Genome-wide expanding of genetic evolution and potential pathogenicity in Vibrio alginolyticus.

Vibrio alginolyticus, an emergent species of Vibrio genus, exists in aquatic and marine environments. It has undergone genetic diversification, but its detailed genomic diversity is still unclear. Here, we performed a multi-dimensional comparative genomic analysis to explore the population phylogeny, virulence-related genes and potential drug resistance genes of 184 V. alginolyticus isolates. Although genetic diversity is complex, we analysed the population structure using three sub-datasets, including the subdivision for three lineages into sublineages and the distribution of strains in the marine ecological niche. Accessory genes, most of which reclassified V. alginolyticus genomes as different but with relatively close affinities, were nonuniformly distributed among these isolates. We demonstrated that the spread of some post-evolutionary isolates (mainly L3 strains isolated from Chinese territorial seas) was likely to be closely related to human activities, whereas other more ancestral strains (strains in the L1 and L2) tended to be locally endemic and formed clonal complex groups. In terms of pathogenicity, the potential virulence factors were mainly associated with toxin, adherence, motility, chemotaxis, and the type III secretion system (T3SS). We also found five types of antibacterial drug resistance genes. The prevalence of ß-lactam resistance genes was 100%, which indicated that there may be a potential risk of natural resistance to ß-lactam drugs. Our study reveals insights into genomic characteristics, evolution and potential virulence-associated gene profiles of V. alginolyticus.

Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages.

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence.

Comprehensive depiction of novel heavy metal tolerant and EPS producing bioluminescent Vibrio alginolyticus PBR1 and V. rotiferianus PBL1 confined from marine organisms.

The current study depicts the isolation of luminescent bacteria from fish and squid samples that were collected from Veraval fish harbour. From Indian mackerel, total 14 and from squid, total 23 bioluminescent bacteria were isolated using luminescence agar medium. Two bioluminescent bacteria with highest relative luminescence intensity PBR1 and PBL1 were selected. These two isolates were subjected to detailed biochemical characterization and were tested positive for 5 out of 13 biochemical tests. Furthermore, both PBR1 and PBL1 were able to ferment cellobiose, dextrose, fructose, galactose, maltose, mannose, sucrose and trehalose with acid production. Based on 16S rRNA partial gene sequence analysis, PBR1 was identified as Vibrio alginolyticus and PBL1 as V. rotiferianus. Antibiotic susceptibility test using paper-disc method showed that PBR1 and PBL1 were sensitive to chloramphenicol, ciprofloxacin, co-trimoxazole, gatifloxacin, levofloxacin, linezolid ad roxithromycin out of 18 antibiotics tested. Moreover, both strains were evaluated for their exopolysachharide (EPS) producing ability where PBR1 and PBL1 were able to yield 1.34 g% (w/v) and 2.45 g% (w/v) EPS respectively from 5 g% (v/v) sucrose concentration. Heavy metal toxicity assessment was carried out using agar well diffusion method with eight heavy metals and both the strains were sensitive to As(III), Cd(II), Ce(II), Cr(III), Cu(II), Hg(II) and while they showed resistance to Pb(II) and Sr(II). Based on these results, a study was conducted to demonstrate bio-removal of Pb and Sr by EPS of PBR1 and PBL1. Fourier transform infrared (FTIR) spectra revealed the functional groups of EPS involved in interaction with the heavy metals. Owing to the sensitivity for the remaining heavy metals, these bioluminescent bacteria can be used further for the development of luminescence-based biosensor.

Genomic variation among closely related Vibrio alginolyticus strains is located on mobile genetic elements.

