Pathogenicity And Molecular Characterization Of Major Virulence Genes Of Vibrio Parahaemolyticus Isolates From Seafoods

Vibrio parahaemolyticus is a bacterium that common contaminant of seafoods worldwide and is ubiquitously distributed in coastal waters. It is the leading cause of seafood-associated bacterial gastroenteritis, wound infections and septicemia. Pathogenicity of V. parahaemolyticus is related to the production of a thermostable direct hemolysin (TDH) and/or a TDH related hemolytic (TRH) encoded by tdh and trh genes respectively. However, not all V. parahaemolyticus isolates are pathogenic, the relationship between their pathogenicity and distribution of major putative virulence genes among isolates from environmental, seafood and clinical sources is not well understood from the molecular epidemiologic perspectives. Most clinical isolates of V. parahaemolyticus exhibit Kanagawa phenomenon (KP) induced by TDH on Wagatsuma agar medium, while only small portion of the environmental origins are KP positive and the presence of trh gene in the isolates is strongly correlated with urease phenotypes. The strains bearing tdh and/or trh genes belong to diverse serotypes. However, a pandemic clone of O3:K6 is a major serotype which was first detected in Culcata, India, and has been responsible for many outbreaks in Asia and the United State since 1996. The other serotypes O4:K48, O4:K68, O1:K25 and 01.KUT (K untypable) have similar potential to cause outbreaks of gastroenteritis, and are genetically closed to the new O3:K6 clone. Therefore, it is essential to characterize a substantial amount of V. parahaemolyticus isolates from clinical samples, seafoods and environments, compare the distribution of major putative virulence genes among those isolates and type the V. parahaemolyticus isolates from different origins for better understanding of the pathogenesis and molecular ecology of this bacterium. Thus, crucial control strategies could be established for reducing risk of V. parahaemolyticus infections associated with seafood consumptionA total of 766 fresh, frozen seafood and seafood processing environmental samples were collected from five different seafood factories and Hangzhau's retail markets. The samples were then examined for the presence of V. parahaemolyticus using alkaline peptone water enrichment and selective agar medium (thiosulfate-citrate-bile salts-sucrose, TCBS) followed by duplex PCR targeting the thermolabile hemolysin (tlh) and Gyrase B (gyrB) genes specific for this bacterium. Finally,163 (21.3%) out of 766 samples were identified as V. parahaemolyticus. The results indicate that V. parahaemolyticus with potential pathogenicity are present in fresh and frozen seafood as well as in seafood processing environments.Fifty-four selected V. parahaemolyticus isolates from clinical samples, seafoods and environments (including reference strain) were characterized by pulsed-field gel electrophoresis (PFGE) using Apal, Smal and Notl for DNA digestion. The enzyme Notl was found more discriminatory power than Smal or ApaⅠfor V. parahaemolyticus typing. Out of 54 strains, only 49 strains were typable, while other 5 strains were untypable. PFGE patterns after Notl digestion could be grouped into 11 distinct pulsetypes (types A to K). Five seafood isolates and the reference strain BJ1997 were grouped as cluster I, while the other seafood and environmental isolates were grouped as A to H, J and K. The clinical strains were grouped into clusters A, B, D, E, G and K respectively. Cluster B was the main cluster and comprised of 4 clinical isolates and 4 seafood isolates. It could be further divided into three subtypes with 2 clinical isolates (strains ZJ1 and ZJ5) belonging to subtype B1 and 2 seafood isolates (strains ZJ6 and ZJ7) grouping into B3. These results implied that the food poisoning in Hangzhou might be due to the consumption of improperly cooked seafoods contaminated with the potentially pathogenic V. parahaemolyticus. Our results also reveal that PFGE typing scheme could be used for the characterization of the high genetic diversity among the V. parahaemolyticus isolates from different origins.Two hundred and sixty-three V. parahaemolyticus (including 4 reference,32 clinical isolates and 227 seafood and environmental