In situ strain-level detection and identification of Vibrio parahaemolyticus using surface-enhanced Raman spectroscopy.

The outer membrane of a bacterium is composed of chemical and biological components that carry specific molecular information related to strains, growth stages, expressions to stimulation, and maybe even geographic differences. In this work, we demonstrate that the biochemical information embedded in the outer membrane can be used for rapid detection and identification of pathogenic bacteria using surface-enhanced Raman spectroscopy (SERS). We used seven different strains of the marine pathogen Vibrio parahaemolyticus as a model system. The strains represent four genetically distinct clades isolated from clinical and environmental sources in Washington, U.S.A. The unique quasi-3D (Q3D) plasmonic nanostructure arrays, optimized using finite-difference time-domain (FDTD) calculations, were used as SERS-active substrates for sensitive and reproducible detection of these bacteria. SERS barcodes were generated on the basis of SERS spectra and were used to successfully detect individual strains in both blind samples and mixtures. The sensing and detection methods developed in this work could have broad applications in the areas of environmental monitoring, biomedical diagnostics, and homeland security.

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus 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. parahaemolyticus 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 evolutionary analysis of the major structural subunit of Vibrio vulnificus type IV pili.

Type IV pili contribute to virulence in Vibrio vulnificus, the bacterium responsible for the majority of fatal seafood-related infections. Here, we performed within- and between-species evolutionary analysis of the gene that encodes the major structural subunit of the pilus, pilA, by comparing it with pilD and gyrB, the genes encoding the type IV prepilin peptidase and beta subunit of DNA gyrase, respectively. Although the diversity in pilD and gyrB is similar to each other and likely to have accumulated after speciation of V. vulnificus, pilA is several times more diverse at both nonsynonymous and synonymous levels. Also, in contrast to pilD and gyrB, there are virtually unrestricted and highly localized horizontal movements of pilA alleles between the major phylogenetic groups of V. vulnificus. The frequent movement of pilA involves homologous recombination of the entire gene with no evidence for intragenic recombination between the alleles. We propose that pilA allelic diversity and horizontal movement is maintained in the population by both diversifying and frequency-dependent selection most likely to escape shellfish innate immunity defense or lytic phages. Other possibilities leading to such selection dynamics of V. vulnificus pilA could involve adaptation to diverse host populations or within-host compartments, or natural DNA uptake and transformation. We show that the history of nucleotide diversification in pilA predates V. vulnificus speciation and this diversification started at or before the time of the last common ancestor for V. vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae. At the same time, it appears that within the various pilA groups of V. vulnificus, there is no positive selection for structural mutations and consequently no evidence for source-sink selection. In contrast, pilD has accumulated a number of apparently adaptive mutations in the regions encoding the membrane-spanning portions of the prepilin peptidase, possibly affecting fimbrial expression and/or function, and is being subjected to source-sink selection dynamics.

The use of polymerase chain reaction for the detection and speciation of bacterial bone and joint infection in children.

We evaluated 36 consecutive patients presenting with signs and symptoms of bacterial bone and joint infection and 10 control patients using bacterial cultures of blood and the presumed site of infection compared with polymerase chain reaction (PCR) techniques using a universal primer and restriction endonuclease digestion. Of the 28 patients with definitive clinical and/or laboratory evidence of bacterial infection, 16 patients had positive bacterial cultures and 12 were PCR-positive. Twenty of 28 patients were either PCR- or culture-positive. Nine of the 16 subjects who had culture-positive samples also had PCR-positive samples (8 positive for the same organism and 1 with 2 organisms identified by culture, but only a single organism by PCR. Six culture positive patients were PCR-negative. Of the 12 patients who were culture-negative, 4 had bacterial genomic material present indicating infection. We conclude that current PCR methods are not superior to standard bacterial culture methods when applied to children with presumed bone or joint infections, but that PCR may complement existing microbiologic cultures for detection of bone and joint infections in children.

Vibrio parahaemolyticus risk assessment in the Pacific Northwest: it's not what's in the water.