BACKGROUND: Species of the genus Vibrio, one of the most diverse bacteria genera, have undergone niche adaptation followed by clonal expansion. Niche adaptation and ultimately the formation of ecotypes and speciation in this genus has been suggested to be mainly driven by horizontal gene transfer (HGT) through mobile genetic elements (MGEs). Our knowledge about the diversity and distribution of Vibrio MGEs is heavily biased towards human pathogens and our understanding of the distribution of core genomic signatures and accessory genes encoded on MGEs within specific Vibrio clades is still incomplete. We used nine different strains of the marine bacterium Vibrio alginolyticus isolated from pipefish in the Kiel-Fjord to perform a multiscale-comparative genomic approach that allowed us to investigate [1] those genomic signatures that characterize a habitat-specific ecotype and [2] the source of genomic variation within this ecotype. RESULTS: We found that the nine isolates from the Kiel-Fjord have a closed-pangenome and did not differ based on core-genomic signatures. Unique genomic regions and a unique repertoire of MGEs within the Kiel-Fjord isolates suggest that the acquisition of gene-blocks by HGT played an important role in the evolution of this ecotype. Additionally, we found that ~ 90% of the genomic variation among the nine isolates is encoded on MGEs, which supports ongoing theory that accessory genes are predominately located on MGEs and shared by HGT. Lastly, we could show that these nine isolates share a unique virulence and resistance profile which clearly separates them from all other investigated V. alginolyticus strains and suggests that these are habitat-specific genes, required for a successful colonization of the pipefish, the niche of this ecotype. CONCLUSION: We conclude that all nine V. alginolyticus strains from the Kiel-Fjord belong to a unique ecotype, which we named the Kiel-alginolyticus ecotype. The low sequence variation of the core-genome in combination with the presence of MGE encoded relevant traits, as well as the presence of a suitable niche (here the pipefish), suggest, that this ecotype might have evolved from a clonal expansion following HGT driven niche-adaptation.

A novel sRNA srvg17985 identified in Vibrio alginolyticus involving into metabolism and stress response.

Vibrio alginolyticus is an opportunistic pathogen that is a threat to the aquaculture industry. Evidence has revealed critical roles for small RNAs (sRNAs) in bacterial physiology and pathology by modulating gene expression post transcription. However, little information about sRNA-mediated regulation in V. alginolyticus is available. We experimentally verified the existence and characterized the function of sRNA srvg17985 in V. alginolyticus ZJ-T. We identified a 179 nt and growth-phase-dependent transcript with a σ70 promoter and a ρ-independent terminator. The transcript consisted of five stem-loops and was conserved in Vibrio spp. Phenotype microarray assays showed that deletion of srvg17985 led to less use of Gly-Glu as a carbon source but a gain in ability to use l-phenylalanine as a nitrogen source. Srvg17985 regulated the osmotic stress response with stronger tolerance to NaCl but weaker tolerance to urea. In addition, srvg17985 inhibited the deamination of l-serine at pH 9.5 and promoted the hydrolysis of X-beta-d-glucuronide, thus affecting the pH stress response. Bioinformatics by IntaRNA and TargetRNA2 identified 45 common target mRNAs, some of which probably contributed to the observed phenotypes. These results indicated that srvg17985 regulated environmental adaptation. The results provide valuable information for in-depth studies of sRNA-mediated regulation mechanisms of the complex physiological processes of V alginolyticus and provide new targets for antibacterial therapeutics or attenuated vaccines for Vibrio spp.

Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes.

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65 °C for 30 min, 63 °C for 30 min, and 63 °C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics.

MALDI-TOF MS as a tool for the identification of Vibrio alginolyticus from Perna perna mussels (Linnaeus, 1758) / MALDI-TOF MS como ferramenta na identificação de Vibrio alginolyticus isolados de mexilhões Perna perna (Linnaeus, 1758)

Vibrio species are ubiquitous in aquatic environments, including coastal and marine habitats. Vibrio alginolyticus is an opportunistic pathogen for fish, crustaceans and mussels and their identification by biochemical tests may be impaired due their nutritional requirements. The study used Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to identify 49 Vibrio spp. isolates associated with mussels (Perna perna) from different locations along the Rio de Janeiro coast. The rpoA gene was used as a genus-specific marker of Vibrio spp. and was positive in all 209 isolates. MALDI-TOF MS confirmed 87.8% of V. alginolyticus when compared to the results of the biochemical tests. Four isolates were identified as Shewanella putrefaciens (8.16%) and one was identified as V. parahaemolyticus (2.0%). Just one isolate was not identified by this technique (2.0%). The pyrH sequencing confirmed 75% of the proteomic technique results. MALDI-TOF MS is an excellent option for characterization of bacterial species, as it is efficient, fast and easy to apply. In addition, our study confirms its high specificity and sensitivity in these marine bacteria identification.(AU)

