PlzD modifies Vibrio vulnificus foraging behavior and virulence in response to elevated c-di-GMP.

IMPORTANCE: Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, Vibrio vulnificus. Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in V. vulnificus.

Quorum sensing and iron-dependent coordinated control of autoinducer-2 production via small RNA RyhB in Vibrio vulnificus.

Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5'-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.

Reduced virulence of the MARTX toxin increases the persistence of outbreak-associated Vibrio vulnificus in host reservoirs.

Opportunistic bacteria strategically dampen their virulence to allow them to survive and propagate in hosts. However, the molecular mechanisms underlying virulence control are not clearly understood. Here, we found that the opportunistic pathogen Vibrio vulnificus biotype 3, which caused an outbreak of severe wound and intestinal infections associated with farmed tilapia, secretes significantly less virulent multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which is the most critical virulence factor in other clinical Vibrio strains. The biotype 3 MARTX toxin contains a cysteine protease domain (CPD) evolutionarily retaining a unique autocleavage site and a distinct ß-flap region. CPD autoproteolytic activity is attenuated following its autocleavage because of the ß-flap region. This ß-flap blocks the active site, disabling further autoproteolytic processing and release of the modularly structured effector domains within the toxin. Expression of this altered CPD consequently results in attenuated release of effectors by the toxin and significantly reduces the virulence of V. vulnificus biotype 3 in cells and in mice. Bioinformatic analysis revealed that this virulence mechanism is shared in all biotype 3 strains. Thus, these data provide new insights into the mechanisms by which opportunistic bacteria persist in an environmental reservoir, prolonging the potential to cause outbreaks.

Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus.

Biofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components' expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins' production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

Sequence analysis of hepcidin in barbel steed (Hemibarbus labeo): QSHLS motif confers hepcidin iron-regulatory activity but limits its antibacterial activity.

Fish hepcidin genes are generally classified into two groups: hamp1-and hamp2-type isoforms. Hamp1-type hepcidin exhibits iron regulatory and antimicrobial activity, while hamp2-type shows a unique role in the immune response against various pathogens. An iron-regulatory motif exists at the N-terminus of hamp1-type hepcidin; however, the functional effect of this motif in fish is not well understood. Here, cDNA of the barbel steed (Hemibarbus labeo) hepcidin gene was cloned and sequenced. The predicted amino acid sequence comprised a signal peptide, a prodomain, and a mature peptide. Phylogenetic tree analysis revealed that barbel steed hepcidin belongs to the fish HAMP1 cluster and is closely related to Chinese rare minnow (Gobiocypris rarus) hepcidin. Barbel steed hepcidin is constitutively expressed in healthy fish tissues, predominantly in the liver. Following iron dextran treatment or Aeromonas hydrophila infection, expression of barbel steed hepcidin increased significantly in tested tissues. In vivo administration of intact hepcidin mature peptide (hep25) significantly and dose-dependently reduced ferroportin 1 expression, while truncated hepcidin mature peptide (hep20) lacking a QSHLS motif had no such effect. In vitro treatment of barbel steed monocytes/macrophages with hep25, but not hep20, increased the labile iron pool levels. Hep25 and hep20 conferred antibacterial activity only against A. hydrophila and Vibrio vulnificus, with greater activity of the latter at low concentrations. Neither hep25 nor hep20 impaired the cell membrane integrity of A. hydrophila, but could hydrolyze its genomic DNA; lack of a QSHLS motif enables hep20 to have a better hydrolytic effect. In summary, we identified an iron-regulatory motif in a fish species and demonstrated that this motif confers hamp1-type hepcidin iron-regulatory activity, but attenuates its antibacterial activity.

rpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus.

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the ß subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus, even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml-1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Novel short peptide tag from a bacterial toxin for versatile applications.

The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection in enzyme-linked immunosorbent, Western blot, flow cytometry, and immunofluorescence staining analyses in Escherichia coli, mammalian cell lines, yeast, and plant. In addition, our RA-tag/47RA mAb combination showed high sensitivity and reliable affinity (KD = 5.90 × 10-8 M) when compared with conventional tags. Overall, our results suggest that the RA-tag system could facilitate the development of a broadly applicable tag system for biological research.