isolates) were examined for their hemolytic and urea-producing phenotypes together with the presence of major putative virulence genes by PCRs. All V. parahaemolyticus isolates were positive for both toxR and toxRS genes. Twenty-eight of clinical isolates (28/32,87.5%) and three seafood isolates (3/226,1.32%) were tdh-positive while only three of the clinical isolates (9.4%) and seven of the seafood isolates (3.09%) were positive for trh gene. Among the isolates,30 (96.77%) out of 31 tdh-positive strains were exhibited Kanagawa phenomenon,7 (70%) out of 10 trh-positive strains were urease positive. One tdh-positive of seafood isolate as well as 22 clinical isolates belonged to the new pandemic clone O3:K6 according to serotyping and sequencing of the toxRS locus. The new O3:K6 clone,01:KUT and O4:K68 shared the same characteristic variations from old O3:K6 clone at six base positions from 576 to 1244 of the toxRS locus. Twenty-six and 17 out of 32 clinical isolates (81.3%) were positive for GS-PCR and orf8-PCR respectively, while only one (0.44%) of the seafood and environmental isolates were positive for GS-PCR and five (2.21%) positive for orf8. These results demonstrate the new pandemic O3:K6 clone and other pathogenic serotypes of V. parahaemolyticus isolated from clinical and seafood sources are also present in the Chinese coastal areas.Sixteen representative V. parahaemolyticus having different phenotypes and genotypes were examined for their pathogenicity in in vitro and in vivo systems. The pathogenicity varied considerably among isolates. The clinical isolates were apparently more enteropathogenic (P<0.01 with I/B ratio,73.82% vs 62.07%) and more virulent than their seafoods counterparts to mice via intraperitoneal route with P<0.05, log LD50 6.62 vs 7.13). They were also more adherent to in vitro cultured cells and of higher cytotoxicity as measured by LDH release of the HeLa cells although there were no statistical differences. The tdh-positive V. parahaemolyticus isolates were of higher enteropathogenicity (P<0.05,74.24% vs 60.55%) and more virulent via intraperitoneal route (P＜0.05, log LD50 6.55 vs 7.21) than tdh-negative isolates. The tdh-positive isolates were generally more cytotoxic and adhesive to the cultured cell lines as well. From the in vitro and in vivo pathogenicity profiles, trh-positive isolates seemed to line between tdh-positive isolates and those without tdh or trh. These results suggest that clinical isolates or isolates having the tdh genotype were more pathogenic in in vitro or in vivo models than seafood or tdh-negative isolates.Tdh gene from 15 selected tdh-positive strains (12 clinical isolates and 3 seafood isolates) was sequenced in order to verify its genetic characteristics. The tdh of ten clinical isolates belong to tdhl (>98%), while tdh of another 3 isolates (1 seafood and 2 clinical) and 2 seafood isolates (F22 and F29) tended to be tdh2 (>99.2%) and tdh3 (>98%) respectively after sequence analysis. Furthermore, a DNA region with full length of 12 kb (corresponding to whole genome VPA1312 to VPA1327; ca.12 kb) was cloned and sequenced via the long and accurate (LA-PCR) from one seafood isolate HZ34. Two major putative virulence associated genes, encoding the cytotoxic necrotising factor and exoenzyme T, respectively, were found to be located on chromosome 2, which were corresponding to VPA1321 and VPA1327. In addition, genome walking kit was used to amplify the tdh-promoter and 3'end regions of tdh gene (ca.5.6 kb, including) from the strain F22 which possessed both tdh and trh genes. The results show that both 5'and 3'end regions contain both two open reading frames (ORFs). Two ORFs in the 5'end region are insertion sequence like element (IS), which were 97% and 99% homologous to V. parahaemolyticus TH3996 and V. parahaemolyticus RIMD2210633, respectively. We found two novel genes in the 3'end region of tdh locus which encode hypothetical proteins VP1767 (80.9% and 55.5%, respectively) on chromosome 1 of V. parahaemolyticus RIMD2210633.In summary, results obtained in these studies have laid good foundation for further exploration of the pathogenicity of V. parahaemolyticus from seafood isolates, regulation of virulence gene expression and the possibility of horizontal gene transfers.