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 Ι and ΙΙ). 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 Ι and ΙΙ) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.     

Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus.

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.     

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.     

Identification by 16S rDNA fragment amplification and determination of genetic diversity by random amplified polymorphic DNA analysis of Pasteurella pneumotropica isolated from laboratory rodents

Pasteurella pneumotropica is an opportunistic bacterium frequently isolated from colonies of various laboratory rodents. Identification of this species, including its differentiation into two distinct biotypes (Jawetz and Heyl), is usually based on the use of conventional bacteriologic methods. In this study, a 16S rDNA fragment amplification procedure was developed for use as an alternative method for identification and differentiation of P. pneumotropica. Polymerase chain reaction (PCR) products were two distinctive fragments of 937 and 564 bp specific for biotypes Jawetz and Heyl, respectively. Specificity of PCR products could be achieved by EcoRI cleavage, leading to 596 plus 341-bp and 346 plus 218-bp fragments for each of the amplification products. Use of this procedure confirmed identification of 34 field isolates and allowed definitive identification of some strains that could not have been done by use of bacteriologic examinations. Field isolates subjected to random amplified polymorphic DNA (RAPD) analysis had high genetic diversity among biotype Jawetz strains in contrast to biotype Heyl strains. In conclusion, RAPD could represent an additional means for identification of ambiguous strains of biotype Heyl and a valuable epidemiologic tool for identification of biotype Jawetz strains of P. pneumotropica.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

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.     

Molecular characterisation and antimicrobial resistance of Vibrio vulnificus and Vibrio alginolyticus isolated from mussels (Mytilus galloprovincialis)

Twelve Vibrio vulnificus biotype 1 and 11 Vibrio alginolyticus isolated from mussels in Italy were analysed by antimicrobial resistance, plasmid profiles, random amplification of polymorphic DNA (RAPD), and single enzyme amplified fragment length polymorphism (sAFLP). Plasmid DNA was detected in three V. vulnificus and four V. alginolyticus cultures. All isolates were resistant to at least two antimicrobial agents: all isolates were resistant to ampicillin, carbenicillin and streptomycin, except one V. alginolyticus which was sensitive to carbenicillin and two V. alginolyticus which were sensitive to streptomycin. No association was detected between the presence of plasmid DNA and antimicrobial resistance. Seven of the twelve V. vulnificus and two of the eleven V. alginolyticus cultures were susceptible to the 10 microg of the vibriostatic compound O/129; all cultures were susceptible to the 150 microg of O/129. Both RAPD and sAFLP was found to be reproducible. Ten sAFLP and seven RAPD profiles were detected amongst the 12 V. vulnificus cultures: three cultures were identified as indistinguishable by both methods. RAPD and sAFLP analysis of V. alginolyticus generated nine and seven profiles respectively, and these two methods were independent. These results demonstrate extreme variability of V. vulnificus and V. alginolyticus isolated from mussels, and both RAPD and sAFLP provided information on intraspecific differences which will be useful for molecular epidemiological or ecological studies. A combination of methods gave optimal discrimination, although a single method could provide sufficient information to characterise V. vulnificus isolates.     

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2

Abstract The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European cels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strain. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus . Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus .     

Intraspecific Differentiation of Vibrio vulnificus Biotypes by Amplified Fragment Length Polymorphism and Ribotyping

The intraspecific genomic relatedness of 80 Vibrio vulnificus isolates, 44 of biotype 1 and 36 of biotype 2, from different geographic origins and sources was evaluated by ribotyping and AFLP (amplified fragment length polymorphism) fingerprinting. Ribopatterns of DNAs digested with KpnI and hybridized with an oligonucleotide complementary to a highly conserved sequence in the 23S rRNA gene revealed up to 19 ribotypes in the species, which were different for the two biotypes. Sixteen different ribotypes were found within biotype 1 strains from clinical and environmental sources, and only three, recovered mainly from diseased eels, were found within biotype 2. Within this biotype, 96% of the strains showed the same ribopattern. The closest similarity was shown by the strains coming from the same eel farm, irrespectively of biotype. AFLP fingerprints obtained by selective PCR amplification of HindIII-TaqI double-restricted DNA fragments exhibited a strain-specific pattern which allowed the finest differentiation of subgroups within the eel-pathogenic isolates sharing the same ribopattern. Both techniques revealed good genetic markers for intraspecific differentiation of V. vulnificus. Ribotyping clearly separated the eel-pathogenic strains from the clinical and environmental isolates, whereas AFLP enabled the monitoring of individual strains and therefore constitutes one of the most discriminative tools for epidemiological and ecological studies.     

Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains ofVibrio vulnificus biotypes 1 and 2 by silver staining and immunoblotting

Lipopolysaccharides (LPS) of 11 strains of Vibrio vulnificus biotypes 1 and 2, isolated from an eel farm, and of 10 reference strains, were examined by SDS-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. LPS samples were obtained from whole-cell lysates, outer membrane fragments, and extracellular products. By silver staining, only a diffuse band of low-molecular weight could be visualized in all cases except for a biotype 1 strain isolated from water. However, immunoblotting with antisera obtained against strains of biotypes 1 and 2 from eels allowed visualization of multiple O-polysaccharide chains. All biotype 2 strains, independently of their origins, belonged to the same serotype and presented the same LPS profile, whereas eel isolates of biotype 1 were serologically identical and different from the rest of tested strains of biotype 1. This is the first report of LPSs with a ladder-like structure in Vibrio vulnificus.     

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.     

Vibrio vulnificus produces quorum sensing signals of the AHL-class

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N -butanoyl-homoserine-lactone (C₄-HL) by both reporter strains and by HPLC-high-resolution MS. C₄-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain–heart infusion) and its production was enhanced when blood factors were added to MSWYE. C₄-HL was detected in vivo , in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.     

Randomly amplified polymorphic DNA analysis of clinical and environmental isolates of Vibrio vulnificus and other vibrio species

Vibrio vulnificus is an estuarine bacterium that is capable of causing a rapidly fatal infection in humans. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed for use in detecting V. vulnificus, as well as other members of the genus Vibrio. The resulting RAPD profiles were analyzed by using RFLPScan software. This RAPD method clearly differentiated between members of the genus Vibrio and between isolates of V. vulnificus. Each V. vulnificus strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous. All of the vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while the V. vulnificus strains had an additional two molecular weight range bands in common. All of the V. vulnificus strains isolated from clinical specimens produced an additional band that was only occasionally found in environmental strains; this suggests that, as is the case with the Kanagawa hemolysin of Vibrio parahaemolyticus, the presence of this band may be correlated with the ability of a strain to produce an infection in humans. In addition, band pattern differences were observed between encapsulated and nonencapsulated isogenic morphotypes of the same strain of V. vulnificus.     

烟粉虱B型和Q型群体遗传结构的RAPD分析

近20年来,烟粉虱B型传入世界各地并暴发成灾,成为一种重要的农业入侵害虫; 烟粉虱Q型则是近几年引起人们高度重视的一种新的入侵生物型,目前已传入许多国家并造成一定危害。本文利用RAPD分子标记对烟粉虱B型和Q型不同地理种群的遗传结构进行了分析。结果表明：（1）引物H16对烟粉虱B型不同种群扩增的特异带,能有效区分烟粉虱B型和Q型、浙江非B/Q型种群;（2）烟粉虱Q型种群各项遗传多样性指数均比烟粉虱B型的要高;（3）我国烟粉虱Q型来自伊比利亚半岛的可能性比来自中东地区的可能性要大。另外,聚类分析结果提示,RAPD分子标记能有效地区分烟粉虱不同生物型,但可能不适用于生物型之间亲缘关系分析。     

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.     

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.     