Calcium-dependent site-switching regulates expression of the atypical iam pilus locus in Vibrio vulnificus.

The opportunistic human pathogen Vibrio vulnificus inhabits warm coastal waters and asymptomatically colonizes seafood, most commonly oysters. We previously characterized an isolate that exhibited greater biofilm formation, aggregation and oyster colonization than its parent. This was due, in part, to the production of a Type IV Tad pilus (Iam). However, the locus lacked key processing and regulatory genes required for pilus production. Here, we identify a pilin peptidase iamP, and LysR-type regulator (LRTR) iamR, that fulfil these roles and show that environmental calcium, which oysters enrich for shell repair and growth, regulates iam expression. The architecture of the iam locus differs from the classical LRTR paradigm and requires an additional promoter to be integrated into the regulatory network. IamR specifically recognized the iamR promoter (PiamR ) and the intergenic iamP-iamA region (PiamP-A ). PiamR exhibited classical negative auto-regulation but, strikingly, IamR inversely regulated the divergent iamP and iamA promoters in a calcium-dependent manner. Moreover, expression of the c-di-GMP and calcium-regulated, biofilm-promoting brp exopolysaccharide was IamA-dependent. These results support a scenario in which the calcium-enriched oyster environment triggers IamP-mediated processing of prepilin amassed in the periplasm for rapid pilin elaboration and subsequent BRP production to promote colonization.

The effects of temperature, pH, and iron on biofilm formation by clinical versus environmental strains of Vibrio vulnificus.

Due to the nature of Vibrio vulnificus infections (i.e., gastroenteritis and septicemia), only very few studies of a biofilm-associated form in this pathogen's life cycle have been conducted. We proposed that biofilm production by clinical strains of V. vulnificus would be higher than by environmental strains. Biofilm formation by clinical and environmental reference strains was tested under different temperatures (24, 30, and 37 °C), pH (5.5, 7.5, and 8.5) and iron concentrations (18, 30, 50, 100, and 200 µM). Biofilm production by clinical strains was consistently higher (p < 0.001) at 24 °C than by environmental strains. Higher biofilm production was observed at pH 5.5 by all strains. Growth rates were lowest at pH 5.5 for environmental strains but for clinical strains there were no differences at pH 5.5, 7.5, and 8.5, demonstrating a tolerance to acidic and alkaline conditions. There was a strong, direct correlation between iron concentration in the growth medium and biofilm production by all strains tested. The current study indicates that biofilm formation might be important for the survival of V. vulnificus in vivo as well as in the marine environment. With regard to temperature and pH, higher biofilm production appears to be a trait of clinical strains and could be considered a virulence determinant in V. vulnificus.

Multi-Factor Regulation of the Master Modulator LeuO for the Cyclic-(Phe-Pro) Signaling Pathway in Vibrio vulnificus.

LeuO plays the role of a master regulator in the cyclic-L-phenylalanine-L-proline (cFP)-dependent signaling pathway in Vibrio vulnificus. cFP, as shown through isothermal titration calorimetry analysis, binds specifically to the periplasmic domain of ToxR. Binding of cFP triggers a change in the cytoplasmic domain of ToxR, which then activates transcription of leuO encoding a LysR-type regulator. LeuO binds to the region upstream of its own coding sequence, inhibiting its own transcription and maintaining a controlled level of expression. A five-bp deletion in this region abolished expression of LeuO, but a ten-bp deletion did not, suggesting that a DNA bending mechanism is involved in the regulation. Furthermore, binding of RNA polymerase was significantly lower both in the deletion of the ToxR binding site and in the five-bp deletion, but not in the ten-bp deletion, as shown in pull-down assays using an antibody against RNA polymerase subunit α. In summary, multiple factors are involved in control of the expression of LeuO, a master regulator that orchestrates downstream regulators to modulate factors required for survival and pathogenicity of the pathogen.

Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species.

The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation.IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

Adaptation to host in Vibrio vulnificus, a zoonotic pathogen that causes septicemia in fish and humans.

