Ecology of \u3ci\u3eVibrio parahaemolyticus\u3c/i\u3e and \u3ci\u3eVibrio vulnificus\u3c/i\u3e in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaetnolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST

Comparative Genomic Analysis of Vibrio diabolicus and Six Taxonomic Synonyms: A First Look at the Distribution and Diversity of the Expanded Species

Vibrio is a diverse genus of Gammaproteobacteria autochthonous to marine environments worldwide. Vibrio diabolicus and V. antiquarius were originally isolated from deep-sea hydrothermal fields in the East Pacific Rise. These species are closely related to members of the Harveyi clade (e.g., V. alginolyticus and V. parahaemolyticus) that are commonly isolated from coastal systems. This study reports the discovery and draft genome sequence of a novel isolate (Vibrio sp. 939) cultured from Pacific oysters (Crassostrea gigas). Questions surrounding the identity of Vibrio sp. 939 motivated a genome-scale taxonomic analysis of the Harveyi clade. A 49-genome phylogeny based on 1,109 conserved coding sequences and a comparison of average nucleotide identity (ANI) values revealed a clear case of synonymy between Vibrio sp. 939, V. diabolicus Art-Gut C1 and CNCM I-1629, V. antiquarius EX25 and four V. alginolyticus strains (E0666, FF273, TS13, and V2). This discovery expands the V. diabolicus species and makes available six additional genomes for comparative genomic analyses. The distribution of the expanded species is thought to be global given the range of isolation sources (horse mackerel, seawater, sediment, dentex, oyster, artemia and polycheate) and origins (China, India, Greece, United States, East Pacific Rise, and Chile). A subsequent comparative genomic analysis of this new eight-genome subclade revealed a high degree of individual genome plasticity and a large repertoire of genes related to virulence and defense. These findings represent a significant revision to the understanding of V. diabolicus and V. antiquarius as both have long been regarded as distinct species. This first look at the expanded V. diabolicus subclade suggests that the distribution and diversity of this species mirrors that of other Harveyi clade species, which are notable for their ubiquity and diversity

A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus

Role of Type IV Pilins in Persistence of Vibrio vulnificus in Crassostrea virginica Oysters▿

Vibrio vulnificus is part of the natural estuarine microflora and accumulates in shellfish through filter feeding. It is responsible for the majority of seafood-associated fatalities in the United States mainly through consumption of raw oysters. Previously we have shown that a V. vulnificus mutant unable to express PilD, the type IV prepilin peptidase, does not express pili on the surface of the bacterium and is defective in adherence to human epithelial cells (R. N. Paranjpye, J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom, Infect. Immun. 66:5659-5668, 1998). A mutant unable to express one of the type IV pilins, PilA, is also defective in adherence to epithelial cells as well as biofilm formation on abiotic surfaces (R. N. Paranjpye and M. S. Strom, Infect. Immun. 73:1411-1422, 2005). In this study we report that the loss of PilD or PilA significantly reduces the ability of V. vulnificus to persist in Crassostrea virginica over a 66-h interval, strongly suggesting that pili expressed by this bacterium play a role in colonization or persistence in oysters

Summary of loci statistics included in the MLST scheme (<i>dna</i>E, <i>gyr</i>B, <i>rec</i>A, <i>dtd</i>S, <i>pnt</i>A, <i>pry</i>C and <i>tna</i>A) such as fragment length, number of alleles and polymorphic sites, nucleotide diversity (π), rates of nonsynonymous (<i>d</i>_N) and synonymous (<i>d</i>_S) mutations, and tests of selection (<i>d</i>_N/<i>d</i>_S).

a<p>Rate of nonsynonymous (<i>d</i>_N) and synonymous (<i>d</i>_S) substitutions where <i>d</i>_N/<i>d</i>_S<1 indicates that the loci is subject to purifying selection.</p

Results of the Pairwise Homoplasy Index (φ_w)^a and Sawyer’s Run Test^b for homologous recombination performed on individual loci included in the MLST scheme (<i>dna</i>E, <i>gyr</i>B, <i>rec</i>A, <i>dtd</i>S, <i>pnt</i>A, <i>pry</i>C and <i>tna</i>A).

a<p>mean Pairwise Homoplasy Index, see reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055726#pone.0055726-Bruen1" target="_blank">[48]</a>.</p>b<p>sum of squared lengths of condensed fragments (SSCF) and sum of squared lengths of uncondensed fragments (SSUF), see reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055726#pone.0055726-Sawyer1" target="_blank">[49]</a>.</p>*<p>significance declared at P<0.05.</p

REP-PCR patterns and representative dendrogram.

<p>The electrophoresis banding patterns of 167 <i>V. parahaemolyticus</i> isolates assayed by REP-PCR is shown. BioNumerics analysis of patterns revealed 39 unique REP-PCR groups comprised of N isolates. The corresponding BioNumerics dendrogram illustrates the genetic relatedness between REP-PCR groups, which we grouped into three major clusters (I, II, III). Groups 27, 28 and 3 comprise cluster I while groups 11, 29 and 34 comprise cluster III and all remaining groups comprise cluster II. Electrophoresis banding patterns shown with scale indicating fragment size in base pairs (bp).</p

MLST majority consensus phylogeny.

<p>A majority consensus phylogeny of 77 <i>V. parahaemolyticus</i> isolates based on 7 concatenated housekeeping loci (<i>dna</i>E, <i>gyr</i>B, <i>rec</i>A, <i>dtd</i>S, <i>pnt</i>A, <i>pry</i>C and <i>tna</i>A) and representing 3,682 total nucleotides was constructed using the Bayesian Markov chain Monte Carlo (MCMC) method as implemented in MrBayes v3.2. The 77 isolates included in this phylogeny were separated into three major clusters (I, II, III) and 12 distinct clades (1–12). Sequence typing (ST) designations for MLST analysis describe the 24 MLST sequence types comprising each of the 12 clades. Distinct clades clearly highlighted by alternating blue and gray shading. Nodes are labeled with posterior probabilities (0–1) while cladogram shading is indicative of branches with weak support (red) and strong support (black).</p