The Gram-negative bacterium Vibrio parahaemolyticus (Vp) is a major cause of illness associated with the consumption of raw or undercooked seafood, primarily oysters. This species is a natural member of the bacterial community in brackish waters and is bioaccumulated by oysters through filter feeding. Only a subset of strains is thought to be pathogenic. Currently known virulence markers include the gene for the thermostable direct hemolysin (tdh). In this work we analyzed water and oysters for total Vp and strains encoding tdh from 26 oyster-growing areas of the Puget Sound and Pacific coast of Washington state in 2007 and 2008. In addition, possible plankton-associated Vp were assessed from net tow samples. The density of both total and tdh+ Vp in the water column were considerably higher in 2008 than 2007. However, the concentrations of both total and tdh+ Vp in the oyster tissue was similar for both years. A high proportion of Vp strains in the water column was found to be tdh+ in both 2007 and 2008; however, tdh+ strains were detected at much lower levels in oysters. The data show that analysis of Vp density in the oysters is a better risk assessment tool than density in the overlying water column.

Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs.

Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

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 real-time PCR assay for the rapid determination of 16S rRNA genotype in Vibrio vulnificus.

In a terminal restriction fragment polymorphism (T-RFLP) study, we recently reported a significant association between the type B 16S rRNA gene and clinical strains of Vibrio vulnificus associated with the consumption of raw oysters. In the present study we describe a real-time PCR assay for the rapid determination of the 16S rRNA type of V. vulnificus isolates. This assay was used to reexamine the 16S rRNA gene type in the strains studied previously by T-RFLP and additional isolates from selected sources. Analyses revealed that 15 of the strains (10 environmental and 5 clinical) previously found to be 16S rRNA type A actually appear to possess both the type A and B genes. The presence of both alleles was confirmed by cloning and sequencing both gene types from one strain. To our knowledge, this is the first report of 16S rRNA sequence heterogeneity within individual strains of V. vulnificus. The findings confirm the T-RFLP data that 16S rRNA type may be a useful marker for determining the clinical significance of V. vulnificus in disease in humans and cultured eels. The real-time PCR assay is much more rapid and less resource-intensive than T-RFLP, and should facilitate further study of the occurrence and distribution of the 16S rRNA genotypes of V. vulnificus. These studies should provide more definitive estimates of the risks associated with this organism and may lead to a better understanding of its virulence mechanism(s).

Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterium salmoninarum adherence and invasion of host cells.

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fishes, is a Gram-positive diplococcobacillus belonging to the family Micrococcaceae. Analysis of the genome sequence of the bacterium demonstrated the presence of a sortase homolog (srtD), a gene specifying an enzyme found in Gram-positive bacteria and required for covalent anchoring of cell surface proteins. Interference of sortase activity is being examined as a target for therapeutic prevention of infection by several pathogenic Gram-positive bacterial species. In silico analysis identified 8 open reading frames containing sortase recognition motifs, suggesting these proteins are translocated to the bacterial cell wall. The sortase and potential sortase substrate genes are transcribed in R. salmoninarum, suggesting they encode functional proteins. Treatment of R. salmoninarum with phenyl vinyl sulfone (PVS) significantly reduced bacterial adherence to Chinook salmon fibronectin. In addition, the ability of the PVS-treated bacteria to adhere to Chinook salmon embryo cells (CHSE-214) in vitro was dramatically reduced compared to that of untreated bacteria. More importantly, PVS-treated bacteria were unable to invade and replicate within CHSE-214 cells (demonstrated by an intracellular growth assay and by light microscopy). When treated with PVS, R. salmoninarum was not cytopathic to CHSE-214 cells, whereas untreated bacteria produced cytopathology within a few days. These findings clearly show that PVS, a small molecule drug and a known sortase inhibitor, can interfere with the ability of R. salmoninarum to adhere and colonize fish cells, with a corresponding decrease in virulence.

Atypical strains of Aeromonas salmonicida contain multiple copies of insertion element ISAsa4 useful as a genetic marker and a target for PCR assay.