Espécies de Vibrio são ubiquitárias em ambientes aquáticos, incluindo habitats costeiros e marinhos. A espécie Vibrio alginolyticus é oportunista para peixes, crustáceos e moluscos e a sua identificação através de testes bioquímicos pode ter a qualidade prejudicada devido às suas exigências nutricionais. O presente estudo utilizou Espectrometria de Massa por Tempo de Vôo de Ionização/Desorção por Laser Assistida por Matriz (MALDI-TOF MS) para identificar diferentes espécies de Vibrio provenientes de mexilhões (Perna perna) coletados em diferentes locais ao longo da costa do Rio de Janeiro. O gene rpoA foi utilizado como um marcador gênero-específico de Vibrio spp. sendo positivo em todos os 209 isolados. MALDI-TOF MS confirmou 87,75% de V. alginolyticus quando comparados com os resultados dos testes bioquímicos. Quatro isolados foram identificados como Shewanella putrefaciens (8,16%), um como V. parahaemolyticus (2,0%) e apenas um (2,0%) não foi identificado pela técnica proteômica. E o sequenciamento do pyrH confirmou 75% dos resultados da técnica proteomica. MALDI-TOF MS tem sido considerada uma excelente opção para a caracterização bacteriana, por ser eficiente, rápida, de fácil aplicação e este estudo confirmou a sua elevada especificidade e sensibilidade na identificação de bactérias marinhas.(AU)

Efficacy of cell free supernatant from Bacillus licheniformis in protecting Artemia salina against Vibrio alginolyticus and Pseudomonas gessardii.

Bacterial diseases are widespread in aquaculture farms and causative agents often adapt to biofilm mode of growth. These biofilms are detrimental to aquaculture species as they resist antibiotics and other agents that are used to control them. Two bacterial pathogens isolated from infected prawn samples were identified as Vibrio alginolyticus and Pseudomonas gessardii on the basis of morphological features, biochemical characteristics, 16S r RNA gene sequencing and phylogenetic analysis. Their pathogenic nature was confirmed by performing in vivo challenge experiments using Artemia salina as a model system. Seven days post infection, the mortality observed with V. alginolyticus and P. gessardii was 97 ±â€¯4.08% and 77.5 ±â€¯5.24%, respectively. The isolates formed extensive biofilms on polystyrene and glass surfaces. These infections could be controlled in an effective manner by using the cell free supernatant (CFS) of a tropical marine epizoic strain of Bacillus licheniformis D1 that is earlier reported to contain an antimicrobial protein (BLDZ1). The CFS inhibited biofilms in an efficient manner (82.35 ±â€¯1.69 and 82.52 ±â€¯1.11% for V. alginolyticus and P. gessardii, respectively) on co-incubation. In addition, pre-formed biofilms of V. alginolyticus and P. gessardii were also removed (84.53 ±â€¯1.26 and 67.08 ±â€¯1.43%, respectively). Fluorescence and scanning electron microscopic studies confirmed the antibiofilm potential of this protein on glass surfaces. The antibiofilm nature was due to the anti-adhesion and antimicrobial properties exhibited by the CFS. Treatment of A. salina with CFS (6 h prior to infections) was effective in protecting larvae against infections by field isolates. This study highlights the significance of marine natural products in providing alternative biofilm controlling agents to tackle infections and decreasing the usage of antibiotics in aquaculture settings.

Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

Vibrio alginolyticus (V. alginolyticus) is a common pathogen for humans and marine aquatic animals. Vibriosis of marine aquatic animals, caused by V. alginolyticus, has become more prevalent globally in recent years. Hence, a safe and effective vaccine is urgently needed for the control of this disease. Here, the strain 16-3 isolated from the large yellow croaker (Larimichthys crocea) suffered from canker was identified as V. alginolyticus based on morphological, biochemical, and 16S rDNA sequencing analysis. Then, recombinant temperature-controlled lysis plasmid pBV220-lysisE was electroporated into the strain 16-3 to generate V. alginolyticus bacterial ghosts (VaBGs) by inducing lysis gene E expression, and the safety and immune effects of VaBGs were further investigated in mice and large yellow croaker. The results showed that VaBGs were as safe as formalin-killed V. alginolyticus cells (FKC) to mice and fish. Compared with FKC and PBS groups, significant elevations of the serum agglutinating antibody titer, serum bactericidal activity, lymphocyte proliferative responses, and levels of four different cytokines (Th1 type: IL-2, TNF-α; Th2 type: IL-4 and IL-6) in serum were detected in the VaBGs group, indicating that a Th1/Th2-mediated mixed immune response was elicited by the VaBGs. More importantly, after challenged with the parent strain 16-3, VaBGs-vaccinated mice and fish showed higher protection than FKC-vaccinated mice, the relative percent of survival (RPS) being 60%, 66.7% and 40%, respectively. Taken together, this is the first demonstration that the newly constructed V. alginolyticus ghosts may be developed as a safe and effective vaccine against V. alginolyticus infection in aquaculture.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 102 to 105CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 102CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively.

Genetic heterogeneity among Vibrio alginolyticus strains, and design of a PCR-based identification method using gyrB gene sequence.

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

Molecular characterization and phylogenetic analysis of highly pathogenic Vibrio alginolyticus strains isolated during mortality outbreaks in cultured Ruditapes decussatus juvenile.

In the summer of 2008 and 2009, a series of mortalities in growing out seeds of R. decussatus juveniles were occurred in the eastern Tunisian littoral. Nine predominant bacterial strains were isolated from dead and moribund juveniles and characterized as Vibrio alginolyticus. These isolates were subjected to biochemical and molecular characterization. All the Vibrio strains were tested for their susceptibility against the most widely used antibiotic in aquaculture as well as, the assessment of the presence of erythromycin (emrB) and tetracycline (tetS) resistance genes among the tested bacteria. The degree of genetic relatedness between V. alginolyticus strains was evaluated on the basis of the Entero-Bacterial Repetitive Intergenic Consensus (ERIC) and the Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) approaches. We also looked for siderophore activity and the ability to grow under iron limitation. Furthermore, the pathogenic potential of the tested isolates was evaluated using R. decussatus larva and juveniles as infection models. On antimicrobial susceptibility test, Vibrio strains exhibited total resistance to at least four antibiotics. The MICs data revealed that flumequine and oxolinic acid were the most effective antibiotics to control the studied bacteria. Results also showed that studied antibiotics resistance genes were widely disseminated in the genome of V. alginolyticus strains. Both ERIC and RAPD-PCR fingerprinting showed the presence of genetic variation among Vibrio isolates. However, RAPD typing exhibited a higher discriminative potential than ERIC-PCR. Besides, we reported here for the first time the co-production of catechol and hydroxamte by V. alginolyticus species. The challenge experiment showed that most of Vibrio isolates caused high mortality rates for both larva and juveniles at 48-h post-exposure to a bacterial concentration of 106 CFU/ml.

Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus.

Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance.IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.

Antifouling Activity towards Mussel by Small-Molecule Compounds from a Strain of Vibrio alginolyticus Bacterium Associated with Sea Anemone Haliplanella sp.

Mussels are major fouling organisms causing serious technical and economic problems. In this study, antifouling activity towards mussel was found in three compounds isolated from a marine bacterium associated with the sea anemone Haliplanella sp. This bacterial strain, called PE2, was identified as Vibrio alginolyticus using morphology, biochemical tests, and phylogenetic analysis based on sequences of 16S rRNA and four housekeeping genes (rpoD, gyrB, rctB, and toxR). Three small-molecule compounds (indole, 3-formylindole, and cyclo (Pro-Leu)) were purified from the ethyl acetate extract of V. alginolyticus PE2 using column chromatography techniques. They all significantly inhibited byssal thread production of the green mussel Perna viridis, with EC50 values of 24.45 µg/ml for indole, 50.07 µg/ml for 3-formylindole, and 49.24 µg/ml for cyclo (Pro-Leu). Previous research on the antifouling activity of metabolites from marine bacteria towards mussels is scarce. Indole, 3-formylindole and cyclo (Pro-Leu) also exhibited antifouling activity against settlement of the barnacle Balanus albicostatus (EC50 values of 8.84, 0.43, and 11.35 µg/ml, respectively) and the marine bacterium Pseudomonas sp. (EC50 values of 42.68, 69.68, and 39.05 µg/ml, respectively). These results suggested that the three compounds are potentially useful for environmentally friendly mussel control and/or the development of new antifouling additives that are effective against several biofoulers.

