Aims:  The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results:  The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions:  The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study:  In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.

Aims:  The aim of this study was to compare different primers for rapid and effective detection of Vibrio parahaemolyticus by polymerase chain reaction (PCR). Methods and Results:  A total of four pairs of primers, three previously published and one based on a newly developed V. parahaemolyticus metalloprotease (vpm) gene, have been assayed for PCR detection of V. parahaemolyticus. They have been tested for specificity and sensitivity on a total of 101 strains including reference and environment isolates belonging to V. parahaemolyticus and other species in Vibrio. Of the four sets of primers tested, the one designed on the basis of the metalloprotease gene (675 bp) gave optimal results with bacterial strains examined as they only amplified the specific fragment in strains that had been genetically and biochemically assessed as V. parahaemolyticus and the limit of detection was 4 pg of purified target DNA. Conclusions:  The primers designed on the metalloprotease gene gave optimal results for specific, sensitive and rapid detection of V. parahaemolyticus by PCR. Significance and Impact of the Study:  PCR amplification with the optimal primer set VPM1/VPM2 could facilitate the rapid diagnosis and surveillance of potentially pathogenic strains of V. parahaemolyticus and reduce food‐borne illness.

Aims: The aim of this study was to investigate functions of flagellar genes fliC2, fliC12, fliA and flhDC in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: In this study, functions of flagellar genes, fliC2, fliC12 (fliC1 + fliC2), fliA and flhDC (flhD + flhC) of Edw. tarda H1 were analysed by constructing in-frame deletion mutants respectively and complementary strains fliC2 + and fliA + . Electron microscopy revealed that in-frame deletion of fliC12, fliA and flhDC significantly impaired the number and length of flagellar filaments, resulting in loss of both swimming and swarming motilities of the bacteria. In addition, compared to the wild-type strain and complementary strains, the flagellum-impaired mutants exhibited reduced biofilm formation ability, showed decreased ability in adherence and internalization to Epithelioma papulosum cyprini (EPC) cells and reduced pathogenicity to zebrafish. Conclusions: These results indicated that fliC12, fliA and flhDC of Edw. tarda played essential roles in flagellar filaments structure, bacteria motility, biofilm formation, adherence, internalization and pathogenicity of this bacterium. Significance and Impact of the Study: This study revealed that flagella function in facilitating virulence and it may provide a new target for vaccines against Edw. tarda infection.

Aims: The aim of this study was to investigate the role of invasin in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: In this study, an in-frame deletion mutant of invasin (Dinv) in Edw. tarda H1 was constructed through double crossover allelic exchange to explore the function of invasin in virulence to fish. Meanwhile, an invasin overexpression strain (inv + ) was obtained by electrotransformation of a low-copy plasmid pACYC184 carrying the intact invasin into the Dinv mutant. Several virulence-associated characters of the mutants and wild-type strain were tested. Compared with the wild-type H1, haemolytic activity and biofilm formation were decreased in Dinv, while increased significantly in inv + . In addition, the invasin overexpressing strain inv + exhibited increased internalization into Epithelioma Papulosum Cyprini (EPC) cells. Moreover, in zebrafish model, Dinv showed decreased virulence compared with H1, while inv + restored the virulence of wild type completely. Conclusions:The results demonstrated that invasin of Edw. tarda plays essential roles in haemolytic activity, biofilm formation, adherence, internalization and pathogenicity of this bacterium. Significance and Impact of the Study: This study revealed the role of invasin in Edw. tarda infection and provided useful information for further unveiling the pathogenesis of Edw. tarda.

Aims:  The aim of this study was to investigate the role of membrane‐bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. Methods and Results:  An mltA in‐frame deletion mutant (ΔmltA) and an mltA overexpression strain (mltA+) of Edw. tarda were constructed through double‐crossover allelic exchange and by transformation of a low‐copy plasmid carrying the intact mltA into the ΔmltA mutant, respectively. Either inactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivity to β‐lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild‐type strain. Autolysis induced by EDTA was reduced in ΔmltA strain, while mltA+ strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA+ strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild‐type strain. Conclusions:  The results indicated that MltA plays essential roles in β‐lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw. tarda. This is the first report that MltA has a virulence‐related function in Edw. tarda. Significance and Impact of the Study:  This study provided useful information for further studies on pathogenesis of Edw. tarda.

the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

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