Comparison of the effects of environmental parameters on growth rates of Vibrio vulnificus biotypes I, II, and III by culture and quantitative PCR analysis

Vibrio vulnificus is a natural inhabitant of estuarine waters. The three known biotypes include (i) most human pathogens, (ii) primarily eel pathogens, and (iii) pathogens associated with fish and with human wound infections in Israel. Despite the frequently lethal consequences of V. vulnificus infections, the growth rates of the various biotypes and their response to environmental changes are not well characterized. We compared the specific growth rates (μ) of a representative of each biotype by culture and quantitative PCR (qPCR) analysis in a defined medium under varied pH, temperature, and salinity. Growth rates based on culturable concentrations were always higher than those based on qPCR estimates; however, both enumeration methods yielded comparable results on the influence of environmental factors on growth rates. Temperature (25°C, 30°C, 37°C), pH (7.0, 8.0), and salinity (5 to 40‰) all had significant effects on the μ of each biotype. Temperature had the greatest effect on the μ of biotype 1 (CMCP6), whereas salinity had the greatest effect on the μ of biotypes 2 (ATCC 33147) and 3 (302/99). The biotypes' growth rates varied significantly; biotype 1 grew most rapidly, while biotype 3 grew most slowly. The highest growth rates were achieved at 37°C, pH 7.0, and salinities of 15 to 30‰ (μ = 4.0, 2.9, and 2.4 generations h(-1) for biotypes 1, 2, and 3, respectively). Other strains of the biotypes yielded comparable results, suggesting that the physiological responses of the biotypes are differentially affected by parameters that are highly variable both in estuarine environments and between the free-living and pathogen states of V. vulnificus.     

A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlate with clinical or environmental isolation

Vibrio vulnificus is an estuarine bacterium which is the causative agent of both food-borne disease and wound infection. Although V. vulnificus is commonly found in molluscan shellfish at high numbers, the incidence of disease is relatively low, leading to the hypothesis that not all strains of V. vulnificus are equally virulent. Unfortunately, there is currently no easy test to identify virulent strains of this species. We have previously identified a 200 bp randomly amplified polymorphic DNA (RAPD) PCR amplicon associated with clinical isolates. DNA sequence data from this locus in six clinical and four environmental isolates showed that the strains could be divided into two groups, which we termed C-type (correlates with clinical origin) and E-type (correlates with environmental origin). We designed PCR primers that could distinguish between the two groups, and typed 55 randomly selected strains. We found that 90% of the C-type strains were clinical isolates, while 93% of environmental isolates were classified as E-type. The region directly downstream of this locus contained a heptanucleotide sequence repeated various times depending on the strain. Using a PCR-based assay to detect the repeat number present in a given strain, we found a statistically significant correlation with the C/E type classification and the number of repeats. The data reported here are consistent with the existence of two genotypes of V. vulnificus, with the C-type being a strong indicator of potential virulence.     

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.     

Biotypes and randomly amplified polymorphic DNA (RAPD) profiles of subgingival Candida albicans isolates in HIV infection

A group of subgingival isolates of C. albicans recovered from Italian HIV-positive (HIV+) subjects were characterized both phenotypically and genotypically. Phenotyping of the isolates was carried out by a biotyping method based on the enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts. Genotyping was performed through randomly amplified polymorphic DNA (RAPD) analysis. Five biotypes were found among the 29 subgingival C. albicans strains examined. The predominant biotypes were A1R (55.17%), A1S (24.14%), and A2R (13.79%), while the biotypes A11R and A13R were represented by a single isolate each. RAPD profiles identified 15 genotypes among the 29 isolates. Almost every individual harboured his/her own specific isolate and in three out of the six subjects with multiple isolates (two to six each) more than one genotype (two to six) was found. The biotype distribution we found is consistent with previous reports on C. albicans isolates from other oral sources, whereas the resistance to boric acid was highly frequent in subgingival strains. RAPD analysis showed high genetic heterogeneity within subgingival isolates, also when isolates were phenotypically identical. These findings, obtained from HIV+ subjects living in Southern Italy, may be useful as baseline information on subgingival C. albicans colonization in the Mediterranean area.