| Vibrio parahaemolyticus, a Gram-negative halophilic bacterium, is an important foodborne pathogen caused disease outbreaks in coastal areas of China, and may cause human sporadic and local epidemic of gastroenteritis. The scale of outbreaks and the crowd exposure in coastal areas and parts of inlands caused by Vibrio parahaemolyticus are rising, having ranked first in the microbial foodborne outbreaks. V. parahaemolyticus has been known to produce either thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), or both. The bacteria could be transmitted not only through seafood but also through freshwater products in food chains. The pathogenic mechanism of V. parahaemolyticus has not unclear, but some virulence genes have been confirmed. Moreover, pulse field gel electrophoresis basing on the structure of the DNA in microbial identification has more advantages on the typing ability, resolution, repeatability, comparability and sensitivity, due to the traditional biochemical and serotyping having not been effective for homology analysis. Therefore, in this study we investigated the distribution of virulence-associated genes of V. parahaemolyticus isolates in China, especially pandemic strains and detected the antimicrobial susceptibility of isolates with K-B method recommended by Clinical and Laboratory Standard Institute in2007. In order to deterimine genetic lineages and the source of V. parahaemolyticus isolates, PFGE typing was conducted, and analyzed the relationship between the PFGE subtypes and virulence-associated genes distribution characteristics, by which providing the scientific foundation for the early warning, monitoring and manipulation to prevent foodborne diseases caused by V. parahaemolyticus.In this study, we first attempted to detect the virulence-associated genes of233V. parahaemolyticus strains isolated in Shanghai, Zhejiang, Fujian, Guangdong, and Jiangsu from2008to2010. The results suggested that there were117pathogenic strains (52.9%),94pandemic strains (42.2%) in233V. parahaemolyticus isolates. Among117pathogenic strains,108of them were tdh positive strains (48.9%), and9were trh positive strains (4.0%). The positive rates of orf8, MTase, HU-a and VP2905of all strains were42.6%,43.0%,41.7%and43.0%, respectively. Moreover, the positive rates of pandemic and pathogenic strains from clinical isolates were significantly higher than those from foodborne. Also, among94pandemic strains, the positive rates of orf8, MTase, HU-a and VP2905were97.8%,97.8%,96.8%and96.8%, respectively, which showed that orf8, MTase, HU-a and VP2905could be the marker genes of pandemic strains.The177clinical strains and139foodborne strains were analyzed for susceptibility to7kinds of antibiotics, and the results showed that these strains were resistant to Ampicillin57%, Streptomycin40.5%and Kanamycin25.3%; the resistance to Norfloxacin, Chloramphenicol, Trimethoprim-sulfamethoxazole and Tetracycline was only0.3%,0.3%,0.9%,1.6%, respectively. These results suggested that the resistant phenomenon of these strains was not serious. Also, the resistance to Tetracycline, Kanamycin and Ampicillin in177clinical isolates was higher than foodbrone isolates, but lower to Streptomycin, Norfloxacin, Chloramphenicol and Trimethoprim-sulfamethoxazole. Pandemic, pathogenic and non-pathogenic strains all had a high resistence to Streptomycin, Kanamycin and Ampicillin, but they had different levels of antibiotic resistance. The results also showed that majority of the pandemic isolates were susceptible to antibiotics commonly used to treat patients’diarrhea, and the resistance phenomenon was not significant. However, our results were different from previous report that all pandemic strains had the same antibiotic profile as described by Okuda. In this study, most of but not all pandemic strains (58.4%) were resistant to Ampicillin. In this study, we applied PFGE to investigate141V. parahaemolyticus isolates fromJiangsu, Shanghai, Zhejiang, Fujian and Guangdong. The results indicated that119strains can be typed and the typed rate was84.4%.119strains can be divided into108PFGE types and5genetic lineages (I, Ⅱ, III, IV and V), and the distribution rates of these5genetic lineages in V. parahaemolyticus isolates tested were14.3%,25.2%,15.1%,13.4%and31.9%, respectively, which suggested that the isolates from mainland China were mainly distributed in the lineage Ⅱand ⅤV.The results of the PFGE lineage classification of isolates from different regions showed that Yangzhou strains were mainly in the lineage II and V Zhejiang strains distributed in I and the rest of the other strains, which relatively concentrated, had their own predominant lineages. The results of the PFGE lineage classification of isolates from different years suggested that isolates in2006,2007,2008,2009and2010were mianly in the lineage IV, V, V, I and III, respectively; isolates from different years had different predominant lineages. The results of the PFGE lineage classification of isolates from different sources indicated that clinical strains concentrated in II and V, and foodbrone strains were mianly in V. These results suggested that distribution of PFGE lineages had some association with areas, separating time and different host. The results of antibiotic resistance of different lineage classification showed that the isolates in all genetic lineages were sensitive to Norfloxacin, Chloramphenicol and Trimethoprim-sulfamethoxazole; isolates resisted to Tetracycline were mianly in I and Ⅱ, and the distribution of isolates resisted to Streptomycin, Kanamycin and Ampicillin in the lineages were not significantly different, which indicated that distribution of PFGE lineages had not correlation with antimicrobial resistance of strains.The results showed that PFGE could be a powerful tool for the determination of dissemination route, molecular epidemiological pattern, tracing the source of V. parahaemolyticus. It was found that9groups of V. parahaemolyticus isolates had the same PFGE profiles (the dice parameter was100%) and the homology among some strains was very high (>80%), indicating that V. parahaemolyticus may spread to human and cause foodborne illness through horizontal transmission, cross-contamination in the process of food marketing, consumption, transportation and so on.The combination analysis between the results of PFGE and virulence-associated genes showed that pandemic, pathogenic and non-pathogenic strains had their own unique genetic lineage. Pandemic strains were mainly present in the pedigree I and V; most non-pathogenic strains distributed in Ⅳ; two kinds of pathogenic strains showed different distribution in the genetic lineages, and the tdh positive strains mainly distributed in the pedigree I, but the trh positive strains mainly distributed in the lineage II and III. The PFGE lineages of V. parahaemolyticus isolates containing orf8, MTase, HU-a and VP2905genes were same as those of pamdemic strains, which may further suggest that these four genes can be the marker genes for pandemic strains.We compared PFGE and MLST for typing V. parahaemolyticus and showed that the two methods yield similar results, although with differences in resolving power and reproducibility. Moreover, PFGE has more power of discrimination than MLST and appeared to be a "Gold standard" technique for V. parahaemolyticus. The ST-3ancestor of pandemic strains and the other members of CC3were only concentrated in the genetic lineage I, II and V; moreover, the major pedigree of pandemic strains was genetic lineage II and V.It is important that the ancestor strain of pandemic strains and the pandemic strains had the same PFGE profile or genetic lineage. |
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