The species Aeromonas salmonicida includes a quite complex group of pathogens that cause a variety of diseases in fishes. Best studied strains of this species are those of the subspecies salmonicida also referred to as 'typical' A. salmonicida, which cause furunculosis in salmonids. Less completely understood are bacteria assigned to other subspecies, e.g. achromogenes and masoucida, or those that cannot be assigned to a recognized subspecies. These strains are referred to collectively as 'atypical' A. salmonicida and cause diseases distinct from furunculosis, primarily affecting non-salmonids. In the course of a study to investigate the suitability of the gene product of tapA as a subunit vaccine, we discovered several atypical strains of A. salmonicida in which the tapA gene was interrupted by an insertion sequence (IS). Subsequent Southern blot analyses indicated that nearly all atypical strains (27 of 29) examined carry many copies of this IS, which we named ISAsa4. Genetic characterization of this IS element revealed it to be a member of the IS5 family, subgroup IS903. Aside from the presence of ISAsa4 in several atypical strains, the nucleotide sequence of tapA was virtually identical to that found in typical strains. This finding suggests that ISAsa4 might be a major source of genetic diversity among atypical strains which, unlike typical strains, are genetically heterogeneous. The presence of ISAsa4 in atypical strains may also help explain the host tropism of atypical strains of this bacterium. Using information on the nucleotide sequences of ISAsa4 from atypical strains of A. salmonicida, primers were designed to selectively amplify genomic DNA from most atypical strains.

Concentrations of erythromycin and azithromycin in mature Chinook salmon Oncorhynchus tshawytscha after intraperitoneal injection, and in their progeny.

A single dose (40 mg kg(-1)) of erythromycin or azithromycin dihydrate was injected intraperitoneally into maturing female fall Chinook salmon 12 to 32 d before spawning to observe the distribution, retention and clearance of the drugs in plasma, kidney, coelomic fluid and egg vitellin, and their persistence in alevins derived from these fish. Salmon administered prophylactic dosages of erythromycin as subadults were also included to investigate potential interactive effects of oral and injected treatments on reproductive performance and antibiotic clearance. Erythromycin was rapidly cleared from plasma and coelomic fluid, but was detected in the kidney (3.52 to 12.40 microg g(-1)) and egg vitellin (5.32 to 8.87 microg ml(-1)) of all fish at spawning. High, stable concentrations of azithromycin were detected in plasma (14.66 to 20.33 microg ml(-1)), kidney (43.16 to 59.96 microg g(-1)), coelomic fluid (2.52 to 5.50 microg ml(-1)) and egg vitellin (12.65 to 23.51 microg ml(-1)). Oral administration of erythromycin to subadult salmon did not significantly affect tissue concentrations of either erythromycin or azithromycin administered by prespawning injection. Reductions in the percentage of eggs that yielded live embryos at the eyed stage of development occurred among eggs derived from females that had received orally administered erythromycin as subadults. Erythromycin was not detected in unfed fry derived from adults injected with the drug prespawning, but azithromycin was present for more than 2 mo after the onset of exogenous feeding.

Accumulation and clearance of orally administered erythromycin and its derivative, azithromycin, in juvenile fall chinook salmon Oncorhynchus tshawytscha.

Fall Chinook salmon Oncorhynchus tshawytscha were fed practical diets medicated with azithromycin (30 mg kg(-1) fish for 14 d) or erythromycin (100 mg kg(-1) fish for 28 d) either 1, 2, or 3 times beginning 14 d after initiation of exogenous feeding (February) and ending at smoltification (June). Average tissue concentrations of azithromycin increased from 19.0 microg g(-1) in fry to 44.9 microg g(-1) in smolts, and persisted in the tissues > 76 d after treatment ceased. Tissue concentrations of erythromycin were comparatively low, ranging from 0.2 microg g(-1) in fry to 10.4 microg g(-1) in smolts. Erythromycin was not detectable 21 d post-treatment. Neither antibiotic caused histopathologically significant lesions in the trunk kidney or other organ tissues. The high tissue concentrations and prolonged retention of azithromycin in Chinook may be factors that increase the efficacy of the antibiotic against Renibacterium salmoninarum, compared with erythromycin, particularly in early life history stages before covertly infected fish show clinical signs of disease.