Comparative genomic analysis of six new-found integrative conjugative elements (ICEs) in Vibrio alginolyticus.

BACKGROUND: Vibrio alginolyticus is ubiquitous in marine and estuarine environments. In 2012-2013, SXT/R391-like integrative conjugative elements (ICEs) in environmental V. alginolyticus strains were discovered and found to occur in 8.9 % of 192 V. alginolyticus strains, which suggests that V. alginolyticus may be a natural pool possessing resourceful ICEs. However, complete ICE sequences originating from this bacterium have not been reported, which represents a significant barrier to characterizing the ICEs of this bacterium and exploring their relationships with other ICEs. In the present study, we acquired six ICE sequences from five V. alginolyticus strains and performed a comparative analysis of these ICE genomes. RESULTS: A sequence analysis showed that there were only 14 variable bases dispersed between ICEValE0601 and ICEValHN492. ICEValE0601 and ICEValHN492 were treated as the same ICE. ICEValA056-1, ICEValE0601 and ICEValHN492 integrate into the 5' end of the host's prfC gene, and their Int and Xis share at least 97 % identity with their counterparts from SXT. ICEValE0601 or ICEValHN492 contain 50 of 52 conserved core genes in the SXT/R391 ICEs (not s025 or s026). ICEValA056-2, ICEValHN396 and ICEValHN437 have a different tRNA-ser integration site and a distinct int/xis module; however, the remaining backbone genes are highly similar to their counterparts in SXT/R391 ICEs. DNA sequences inserted into hotspot and variable regions of the ICEs are of various sizes. The variable genes of six ICEs encode a large array of functions to bestow various adaptive abilities upon their hosts, and only ICEValA056-1 contains drug-resistant genes. Many variable genes have orthologous and functionally related genes to those found in SXT/R391 ICEs, such as genes coding for a toxin-antitoxin system, a restriction-modification system, helicases and endonucleases. Six ICEs also contain a large number of unique genes or gene clusters that were not found in other ICEs. Six ICEs harbor more abundant transposase genes compared with other parts of their host genomes. A phylogenetic analysis indicated that transposase genes in these ICEs are highly diverse. CONCLUSIONS: ICEValA056-1, ICEValE0601 and ICEValHN492 are typical members of the SXT/R391 family. ICEValA056-2, ICEValHN396 and ICEValHN437 form a new atypical group belonging to the SXT/R391 family. In addition to the many genes found to be present in other ICEs, six ICEs contain a large number of unique genes or gene clusters that were not found in other ICEs. ICEs may serve as a carrier for transposable genetic elements (TEs) and largely facilitate the dissemination of TEs.

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control.

A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.

First characterization of bacterial pathogen, Vibrio alginolyticus, for Porites andrewsi White syndrome in the South China Sea.

