Analysis Of The Distribution Of Virulence-related Genes And Antibiotic Resistance Of Vibrio Parahaemolyticus And Vibrio Alginolyticus

Foodborne Vibrio parahaemolyticus and Vibrio alginolyticus are common aquatic microorganisms in marine environment. They are also pathogens to aquaculture animals and human being by causing food poisoning as well as gastrointestinal infection in humans and vibriosis in aquaculture that would lead to huge economic loss. Although V. parahaemolyticus is highly homologous to V. alginolyticus, researches were mainly focused on V. parahaemolyticus, the pathogenesis of V. alginolyticus remains mianly unknown. This study focused on the research of the distribution of virulence-related genes and antibiotic resistance of V. parahaemolyticus and V. alginolyticus isolated from environment and aquatic products. This study provided guidance for pathogenesis, antibiotic resistance mechanism as well as practical benefits of epidemiology and food safety risk assessment of V. parahaemolyticus and V. alginolyticus. Main results were showed as the following:1. Two sets of multiplex PCR assays that can simultaneously detect 11 virulence-related genes were developed and applied to investigate the distribution of virulence genes among 248 V. parahaemolyticus and 128 V. alginolyticus isolatated from environment and aquatic products like Perna viridis, Meretrix lusoria, Royal Mussel and etc. Target genes included hemolysin genes(trh, tdh and tlh), toxic regulation genes(tox R and tox S) and other virulence associated genes(Collagenase, Ure R, Fla A, omp W, Asp A and fur). Results showed that tlh, Collagenase, Fla A, omp W, fur, Asp A, tox R and tox S were widely distributed in both species with distinctive characteristics: more than 90% V. parahaemolyticus carried Asp A, tox R and tox S genes but none of them carry trh and tdh, while 38.28% V. alginolyticus carried trh and tdh and fewer isolates carried Asp A, tox R and tox S.2. With the combination of serotyping and molecular typing, high pathogenicity serotype such as O3(8) and O4(9) Vibrio parahaemolyticus strains were found in 248 V. parahaemolyticus. They were digested by Sfi I and fingerprinted by pulsed field gel electrophoresis(PFGE).3. Kirby-Bauer disk diffusion method was applied to test the antibiotic resistance of 18 commonly used antibiotics( Ampicillin, Amoxycillin, Carbenicillin, Cephalothin, Cefoperazone, Cefuroxime sodium, Cephazolin, Gentamicin, Streptomycin, Amikacin, Vancomycin, Polymyxin B, Tetracycline, Trimethoprim/sulphamethoxazole, Erythromycin, Ciprofloxacin, Rifampicin and Clindamycin) in aquaculture against V. parahaemolyticus and V. alginolyticus. Both species showed resistance to antibiotics in varying degrees and the significant multiple resistance characteristic of both species indicates the serious rational application of antibiotics in aquaculture industry as well as the importance of research on drug resistance mechanism of Vibrio, especially the location of resistance gene of Vibrio alginolyticus.In summary, this study has developed two sets of multiplex PCR assays applied to investigate the distribution of virulence related genes among V. parahaemolyticus and V. alginolyticus, fingerprinted high pathogenicity serotype O3 and O4 Vibrio parahaemolyticus strains via PFGE, analysed the antibiotic resistance status of 248 V. parahaemolyticus and 128 V. alginolyticus isolated from environment and aquatic products, in order to explore the distribution and epidemiological characteristics and the pathogenesis, antibiotic resistance mechanism of V. parahaemolyticus and V. alginolyticus, provid guidance for epidemiology and food safety risk assessment.