| Background Vibrio parahaemolyticus was first discovered during an outbreak of food poisoning in Japan in 1950. It has been implicted as a major cause of foodborne disease around the world. In China, Vibrio parahaemolyticus has become the most common and important food pathogenic bacteria. It’s very important to investigate the characteristics of phenotype, genotype and pathogenicity of Vibrio parahaemolyticus.Objective Gain insight into phenetic and genetic polymorphism of Vibrio parahaemolyticus by biochemical identification, antibiotic susceptibility test, serological detection, screening virulence genes and molecular typing. To explore pathogenic mechanisms of Vibrio parahaemolyticus by interacting with cells in vitro, Pathogenicity evaluation system of Vibrio parahaemolyticus was preliminarily established in the laborary.Methods All 500 food strains and 135 clinical strains were identified by API 20E, which were collected from National Foodborne Disease Surveillance Network. The occurrence of virulence genes, virulence-related genes and species-specific genes in Vibrio parahaemolyticus was studied by PCR. The antimicrobial susceptibilities of experiments strains were determined by the broth microdilution method as recommended by CLSI, eight antimicrobial agents were examined. A selected subset of 71 food strains and 135 clinical strains were analyzed with pulsed-field gel electrophoreses, PFGE patterns were imported and analyzed with BioNumerics software. Based on National food microbiological examination standard GB/T 4789.7-2008,71 food strains and 135 clinical strains were serotyped by using commercial antisera. A diverse panel of 20 food isolates and 14 clinical isolates representing various serotypes, antibiogram and genotypes, were selected to study pathogenicity by interaction with HeLa cells in vitro. Vibrio parahaemolyticus adherence to cultured cells was measured by staining with Giemsa stain. The cytotoxic assay was performed by quantifying the release of lactate dehydrogenase with the Cyto Tox96 kit. To asses the invasive ability of selected experiment strains, quantitative study was done using antibiotic survival assays.Results Of 500 strains isolated from fodd,5 strains were not Vibrio parahaemolyticus, All 135 clinical strains were Vibrio parahaemolyticus by chemical identification.The results of antibiotics susceptibility test showed that 66 strains were resistant to one or two antibiotic agents, accounting for 13.33% food strains.11.31% food strains were resistant to ampicillin,1.62% for trimethoprim and sulphame-thoxazole,2, and 1 strain for chloromycetin and ciprofloxacin. All food strains were sensitive to cefotaxmine, ceftazidime and gentamicin. Of 135 clinical strains,11 strains were resistant to one or two antibiotic agents, accounting for 8.15%.6.67% clinical strains were resistant to ampicillin, 2 strains were resistant to trimethoprim and sulphame-thoxazole,1strain to tetracycline. All clinical strains were sensitive to cefotaxmine, ceftazidime, gentamicin, ciprofloxacin and chloromycetin.Serological analysis of the O and K antigens claimed that a total of 30 serotypes were identified among 71 food strains, belonging to seven O groups.3 strains were untyped, 46.48% food strains were identified O1 and 05.22 K serum were agglutinated,37 strains were untyped for K serum.29 serotypes were identified for clinical strains, predominantly 03,04 and O1 groups, accounting for 89.26% clinical strains.22 K serum were agglutinated,12 strains were untyped for K serum. O3:K6 was dominant serotype, accounting for 56.30% clinical isolates.The results of PCR methods claim that all experiment strains carry species-specific genes such as tlh, toxR, gyrB, vpm. Most of clinical strains (85.92%) carry tdh and/or trh. 85.19% were positive for the tdh gene,3 isolates for both tdh and trh genes, and 2.96% for trh gene.66.67%、80.74%、65.19%、66.67% clinical strains are positive for the gene of GS-PCR、PGS-PCR、orf8、HU-α, respectively. None of food strains are positive for tdh gene,2.08% food strains carry trh gene.Genomic DNAs of 71 food and 135 clinical strains were digested with Sfi I and Not I, the molecular size of PFGE restriction fragments used for analysis ranged from 30kb～ 700kb. When subjected to UPGMA clustering using the Dice coefficient, two enzymes showed a similar discrimationary power. Both Sfi I and Not I methods allowed differentiations of 71 food strains and135 clinical strains into 15 groups. Strains in K group were all clinical isolates by digested with Sfi I, accounting for 43.20% experiment strains. Pandemic clone and sporadic strains were divided into different groups.78 strains were analyzed with the Ribotyping,7～10 bands were endonucleased by EcoR I. Molecular size of bands lied between 4kb～50kb. Ribotyping with restriction endonuclease EcoR I gave 64 kinds of RP patterns, which divided into 7 groups. Molecular size of bands analyzed by PFGE lied between 25kb～1200kb. PFGE with Sfi I and Not I gave 68 and 71 kinds of PFGE patterns respectively.Pathogenicity of 20 food strains and 14 clinical strains were studied in vitro.13 food strains possess the ability to adherence to epithelial cells, adhesion index’s about 10.9 clinical strains exhibited stronger adherence intensities, adhesion index of 5 strains were above 30. Clinical strains carrying both or neither viulence genes don’t possess the adherence ability. Vibrio parahaemolyticus apparently induced LDH release of HeLa cells in vitro.The difference between clinical and food isolates in cytotoxicity is not statistically significant.13 clinical isolates examined in antibiotic survival test had an invasive phontype. 1%o～2%o of the original inoculum of strains was internalized into HeLa cells, while 2 food strains had the invasion potential.Conclusions In China, almost of Vibrio parahaemolyticus strains isolated from clinical scources are pathogenic strains, while only<3% food isolates are pathogenic strains. Food and clinical strains are susceptible to the majority of antimicrobials examined. However, the examination confirms the high prevalence of Vibrio parahaemolyticus resistance to ampicillin. The first-line drugs remain effective against Vibrio parahaemolyticus infection. Just as Southeast countries, O3:K6 is the prevalent serotype among strains isolated from diarrheal patients in China, while among the food strains there are various serotypes. Food and clinical strains can be separated by both enzymes using PFGE. For clinical strains, pandemic clone and sporadic strains can be divided into different groups by either enzyme. GS-PCR and HU-a are the reliable tools to identify pandemic clone. The pathogenic mechanisms of Vibrio parahaemolyticus are very complicated, adherence, cytotoxicity, invasion potential were indispensable during pathogenic processes. Virulent strains involved in food poisoning, were observed to have higher adherence ability and invasion potential than food strains.The cytotoxicity experiments haven’t shown statistical difference between food and clinical strains. Invasion potential of Vibrio parahaemolyticus may be correlated with virulence gene. |
PDF Full Text Request