Molecular Typing And Virulence Gene Screening Of Vibrio Parahaemolyticus From Different Sources

Vibrio parahaemolyticus is an important foodborne pathogen, oftenisolated from fish, shellfish and other marine products. In recent years, withthe increased consumption of seafood, more and more cases of foodborneillnesses have been reported. V. parahaemolyticus has become the mainfoodborne pathogenic bacterium in coastal cities. So it is very important tofind out its epidemiologic characteristics and establish some techniques forthe source tracking in outbreak investigations.In this study,109V. parahaemolyticus isolates from patients, food andenvironment were involved. They were firstly serotyped by conventionalserological method, and four of their virulence genes were identified byPCR amplification; after that,109V. parahaemolyticus isolates were typedwith two molecular typing methods: ERIC-PCR and MLST to enclose theirgenotypic polymorphism and genetic correlation of V. parahaemolyticusfrom different sources.Serotyping was conducted by slide agglutination with11O-antibodiesand8-K antibodies. It showed that the isolates belonged to26serotypes,and among them, O3:K6and O4:K8were the most popular serotypes,accounting for20.7%and19.5%, respectively. Other serotypes wereO4:K68, O1:K25, O3:K68, O3:K25, O3:K8, O1:K6and so on. V.parahaemolyticus isolated from environment demonstrated more significant diversity in both K and O serotype distribution than those frompatients and food.Four virulence-related genes: tdh, trh, toxRS/new, and ORF8wereanalyzed by PCR method. It demonstrated that64.2%isolates carried tdhgene, and only7.3%had trh gene, and the positive rate of toxRS/new andORF8were39.4%and30.3%, respectively. Among the109V.parahaemolyticus isolates,71isolates were potentially pathogenic strains:63were tdh+/trh-,7were tdh+/trh+, and only1was tdh-/trh+that is anenvironment isolate.The ERIC-PCR analysis of the109isolates was conducted and theseisolates were subtyped based on their fingerprints. It demonstrated that the109V. parahaemolyticus isolates were grouped into65patterns and31clusters with the discriminative index of0.942. Among the31clusters,ERIC-19was the most pandemic cluster with38isolates,97.4%(37/38) ofwhich carrying tdh gene. There were8clusters that consisted of isolatesfrom two different sources; it showed that the isolates had certain relevancein genetic level. The study also revealed that it was a suitable andconvenient method to combine serotyping and ERIC-PCR genotypingtogether for the rapid epidemiological source tracking.In addition, MLST was also applied for further molecular typing toidentify the genetic correlation of the isolates from different sources. Atotal of55sequence types (STs) were found, among them,40novel STsand78novel allelic profiles accounted for77.7%(40/55). All these novelallelic profiles and STs had been confirmed and deposited in the MLSTwebsite. Among the55STs, ST-3was aggregated by34strains including28clinical isolates and6food isolates, and all the34isolates were all in theERIC-19cluster, which confirmed that ST-3was the most pandemic ST. The phylogenetic analysis indicated that ST-305/ST-830and ST-3/ST-155/ST-794had a fairly close genetic relationship. The discriminativeindex of MLST was0.99,0.96and0.76for the environmental, food andclinical isolates, respectively, which demonstrated that MLST subtypingwas more suitable for non-clinical isolates of V. parahaemolyticus.