BACKGROUND: White syndrome, a term for scleractinian coral disease with progressive tissue loss, is known to cause depressed growth and increased morality of coral reefs in the major oceans around the world, and the occurrence of this disease has been frequently reported in the past few decades. Investigations during April to September in both 2010 and 2011 identified widespread Porites andrewsi White syndrome (PAWS) in Xisha Archipelago, South China Sea. However, the causes and etiology of PAWS have been unknown. METHODOLOGY/PRINCIPAL FINDINGS: A transmission experiment was performed on P. andrewsi in the Qilianyu Subgroup (QLY). The results showed that there was a significant (P ≤ 0.05) difference between test and control groups after 28 days if the invalid replicates were excluded. Rates of tissue loss ranged from 0.90-10.76 cm(2) d(-1) with a mean of 5.40 ± 3.34 cm(2) d(-1) (mean ± SD). Bacterial strains were isolated from the PAWS corals at the disease outbreak sites in QLY of the Xisha Archipelago, South China Sea, and included in laboratory-based infection trials to satisfy Koch's postulates for establishing causality. Following exposure to bacterial concentrations of 10(5) cells mL(-1), the infected colonies exhibited similar signs to those observed in the field. Using phylogenetic 16S rRNA gene analysis, classical phenotypic trait comparison, Biolog automatic identification system, MALDI-TOF mass spectrometry and MALDI Biotyper method, two pathogenic strains were identified as Vibrio alginolyticus . CONCLUSION/SIGNIFICANCE: This is the first report of V. alginolyticus as a pathogenic agent of PAWS in the South China Sea. Our results point out an urgent need to develop sensitive detection methods for V. alginolyticus virulence strains and robust diagnostics for coral disease caused by this and Vibrio pathogenic bacterium in the South China Sea.

Genotypic diversity, antimicrobial resistance and screening of Vibrio cholerae molecular virulence markers in Vibrio alginolyticus strains recovered from a Tunisian Ruditapes decussatus hatchery.

In this study, a total of 54 Vibrio alginolyticus strains were analyzed. The isolates were recovered from different compartments of the Ruditapes decussatus hatchery in the National Institute of Marine Sciences and Technologies, Monastir, Tunisia. All isolates were biochemically identified (API 20E and API ZYM strips), characterized by amplification of the Hsp-40 gene polymerase chain reaction (PCR) and analyzed by enterobacterial repetitive intergenic consensus (ERIC)-based genotyping to evaluate genetic relationship between the isolated strains. We also looked for the presence of ten V cholera virulence genes (toxRS, toxR, toxT toxS, tcpP, tcpA, ace, vpi, zot and ctxA) in the genomes of Vibrio isolates. The antibiotics susceptibility, exoenzymes production and in vitro cytotoxic activitiy against HeLa cell line were also carried out for all tested bacteria. Most of V alginolyticus isolates showed significant antimicrobial resistance rates to at least ten antibacterial agents. For most isolates, the minimum inhibitory concentration (MIC) data showed that tetracydclin and streptomycin were the most effective antibiotics. Construction of the phylogenetic dendogram showed that studied isolates were in general genetically heterogeneous; however some Vibrio strains were present in different structures of the R. decussatus hatchery. The V cholerae virulence genes investigation showed a wild distribution of toxS (49/54), toxaR (45/54) and toxT (22/54) genes among V alginolyticus strains isolated from the R. decussatus rearing system. Cytotoxic effects of several Vibrio extracellular products (28154) were also observed on HeLa cells.

Occurrence of Vibrio parahaemolyticus and Vibrio alginolyticus in the German Bight over a seasonal cycle.

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10(3) N l(-1) and MPNs up to 240 N g(-1) were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.

Genotype analysis of collagenase gene by PCR-SSCP in Vibrio alginolyticus and its association with virulence to marine fish.

In this article, the genotypes for Vibrio alginolyticus were analyzed based on collagenase gene by PCR-single strand conformation polymorphism (SSCP) method and their relationship to the virulence for marine fish were studied by a bacteria challenging experiment. Results showed total 13 genotypes designated as A1, A2, A3, A4, B, C, D, E, F, G1, G2, H, and I were identified. Among them, isolates from type A1 were dominant in abundance (48.1%) and the isolates from Nanyou had the highest polymorphisms, in which 11 genotypes were identified from 25 isolates. Moreover, the bacteria challenging experiment showed the virulence of V. alginolyticus was correlated to that of the collagenase-SSCP type (r = 0.438, P < 0.01), which demonstrated the strains from type A1, A2, A3, B, C, F, G1, and I were low-virulent isolates, while those from type A4, D, E, G2, and H were high virulence. These data will provide a new understanding of the relationship between virulence of V. alginolyticus and its molecular marker.