associated with pathogenicity of V. parahaemolyticus. The Kanagawa phenomenon was assayed as phenotypic marker of thermostable direct hemolysin (TDH) toxin, the urease activity was assayed as phenotypic marker of trh gene; the protease and gelatinase activity of individual strains were examined to identify other potential virulence factors of V. parahaemolyticus. The tlh+ positive of 1,457 isolates was found but none V. parahaemolyticus strains carried out of the tdh and trh genes that also showed the negative for TDH and urease activity tests. V. vulnificus of 609 isolates were vvhA positive. From the study, prevalence of pathogenic strains of V. parahaemolyticus were 172 (47.78%), V. vulnificus were 15 (4.17%) and 84 (23.33%) were contaminated both organisms from 360 oyster. Quantitative study of V. parahaemolyticus in oysters from all harvested regions found the highest densities in May. The highest densities were observed in oysters harvested from Chaiya, Cha-ngor and Kadae which mostly exceeded 107 MPN/g. While V. vulnificus found from the oysters of all locations were below the detectable level 3.0 MPN/g in August, February and March. In December and January, V. vulnificus densities of all location showed exceeding of 104 MPN/g. V. parahaemolyticus densities were greater than those of V. vulnificus in lots from these same areas, except in December and January. In this study period, the total average densities of V. parahaemolyticus and V. vulnificus were 3.2±2.5 log MPN/g and 1.5±2.2 log MPN/g, respectively. There were some evidences showing that the mean log MPN values were not same among these five locations (p < 0.05). The densities of V. parahaemolyticus lower than FDA/WHO (2001) concerned value of <10,000 MPN/g and none of V. vulnificus was found in August, February and March which probable to appropriate for raw oyster consumption. The 297 isolates of V. vulnificus and 768 of V. parahaemolyticus isolates produced of the gelatinase and protease. All of V. vulnificus strains were positive for DNase and lipase (100%), hemolysin (14.8%), lecithinase (52.4%). All strains of V. vulnificus exhibited negative results either mucinase nor elastinase activity. V. parahaemolyticus strains were susceptible to doxycycline (98.4%), norfloxacin (96.1%), ciprofloxacin (76.6%) and cefotaxime (70.7%). While V. vulnificus strains found from Bandon bay were susceptible to doxycycline, norfloxacin, cipofloxacine (100%) and cefotaxime (84.5%). This study revealed that the pacific oyster at Bandon bay found high prevalence and densities V. parahaemolyticus than V. vulnificus. However, both bacteria were susceptible to tested antimicrobial agents.