Vibrio vulnificus is a siderophilic pathogen spreading due to global warming. The zoonotic strains constitute a clonal-complex related to fish farms that are distributed worldwide. In this study, we applied a transcriptomic and single gene approach and discover that the zoonotic strains bypassed the iron requirement of the species thanks to the acquisition of two iron-regulated outer membrane proteins (IROMPs) involved in resistance to fish innate immunity. Both proteins have been acquired by horizontal gene transfer and are contributing to the successful spreading of this clonal-complex. We have also discovered that the zoonotic strains express a virulent phenotype in the blood of its main susceptible hosts (iron-overloaded humans and healthy eels) by combining a host-specific protective envelope with the common expression of two toxins (VvhA and RtxA1), one of which (RtxA1) is directly involved in sepsis. Finally, we found that both IROMPs are also present in other fish pathogenic species and have recently been transmitted to the phylogenetic lineage involved in human primary sepsis after raw seafood ingestion. Together our results highlight the potential hazard that the aquaculture industry poses to public health, which is of particular relevance in the context of a warming world.

Unveiling the acid stress response of clinical genotype Vibrio vulnificus isolated from the marine environments of Mangaluru coast, India.

Gastric acidity is one of the earliest host defences faced by ingested organisms, and successful pathogens need to overcome this hurdle. The objective of this study was the systematic assessment of acid-stress response of Vibrio vulnificus isolated from coastal regions of Mangaluru. Acid-shock experiments were carried out at pH 4.0 and pH 4.5, with different experimental conditions expected to produce a varied acid response. Exposure to mild acid before the acid shock was favourable to the bacteria but was dependent on cell population and pH of the media and was independent of the strains tested. Lysine-dependent acid response was demonstrated with reference to the previously identified lysine decarboxylase system. Additionally, the results showed that inoculation into oysters provided some level of protection against acid stress. Increased expression of lysine/cadaverine genes was observed upon the addition of ground oyster and was confirmed by quantitative real-time PCR. The potential role of ornithine was analyzed with regard to acid stress, but no change in the survival pattern was observed. These findings highlight the physiology of bacteria in acid stress.

Ability of Vibrio vulnificus isolated from fish of the Lagoa dos Patos estuary in south Brazil to form biofilms after sublethal stress and bacterial resistance to antibiotics and sanitizers.

The present study aimed to investigate the presence of Vibrio vulnificus in fish captured at the Lagoa dos Patos estuary (RS, Brazil), to establish a correlation between bacterial biofilm formation and sublethal stress, and to assess the resistance of the isolates to antimicrobials and sanitizers. A total of 217 isolates characteristic of Vibrio sp. were analyzed. Isolates were identified and subsequently their ability to form biofilm, the impact of exposure to sublethal stress on their biofilm formation ability, and their resistance to antimicrobial and to sodium hypochlorite and chlorine dioxide sanitizers were evaluated. V. vulnificus was isolated from the fish Paralichthys orbignyanus and Micropogonias furnieri. The bacterial isolates examined were able to form biofilms. Biofilm formation ability of these strains was decreased or inhibited after being exposed to sublethal stress. The isolates were resistant to most antimicrobials. The sanitizer concentrations necessary to eliminate V. vulnificus were higher than those usually used in the fishing industry.

Molecular identification and expression analysis of four Lysin motif (LysM) domain-containing proteins from turbot (Scophthalmus maximus).

Lysin motif (LysM) is involved in chitin, peptidoglycan and other structurally-related oligosaccharides recognition and binding, and it is important for the biological processes of responsing to bacterial and viral infections and pathogen defense. LysM is also a widely spread protein, ranging from prokaryotes to eukaryotes, including bacteria, plants and mammals. However, research of LysM in teleosts especially in marine fish was rarely scarce. In the present study, four novel LysM domain-containing proteins in turbot (Scophthalmus maximus), named as SmLysMd1, SmLysMd2, SmLysMd3, and SmLysMd4, were cloned and identified firstly. The full-length cDNA of SmLysMd1 was 1235 bp with a 678 bp ORF, capable of encoding a peptide of 225 amino acids. The complete cDNA sequence of SmLysMd2 was 1273 bp, and contained a 675 bp ORF, encoding a predicted protein of 224 amino acids. The full-length of SmLysMd3 cDNA was 2132 bp, containing a ORF of 987 bp, with a ORF of encoding 328 amino acids. The full-length SmLysMd4 cDNA was 1115 bp contained a 888 bp ORF, encoding 295 amino acids. And all the four predicated proteins contained a specific LYSM domain. Moreover, SmLysMd1 and SmLysMd2 belong to the intracellular non-secretory types, and SmLysMd3 and SmLysMd4 belong to the anchored transmembrane types. In addition, the four SmLysMd were ubiquitously expressed in all the examined tissues. Moreover, the SmLysMds levels were up-regulated in muscle and liver, and had a reduce tendency immediately in different degree following Vibrio vulnificus challenge, indicating that the turbot LysM could be participant in the immune responses to bacterial infections. The present result of LysM in turbot for the first time in a marine fish will provide foundation knowledge for the functions studies of LysM in immune responses. Further studies should be carried out to better understand their immune mechanism in turbot and other teleosts.

