Evaluation of the virulence of Vibrio parahaemolyticus strains isolated in Russia and contiguous states
О А ШалуШалу Оксана АндреевнаЕ Н ГоленищеваЕ Н ГоленищеваЛ М СмоликоваЛ М СмоликоваГ В ГальцеваГ В ГальцеваН В БожкоН В БожкоE. V. MonakhovaE. V. MonakhovaО А ШалуО А ШалуYe N GolenishchevaGolenishcheva Ye NЛ М СмоликоваГ В ГальцеваН В БожкоYe V MonakhovaMonakhova Ye
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Abstract:
One hundred and ninety-six Vibrio parahaemolyticus strains that are etiological agents causing outbreaks and sporadic cases of food toxicoinfections and isolated from the environments of Russia, Ukraine, and Turkmenia are characterized by virulence-associated phenotypic and genotypic signs. Most clinical strains were found to contain the genes of the major pathogenicity factors - thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH); however, a large number of pathogens lack these genes. In all likelihood, these strains can cause human diseases, by producing other toxic substances. The presence of the trh gene correlated with that and expression of the urease genes. Comparative analysis of the findings has demonstrated that the results of polymerase chain reaction detection of the tdh gene do not always agree with the signs of hemolytic activity on the Wagatsum medium (Kanagawa phenomenon). Therefore, both tests that complement each other are recommended for a fuller evaluation of the potential danger of Vibrio parahaemolyticus strains.Keywords:
Hemolysin
Vibrio Infections
The Kanagawa phenomenon induced by the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus is almost exclusively associated with clinical strains, and TDH has been considered an important virulence factor. However, Kanagawa phenomenon-negative strains isolated from patients with diarrhea have recently been shown to produce TDH-related hemolysin (TRH). We studied the distribution of the tdh gene encoding TDH and the trh gene encoding TRH in vibrios by hybridization analyses. The presence or absence of the tdh gene and the trh gene in 285 strains of V. parahaemolyticus was examined by the DNA colony hybridization test with a tdh gene-specific probe and a newly constructed trh gene-specific probe. For assessment of the importance of TRH, many Kanagawa phenomenon-negative clinical strains (35.4% of all strains) were included. Of 214 clinical strains of V. parahaemolyticus, 112 strains (52.3%) had the tdh gene only, 52 strains (24.3%) had the trh gene only, and 24 strains (11.2%) carried both the tdh and the trh gene. The coexistence of the tdh and trh genes in these 24 strains was confirmed by Southern blot hybridization analysis. Of 71 environmental strains, 5 strains (7.0%) hybridized very weakly with the trh gene probe and none hybridized with the tdh gene probe. These results suggest that TRH as well as TDH is an important virulence factor of V. parahaemolyticus. Among 118 strains of other Vibrio species examined for the trh gene, only 1 strain of Vibrio furnissii gave a very weak hybridization signal. Among 48 representative trh gene-positive strains of V. parahaemolyticus, only 18 strains (37.5%) were found to produce TRH in culture medium when examined by a sensitive enzyme-linked immunosorbent assay method.
Hemolysin
Hybridization probe
Southern blot
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Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that inhabits the marine and estuarine environments. It is an important human pathogen causing gastroenteritis when raw or partially-cooked seafoods are consumed. Its pathogenicity is believed to be related to hemolysins such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). PCR method was used to examine three different hemolysin genes in isolates from clinical and seafood samples in Zhejiang province. The tlh gene was found in all isolates. The tdh gene was positive in all eleven clinical strains but only in one out of a total of 42 seafood isolates. The Kanagawa phenomenon was positive for all tdh-positive isolates. None of the isolates was positive for the trh gene. The urease test was negative for all isolates. Thus, it was assumed that the urease gene could be linked with trh gene. Further research is required to examine the relationship between low prevalence of the major virulence factor TDH and the high incidence of foodborne V. parahaemolyticus infections,and its pathogenesis.
Hemolysin
Thermolabile
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Hemolysin
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Loop-mediated isothermal amplification(LAMP) was used to detect and analyze virulence-associated genes, thermostable direct hemolysin(tdh) and thermostable-related hemolysin(trh), in Vibrio parahaemolyticus strains isolated from commercial shellfish in Shanghai from August to October, 2014. A total of 180 samples of six common species of shellfish were studied and results were compared between LAMP and polymerase chain reaction(PCR) assays. The detection rates of tdh and trh genes in Vibrio parahaemolyticus strains from commercial shellfish were 12.77% and 11.66% using LAMP as compared to 10.55% and 7.77% by PCR, respectively. Serotyping the isolated virulent strains revealed two O3: K6 serotype Vibrio parahaemolyticus strains, including one carrying double-positive virulence genes(tdh+/trh+). Additionally, high similarity was observed between pulsed-field gel electrophoresis(PFGE) bands of two O3: K6 serotype Vibrio parahaemolyticus strains(similarity 90%). The results indicate contamination of commercial shellfish in Shanghai by virulent strains of Vibrio parahaemolyticus, where the emergence of tdh+/trh+ O3:K6 serotype Vibrio parahaemolyticus is a cause for concern. Surveillance needs to be strengthened, for Vibrio parahaemolyticus strains of different serotypes, especially the O3: K6 serotype. Results from the comparison with PCR assay indicated that LAMP method was suitable for the detection of virulent strains of Vibrio parahaemolyticus in shellfish.
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A total of 132 strains of V. parahaemolyticus isolated from patients and from the suspected causal food items of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were examined for the ability to hydrolyze urea, with specific reference to the presence of the thermostable direct hemolysin gene (tdh) and the gene for thermostable direct hemolysin-related hemolysin (trh). Ten strains belonging to five different O-antigen serotypes were positive for urea hydrolysis (UH+), and four of these strains did not carry tdh. A total of 106 strains carried tdh, but less than 6% of them were UH+, whereas all trh-carrying strains were UH+. The evidence suggests that urea hydrolysis is not a reliable marker for identifying tdh-carrying V. parahaemolyticus strains in Japan (the Pacific Northeast) but may be a marker for trh-carrying strains.
Hemolysin
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Hemolysin
Vibrio Infections
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Objective The study was conducted to identify the etiological agent that induced food poisoning by analyzing the victims' stool samples and/or anal swabs,food remains,condiments,and additives and spreads used during food processing to determine its biological characteristics.Methods The isolated strains of Vibrio parahaemolyticus were subject to serotyping,drug resistance test,species-specific gene tdh(Thermostable Direct Hemolysin) and virulence gene trh(Thermostable Direct Hemolysin-Related Hemolysin) detection per GB/T4789-2006.Results A total of 74 strains of VP were isolated from 153 stool samples and/or anal swabs,and other three were found one each from condiments,food-processing spreads and food remains in trash bags.The serotyping suggested that all strains of VP were serovar O3∶K6,Kanagawa positive.Amplification of the species-specific gene from the 77 strains resulted in an identical 320 bp band.The virulence gene multi-PCR test of them suggested tdh(434 bp) positive and trh(250 bp) negative.Conclusion Vibrio parahaemolyticus Serovar O3∶K6 was the cause of food poisoning;its strong virulence was confirmed.
Hemolysin
Food poisoning
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