Distribution And Population Structure Of Vibrio Vulnificus In Zhejiang Coastal Area

Vibrio vulnificus is a gram-negative halophilic marine bacterium, which is distributed in the estuaries, marine and coastal environments worldwide, and found in a variety of seafood, including shrimp, fish, oysters and clams. This bacterium was recognized in 1976 in the United States, and it is the leading cause of reported death due to the consumption of seafood in the United States, with approximately 40 cases per year.A lot of coastal countries around the world have reported the acute gastroenteritis, primary septicemia, and wound infections cases due to the V. vulnificus, and the serious cases need amputation or even death. The first V. vulnificus infection case in China was reported in Zhejiang Province, and most cases were found in this area. V. vulnificus infection cases were generally considered to be a result of eating or handling the seafoods contaminated with V. vulnificus, and occurred in the month with high temperature. There are many kinds of seafoods were harvested along the coast of Zhejiang Province, and V. vulnificus presents a significant public health risk to certain persons consuming raw or undercooked seafood, or who acquire hepatitis, hypertension or other immunocompromised diseases.The objectives of this study were to investigate the prevalence of V. vulnificus in seafoods, detect and analyze the molecular characteristics of the clinical isolates, analyze the virulence characteristics and genetic relationship of these V. vulnificus strains from different sources in Zhejiang Province, to provide the data for the food safety risk assessment, monitoring and early warning, and molecular tracing. The main results obtained in this study are as follows:(1) Through the investigation of the prevalence of V. vulnificus in retail seafoods in Zhoushan and Hangzhou, the contamination of V. vulnificus were found in the seafoods, and the highest positive rate and concentration of V. vulnificus were found in the summer, and the high positive rate were found in clams, shrimps and oysters. Both biotype 1 and biotype 2 V. vulnficus strains were isolated, genetic diversity were showed between these isolates, and vcg C-16S rRNA B type was the main genotype, and it prompts the pathogenic potential of these isolates. The antibiotic susceptibility results showed that these isolates were sensitive to most antibiotics, but resistant to some antibiotics belong to aminoglycosides and cephalosporins antibiotics.(2) Three V. vulnificus strains were isolated from the foodborne disease surveillance, these strains were biotype 1 and vcg C-16S rRNA B genotype. The antibiotic susceptibility results showed that three strains were resistant to tobramycin. It is interesting that two cases were coinfection with V. vulnificus and V. parahaemolyticus.(3) A new MLST type V. vulnificus strain was isolated from the wound infection case, it was named ST136 and was biotype 1 with vcg C-16S rRNA B genotype. The antibiotic susceptibility results showed that it was intermediate to cefazolin and cefotetan.(4) The different molecular typing methods including MLVA, MLST and PFGE were performed to subtype the V. vulnificus strains from different sources, both advantages and disadvantages were found among the various molecular typing methods. PFGE is the -gold standard" molecular typing method, but DNA degradation of V. vulnificus was occurred in the process of PFGE. Under the Not Ⅰ-digested condition, the Simpson index D value is 1, and in Sfi Ⅰ-digested condition, the D value is 0.998. The results showed that the Sfi Ⅰ was the priority restriction enzyme for V. vulnificus PFGE, and Not Ⅰ as an alternative option. It is a powerful tool to study the V. vulnificus infection and transmission combined with the epidemiological information. The D value of molecular typing method MLVA is 0.994, and it can be used to the epidemiological investigation and traceability study. The D value of MLST is 0.983, and it can be used to study the population structure and genetic relationship among V. vulnificus strains.(6) Sixty-one V. vulnificus strains isolated from shrimps and clinical were collected from Beijing, Shanghai, Guangdong, Zhejiang and Fujian, MLST analysis was performed in these strains, and 46 new ST types was identified. Abundant genetic diversity was found in these strains, and its population structure, phylogenetic and genetic relationships among strains were analyzed.In summary, the contamination situation of V. vulnificus in retail seafoods is serious in Zhejiang Province, and the virulent genotype isolates was accounted for the majority.Although few direct cause-related cases was reported, but it can’t be excluded the probability of a new virulent strains occurring through the genetic recombination. This study will give us a better understanding of the V. vulnificus in Zhejaing Province, and laid a good foundation for the disease control and prevention.