The virulence factors of Vibrio harveyi, the causative agent of luminous vibriosis, are not completely understood. We investigated the correlations between shrimp mortality, hemolysis, the presence of a hemolysin gene (vhh), and a gene involved in the type III secretion system (the Vibrio calcium response gene vcrD). V. harveyi HY01 was isolated from a shrimp that died from vibriosis, and 36 other V. harveyi isolates were obtained from fish and shellfish in Hat Yai city, Thailand. An ocean isolate of V. harveyi BAA-1116 was also included. Thirteen isolates including V. harveyi HY01 caused shrimp death 12 h after injection. Most V. harveyi isolates in this group (designated as Group A) caused hemolysis on prawn blood agar. None of the shrimp died after injection with V. harveyi BAA-1116. Molecular analysis of all V. harveyi isolates revealed the presence of vcrD in both pathogenic and non-pathogenic strains. Although vhh was detected in all V. harveyi isolates, some isolates did not cause hemolysis, indicating that vhh gene expression might be regulated. Analysis of the V. harveyi HY01 genome revealed a V. cholerae like-hemolysin gene, hlyA (designated as hhl). Specific primers designed for hhl detected this gene in 3 additional V. harveyi isolates but the presence of this gene was not correlated with pathogenicity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity in all V. harveyi isolates, and there were no correlations among the hhl-positive isolates or the pathogenic strains.KEY WORDS: Vibrio harveyi · Type III secretion system · TTSS · vcrD · vhh · hhl · Random amplified polymorphic DNA analysis · RAPD Resale or republication not permitted without written consent of the publisherDis Aquat Org 86: [113][114][115][116][117][118][119][120][121][122] 2009 against sheep and fish erythrocytes compared with non-virulent isolates from sea water or diseased Talorchestia sp. (Liu et al. 1996). Investigations of the pathogenicity of V. harveyi isolates in fish (Atlantic salmon and rainbow trout) have demonstrated that both pathogenic and non-pathogenic V. harveyi isolates induced hemolysis against erythrocytes from sheep, rabbit, donkey, and horse, and the presence of the hemolysin gene vhh has been demonstrated in V. harveyi (Zhang & Austin 2000, Zhang et al. 2001). An investigation of the mortality of Artemia franciscana nauplii after inoculation with V. harveyi isolates from healthy and diseased penaeid shrimp from Asia and the Americas indicated that particular exoenzymes were associated with virulent strains (Soto-Rodriguez et al. 2003). No correlation between the hemolytic activity against sheep erythrocytes and the death of infected shrimps was detected (Soto-Rodriguez et al. 2003). Further research is needed to resolve these controversies between the pathogenicity of V. harveyi and its ability to cause hemolysis.Recent studies have shown that many bacteria use a cell-cell communication process known as quorum sensing to control cell population density and...

Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae , V. parahaemolyticus , V. alginolyticus , V. harveyi and V. vulnificus . We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp ( Litopenaeus vannamei ). Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (σ E ), was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030) to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN) and an upregulated protease (DegQ) which have been associated with σ E in other organisms. Our study is the first report linking hemolytic activity to the σ E regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi .

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BackgroundVibrio parahaemolyticus is a causative agent of gastroenteritis. Most of the clinical isolates carry either tdh and/or trh genes which are considered as the major virulence genes of this pathogen. In this study, the clinical isolates of V. parahaemolyticus carrying trh gene (n = 73) obtained from 1886 to 2012 from various countries were investigated for the urease production, haemolytic activity, and biofilm formation. In addition, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping among these isolates was investigated.ResultsIn this study, no significant differences were observed in the urease production between tdh+ trh1+ and tdh+ trh2+ isolates (p = 0.063) and between the tdh− trh1+ and tdh− trh2+ isolates (p = 0.788). The isolates carrying only the trh gene showed variation in their haemolytic activity. The ratio of urease production and haemolytic activity between the trh1+ and trh2+ isolates and biofilm formation of trh+ V. parahaemolyticus isolates were not significantly different. Sixteen of thirty-four tested isolates (47.0%) of trh+ V. parahaemolyticus were positive for CRISPR detection. The discriminatory power index (DI) of CRISPR-virulence typing was higher than the DI obtained by CRISPR typing alone.ConclusionThe tdh and trh genes were not involved in urease production in the trh+ V. parahaemolyticus, and variation of haemolytic activity detected in V. parahaemolyticus carrying only the trh gene might be correlated to the sequence variation within trh1 and trh2 genes. Additionally, biofilm production of V. parahaemolyticus was not associated with harboring of virulence genes. For genotyping, CRISPR sequences combined with virulence genes can be used as genetic markers to differentiate trh+ V. parahaemolyticus strains.Electronic supplementary materialThe online version of this article (10.1186/s13099-018-0275-4) contains supplementary material, which is available to authorized users.

Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae, V. parahaemolyticus, V. alginolyticus, V. harveyi and V. vulnificus. We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp (Litopenaeus vannamei). Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (s E ), was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030) to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN) and an upregulated protease (DegQ) which have been associated with s E in other organisms. Our study is the first report linking hemolytic activity to the s E regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi.

More than half the world’s population is thought to be infected with Helicobacter pylori . Although the majority of infected people are asymptomatic, H. pylori infection may cause gastric ulcers and deadly gastric cancer. Owing to the difficulty and invasiveness of current routine culture and diagnostic methods, a highly sensitive and specific noninvasive assay for H. pylori is of interest. This study highlighted the design and performance of a colorimetric magneto loop-mediated isothermal amplification (CM-LAMP) assay to detect H. pylori in spiked saliva samples. LF primers were coated on magnetic nanoparticles by carbodiimide-induced immobilization and functionally used for solidphase amplification. During the LAMP reaction at 66°C, biotin-tagged FIPs were incorporated into LAMP amplicons. The colorimetric signal developed after the addition of NeutrAvidin horseradish peroxidase conjugate (NA-HRP) and ABTS. None of the tested microorganisms, including closely related bacteria, was shown positive by the CM-LAMP assay except H. pylori isolates. This novel platform was highly specific and 100-fold more sensitive (40 CFU/ml or 0.2 CFU per reaction) than the PCR and conventional LAMP assays for the detection of H. pylori in spiked saliva. Our results demonstrated the feasibility of using this noninvasive molecular diagnostic test to detect H. pylori in saliva samples.