Microneedle Patch-Mediated Treatment of Bacterial Biofilms.

Current treatments of bacterial biofilms are limited by the poor penetration of antibiotics through their physical barrier as well as significant off-target toxicity of antibiotics and the induction of antibiotic resistance. Here we report a microneedle patch-mediated treatment for the effective elimination of biofilms by penetrating the biofilm and specifically delivering antibiotics to regions of active growth. We fabricated patches with self-dissolvable microneedles and needle tips loaded with chloramphenicol (CAM)-bearing and gelatinase-sensitive gelatin nanoparticles (CAM@GNPs). During the microneedle patch-mediated treatment, arrays of 225 microneedles simultaneously penetrate the biofilm matrix. Once inside, the microneedles dissolve and uniformly release CAM@GNPs into the surrounding area. In response to the gelatinase produced by the active bacterial community, the CAM@GNPs disassemble and release CAM into these active regions of the biofilm. Moreover, CAM@GNPs exhibited minimal off-target toxicity compared to direct CAM administration, which in turn favors wound healing. Importantly, we found that our microneedle-mediated treatment is more effective in treating Vibrio vulnificus biofilms than drug in free solution. We believe this new treatment strategy can be used to improve the delivery of a wide range of antimicrobial agents to biofilm-contaminated sites.

Potentially human-virulent Vibrio vulnificus isolates from diseased great pompano (Trachinotus goodei).

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish-related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health.

Bacteriophages Against Pathogenic Vibrios in Delaware Bay Oysters (Crassostrea virginica) During a Period of High Levels of Pathogenic Vibrio parahaemolyticus.

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.

Molecular identification and function analysis of bactericidal permeability-increasing protein/LPS-binding protein 1 (BPI/LBP1) from turbot (Scophthalmus maximus).

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) play important roles in host antimicrobial defense. In the present study, we identified one isoform of BPI/LBP gene from turbot (Scophthalmus maximus), designated as SmBPI/LBP1. The full-length cDNA sequence of SmBPI/LBP1 was 1826 bp, which encoding one secreted protein with 480 amino acid residues. Structurally, the SmBPI/LBP1 showed high similarity to its homologs from other vertebrates or invertebrates, which all contained a signal peptide, a BPI/LBP/CETP N-terminal with a LPS-binding domain, and a BPI/LBP/CETP C-terminal domain. The deduced amino acid sequences of SmBPI/LBP1 shared significant similarity to BPI/LBP of Seriola lalandi dorsalis (71%) and Paralichthys olivaceus (69%). Phylogentic analysis further supported that SmBPI/LBP1 act as a new member of vertebrate BPI/LBP family. SmBPI/LBP1 was ubiquitously expressed in all tested tissues, with the highest expression level in spleen tissue. The mRNA expression of SmBPI/LBP1 in spleen and kidney were significantly up-regulated after Vibrio vulnificus challenge. Finally, the recombinant SmBPI/LBP1 showed high affinity to lipopolysaccharide, followed by peptidoglycan and lipoteichoic acid, which is the ubiquitous component of Gram-negative or Gram-positive bacteria. These results indicated that SmBPI/LBP1 probably played important roles in immune response against bacteria infection.

Evolutionary Model of Cluster Divergence of the Emergent Marine Pathogen Vibrio vulnificus: From Genotype to Ecotype.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel "chromids," or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.