Characterization Of Transmission And Molecular Epidemiology Of Vibrio Parahaemolyticus And Multilocus Sequences Typing Of O3:K6 Pandemic Clone

Vibrio parahaemolyticus, a gram-negative halophilic bacterium, is widely disseminated in estuarine, marine, and coastal environments throughout the world. Water temperature, salinity, zooplankton blooms, tidal flushing, and dissolved oxygen play an important role in dictating its spatial and temporal distribution. It was first identified as a cause of food-borne illness in Japan in 1950, having caused over half of all food poisoning outbreaks of bacterial origin since 1996. Most V. parahaemolyticus-mediated outbreaks occurred during the warmer months and were attributed to seafood.The known virulent genes of V. parahaemolyticus are tdh and trh, encoding thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH). TDH has been shown to have hemolytic, enterotoxic, cardiotoxic, and cytotoxic activities. After 1996, there existed worldwide pandemic of V. parahaemolyticus new O3:K6 serotype, which derived many other serovariants and formed so-called pandemic O3:K6 clone that became main paghogen causing the rapidly increase of hospitalizations. The O3:K6 clone strains were fist found in Calcutta, India, then in Taiwan, China, and Japan etc, pervading many coastal countries in the world. The publishment of the whole genomic sequence of RIMD2210633 (a pandemic O3:K6 strain collected from Japan in 1996) offered a good source for the studies of V. parahaemolyticus. The chromosomal regions acquired from other Vibrio species by horizontal gene transfers and recombination such as VPaIs, systemic virulent genes, toxRS/new, orf8 were the main factors that caused the forming of O3:K6 clone, which may increase the fitness of the bacterium in a particular environment and enhance the invasive ability of the strain. There are two sets of T3SSs in V.parahaemolyticus, T3SS1 and T3SS2. It was showed that T3SS1 involved in the cytotoxicity in HeLa cells, while T3SS2 has a role in enterotoxicity in a rabbit model. Both T3SS1 and T3SS2 might act synergistically in the pathogenesis of V. parahaemolyticus. So far, the overall mechanism of pathogenesis of V. parahaemolyticus remains unclear.Although serotyping has been the main method to track the spread of V. parahaemolyticus, there are many technologies such as ribotyping, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) using for the studies of molecular pathogenic epidemiology.In recent years, it was found that most foodborne outbreaks were not only directly related with the consumption of marine food products but also closely with the consumption of freshwater food products. Thus, we hypothesize that the ways of V. parahaemolyticus transmitted to humans have been changed. On the other hand, V. parahaemolyticus was the most important pathogen of all foodborne pathogens causing . outbreaks. In this study, it was mainly analyzed the diffusing routes of V. parahaemolyticus and the status of pathogenic strains and pandemic strains in food chains; and also detected the pathogenic genes and genomic islands (GIs), and analyzed the relationship of them, the evolution of pandemic clone. Based on MLST of V. parahaemolyticus, the ancestor strain of O3:K6 pandemic clone was traced.1. Epidemiological transmission characteristics, molecular typing, and serodiversity of V. parahaemolyticus in food chainsIn this chapter, it was investigated that the distribution, prevalence, molecular characteristics of RAPD, the status of virulent genes, and serodiversity of V. parahaemolyticus from aquatic products, cooked food and clinical patients at active surveillance hospitals and patients of food poisoning outbreaks.Based on the results of molecular biological and epidemiological analysis, it was clearly raised that V. parahaemolyticus could be transmitted not only through seafood but also through freshwater products in food chains, which was different with the traditional definition that V. parahaemolyticus carried only in seafood. Aquatic products including sold widely at markets, hotels and restaurants caused the cross-contamination of V. parahaemolyticus between seafood and freshwater products and other food products, thus, the markets, hotels and restaurants became the critical points causing the cross-contamination and resulting in food poisoning cases in the end. The results gave us good guide in applying HACCP for controlling foodborne diseases caused by V. parahaemolyticus.The results of RAPD with P4 primer showed that the main types of V. parahaemolyticus were type A and type B, the main toxigenic subtypes mainly belonged to A8 and A6 in China. Results showed that the serotypes in foodborne strains were abundant and highly diversity; there existed 9O serogroups and 61 serovars, among them no 07 and 09 groups appeared, but 30 serotypes are novel serovars that were not listed by Japanese Committee on the Serological Typing of V. parahaemolyticus. Serotypes O3:K6, O4:K68 found in foodborne strains, closely relative with pandemic clone, expressed potential dangers of pandemic strains in food chains.2. Epidemiological characterization of O3:K6 pandemic clone and its systemic functional genes of V. parahaemolyticusIn this chapter, the status were investigated whether or how the pandemic clone existed in food chains, the