Environmental influences on the seasonal distribution of Vibrio parahaemolyticus in the Pacific Northwest of the USA.

Populations of Vibrio parahaemolyticus in the environment can be influenced by numerous factors. We assessed the correlation of total (tl+) and potentially virulent (tdh+) V. parahaemolyticus in water with three harmful algal bloom (HAB) genera (Pseudo-nitzschia, Alexandrium and Dinophysis), the abundance of diatoms and dinoflagellates, chlorophyll-a and temperature, salinity and macronutrients at five sites in Washington State from 2008-2009. The variability in V. parahaemolyticus density was explained predominantly by strong seasonal trends where maximum densities occurred in June, 2 months prior to the highest seasonal water temperature. In spite of large geographic differences in temperature, salinity and nutrients, there was little evidence of corresponding differences in V. parahaemolyticus density. In addition, there was no evident relationship between V. parahaemolyticus and indices of HAB genera, perhaps due to a lack of significant HAB events during the sampling period. The only nutrient significantly associated with V. parahaemolyticus density after accounting for the seasonal trend was silicate. This negative relationship may be caused by a shift in cell wall structure for some diatom species to a chitinous substrate preferred by V. parahaemolyticus. Results from our study differ from those in other regions corroborating previous findings that environmental factors that trigger vibrio and HAB events may differ depending on geographic locations. Therefore caution should be used when applying results from one region to another.

Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes.

We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture. The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome. Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag. The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection. Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level. Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented. The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca. 30 CFU mg(-1) of tissue was estimated. A similar detection limit was observed for several other gram-negative pathogens. This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.

An Aeromonas salmonicida type IV pilin is required for virulence in rainbow trout Oncorhynchus mykiss.

Aeromonas salmonicida expresses a large number of proven and suspected virulence factors including bacterial surface proteins, extracellular degradative enzymes, and toxins. We report the isolation and characterization of a 4-gene cluster, tapABCD, from virulent A. salmonicida A450 that encodes proteins homologous to components required for type IV pilus biogenesis. One gene, tapA, encodes a protein with high homology to type IV pilus subunit proteins from many gram-negative bacterial pathogens, including Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio vulnificus. A survey of A. salmonicida isolates from a variety of sources shows that the tapA gene is as ubiquitous in this species as it is in other members of the Aeromonads. Immunoblotting experiments demonstrate that it is expressed in vitro and is antigenically conserved among the A. salmonicida strains tested. A mutant A. salmonicida strain defective in expression of TapA was constructed by allelic exchange and found to be slightly less pathogenic for juvenile Oncorhynchus mykiss (rainbow trout) than wild type when delivered by intraperitoneal injection. In addition, fish initially challenged with a high dose of wild type were slightly more resistant to rechallenge with wild type than those initially challenged with the tapA mutant strain, suggesting that presence of TapA contributes to immunity. Two of the other three genes identified, tapB and tapC, encode proteins with homology to factors known to be required for type IV pilus assembly in P. aeruginosa, but in an as yet unidentified manner. TapB is a member of the ABC-transporter family of proteins that contain characteristic nucleotide-binding regions, and which may provide energy for type IV pilus assembly through the hydrolysis of ATP. TapC homologs are integral cytoplasmic membrane proteins that may play a role in pilus anchoring or initiation of assembly. The fourth gene, tapD, encodes a product that shares homology with a family of proteins with a known biochemical function, namely, the type IV prepilin leader peptidases. These bifunctional enzymes proteolytically cleave the leader peptide from the pilin precursor (prepilin) and then N-methylate the newly exposed N-terminal amino acid prior to assembly of the subunits into the pilus structure. We demonstrate that A. salmonicida TapD is able to restore type IV pilus assembly and type II secretion in a P. aeruginosa strain carrying a mutation in its type IV peptidase gene, suggesting that it plays the same role in A. salmonicida.

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.

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.

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.

Expression of duplicate msa genes in the salmonid pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R. salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R. salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R. salmoninarum.