Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization

ABSTRACT A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.

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Application of the VPp1 bacteriophage combined with a coupled enzyme system in the rapid detection of Vibrio parahaemolyticus

Journal of Microbiological Methods ◽

10.1016/j.mimet.2014.01.005 ◽

2014 ◽

Vol 98 ◽

pp. 99-104 ◽

Cited By ~ 10

Author(s):

Yong Peng ◽

Yanqiu Jin ◽

Hong Lin ◽

Jingxue Wang ◽

Muhammad Naseem Khan

Keyword(s):

Vibrio Parahaemolyticus ◽

Rapid Detection ◽

Enzyme System

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Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

Applied and Environmental Microbiology ◽

10.1128/aem.02284-09 ◽

2009 ◽

Vol 76 (3) ◽

pp. 820-828 ◽

Cited By ~ 35

Author(s):

Wataru Yamazaki ◽

Yuko Kumeda ◽

Naoaki Misawa ◽

Yoshitsugu Nakaguchi ◽

Mitsuaki Nishibuchi

Keyword(s):

Vibrio Parahaemolyticus ◽

Rapid Detection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Virulence Determinants ◽

Bacterial Strains ◽

Phenotypic Differences ◽

Loop Mediated Isothermal Amplification ◽

Vibrio Mimicus ◽

Thermostable Direct Hemolysin

ABSTRACT Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

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Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b14-00582 ◽

2015 ◽

Vol 38 (1) ◽

pp. 82-87 ◽

Cited By ~ 12

Author(s):

Huiling Di ◽

Lei Ye ◽

Sucharit Basu Neogi ◽

Hecheng Meng ◽

He Yan ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Rapid Detection ◽

Enrichment Culture ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Amplification Assay

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Selection of specific cell wall antigen for rapid detection of fish pathogenic Vibrio parahaemolyticus by enzyme immunoassay

Indian Journal of Fisheries ◽

10.21077/ijf.2016.63.2.44334-10 ◽

2016 ◽

Vol 63 (2) ◽

Author(s):

P. S. Shyne Anand ◽

K. S. Sobhana ◽

K. C. George ◽

R. Paul Raj

Keyword(s):

Cell Wall ◽

Enzyme Immunoassay ◽

Vibrio Parahaemolyticus ◽

Rapid Detection ◽

Specific Cell ◽

Pathogenic Vibrio ◽

Selection Of

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Characterization of Vibrio parahaemolyticus strains isolated from clinically asymptomatic seafood workers

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa209 ◽

2020 ◽

Author(s):

Kaknokrat Chonsin ◽

Neunghatai Supha ◽

Chie Nakajima ◽

Yasuhiko Suzuki ◽

Orasa Suthienkul

Keyword(s):

Phylogenetic Analysis ◽

Food Safety ◽

Vibrio Parahaemolyticus ◽

Environmental Samples ◽

Genetic Characterization ◽

Processing Plants ◽

Pandemic Strains ◽

Phenotypic And Genetic Characterization ◽

Seafood Processing

Abstract Vibrio parahaemolyticus (VP) is a major cause of gastroenteritis outbreaks in Thailand and other countries due to the consumption of contaminated and undercooked seafood. However, there have been few reports of the molecular epidemiology of VP isolates from asymptomatic seafood handlers. Here, we report the phenotypic and genetic characterization of 61 VP isolates obtained from asymptomatic workers in two seafood processing plants. We found 24 O:K serotypes of which O11:KUT, O1:KUT and O3:KUT were the dominant serotypes. Analysis by PCR showed 12 isolates harbored either tdh or trh genes with the potential to be pathogenic VP strains. The presence of T3SS2α and T3SS2β genes was correlated with the presence of tdh and trh, respectively. Four tdh+ isolates were positive for pandemic marker. In this study, VP isolates were commonly resistant to ampicillin, cephazolin, fosfomycin and novobiocin. Phylogenetic analysis of VP1680 loci in 35 isolates from 17 asymptomatic workers, six gastroenteritis patients, seven environmental samples and five genomes from a database showed 22 different alleles. Gene VP1680 was conserved in tdh+ isolates and pandemic strains, that of trh + isolates was diverse. Asymptomatic workers carrying VP were the most likely source of contamination, which raises concerns over food safety in seafood processing plants.

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