epidemioloigcal characterization and serotypes distribution of pandemic clone in V. parahaemolyticus strains collected from aquatic products, food, clinical patients at active surveillance hospitals and patients of food poisoning outbreaks. The status of systemic functional genes such as tdh, toxRS/new, orf8, HU-a, VP2905, Mtase genes occuring in the pandemic strains were also studied.Results showed there did exist pandemic strains in food chains that could adapt the environments through genes recombination and transmitted through the food chains, caused foodborne outbreaks. O3:K6 was the main serovar of the pandemic strains and other serovars included 01:KUT, O1:K25, O1:K26, O4:K68 and the novel serovars 01:K36, O3:K25,03:K68 emerged in the study. O3:K6 pandemic clone in China belongs to the O3:K6 clone ecological niche in Asian. Though serovar O3:K6 was the main serotype of pandemic clone, some new serovar of the clone could cause local pandemic and become the main serovar in some local area.Different from other previous studies, V. parahaemolyticus could exsited in culturable state in winter. Our results also showed that the infected model of V. parahaemolyticus could have changed that it could infect human and cause diarrhea even in cold condition, such as in winter. It is possible that controlling the transmission of pathogenic and pandemic strains in food chain and especially not eating raw aquatic foods (including freshwater food) could reduce the foodborne diseases largely.The systemic functional genes, toxRS/new, orf8, HU-a, VP2905, Mtase genes play great roles in the pathogenicity and the evolution of pandemic clone. Though not all pandemic strains harboring these genes at the same time, the four genes could be as markers of pandemic clone. As for quickly testing pandemic strains (including pathogenic strains), these four genes and tdh, toxRS/new could be good used as diagnostic markers.3. Genomic islands in Vibrio parahaemolyticus isolatesIn this chapter, the genomic islands, VPaI-1-VPaI-7, T3SSs and other genomic segments related with pathogenicity, were analyzed in 302 isolates.Among 7 genomic islands, also different from pandemic clone of V. parahaemolyticus reported previously, the genomic islands VPaI-1, VPaI-4 and VPaI-5 were unique to pandemic clone in China; VPaI-1 and VPaI-5 played great roles and the VPaI-4 could be the last genomic island during the forming of pandemic clone. It is very interesting that VPaI-6 was not unique to pandemic clone, for most of V. parahaemolyticus including nonpathogenic, pathogenic and pandemic strains harbored this island. VPaI-7 (includinge T3SS2) was closely associated with tdh gene, all tdh positive strains harbored VPaI-7 and T3SS2. Most of the pandemic strains harbored VPaI-2 and VPaI-3; but these two VPaIs were not unique to pandemic clone, for a part of tdh positive strains and a few of nonpathogenic strains also harboring VPaI-2, and nonpathogenic O3:K6 strains also harbored VPaI-3; it could be speculated that the nonpathogenic strains harboring VPaI-2 and VPaI-3 might be the intermediate strains during the transition from nonpathogenic to pandemic strains, that is, VPaI-2 and VPaI-3 were the first VPaIs got by nonpathogenic strains to become pandemic strains.Results of this study showed that all isolates harbored T3SS1. It is important for pandemic clone's pathogenicity to harbor the whole T6SS, and type I pilus. As most of strains harbored biofilm genes, these genes should be inherence for V. parahaemolyticus adaptting for the environments variety.4. Multilocus sequence typing and origin of O3:K6 pandemic cloneIn this chapter,120 strains of V. parahaemolyticus were analyzed with MLST. Based on the systemic genes and 7 VPaIs discussed in above chapters, we analyzed the evolution characterization and explored which kind of strain was the ancestor of O3:K6 pandemic clone, the first O3:K6 pandemic strain found in Asian.It is showed that there were all 45 STs among 112 strains identified by MLST including one complex clone, one doublet and 38 singletons. O3:K6 pandemic clone in China and the international pandemic clone was the same origin, all belong to the CC3, whose ancestor ST was ST3, an worldwide distribution ST.Markedly, the ancestor strain of O3:K6 pandemic clone was found. Results showed that there were no inevitable relationship between ST3 strains and the strains carried all/parts/no systemic functional genes and VPaIs. The ancestor ST3 of O3:K6 pandemic clone originated from the environmental strain:O3:K6, ST3 (not harbor systemic functional genes and VPaIs, except VPaI3). Through the chromosomal regions acquired from other Vibrio species by horizontal gene transfers and recombination such as VPaIs, systemic virulent genes, the environmental strain (ancestor strain) got VPaI-3 firstly, then others, and became the pandemic O3:K6 strain emerged in 1996. After 1996, new pandemic serotypes appeared by recombination of O:K genes for adapted environment varieties.Furthermore, it is showed that the house keeping genes in pandemic CC3 evolved quickly by newly emerging SLVs of CC3, ST2, ST192, ST227, ST196 and ST220. There was no relation between CC3 and the pathogenic strains carried with tdh and/or trh genes. The MLST method offered an effective tool for the surveivallence of new pathogenic complex clone(s).