Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone

Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions I and II). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions I and II) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Occurrence of Vibrio vulnificus Biotypes in Danish Marine Environments

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 10⁴ U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolated V. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificus in European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.     

PCR-Based Genotyping of Epidemic and Preepidemic Trichoderma Isolates Associated with Green Mold of Agaricus bisporus

We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of Trichoderma associated with green mold on the cultivated mushroom Agaricus bisporus. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s. Trichoderma harzianum biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to T. harzianum biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and Trichoderma atroviride (formerly T. harzianum biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and T. atroviride from Europe. Biotype 4 was not represented by the preepidemic isolates of Trichoderma as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.     

Genome-Wide SNP-Genotyping Array to Study the Evolution of the Human Pathogen Vibrio vulnificus Biotype 3

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae, horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen.     

Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

Vibrio vulnificus can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by V. vulnificus and in the detection of biotype 2 serovar E strains.     

Intraspecific Diversity of Vibrio vulnificus in Galveston Bay Water and Oysters as Determined by Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Molecular Typing of Environmental and Clinical Strains of Vibrio vulnificus Isolated in the Northeastern USA

Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as ‘LI’ and ‘LII’), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

Genotypes Associated with Virulence in Environmental Isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.     

Antimicrobial Susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus Isolates from Louisiana Gulf and Retail Raw Oysters

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC ≥ 16 μg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.     

Randomly Amplified Polymorphic DNA Analysis of Starved and Viable but Nonculturable Vibrio vulnificus Cells

Vibrio vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism’s natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of V. vulnificus strains grown in rich media and has been applied to starved and VBNC cells of V. vulnificus in the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify V. vulnificus DNA in these states.     

Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: Evaluation by two molecular markers

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.     

Sediment and Vegetation as Reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.     

Heterogeneity among Isolates of Vibrio vulnificus Recovered from Eels (Anguilla anguilla) in Denmark

The findings of this study demonstrate that Vibrio vulnificus isolates recovered from diseased eels in Denmark are heterogeneous as shown by O serovars, capsule types, ribotyping, phage typing, and plasmid profiling. The study includes 85 V. vulnificus isolates isolated from the gills, intestinal contents, mucus, spleen, and kidneys of eels during five disease outbreaks on two Danish eel farms from 1995 to 1997, along with a collection of 12 V. vulnificus reference strains. The results showed that more than one serovar may be capable of causing disease in eels and that these isolates are genetically heterogenous as shown by ribotyping. Ribotyping also showed that the same isolates may persist in an eel farm and cause recurrent outbreaks. Phage typing did not correlate with ribotyping or serotyping. However, we observed that 26 of 28 isolates, which were not susceptible to any of the phages, showed the same ribotype, O serovar, and capsule type. This suggests that these isolates may possess features that make them resistant to lysis by the phages used in this study. Ninety-three of 97 isolates harbored between one and three high-molecular-weight plasmids which previously had been suggested to be associated with eel virulence. The subdivision of V. vulnificus into two biotypes based on the indole reaction can no longer be supported, since 82 of 97 isolates in this study were indole positive, and a subdivision into serovars appears to be more correct.     

Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.