Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy

The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty-two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results.     

Ribotyping and plasmid profiling of Vibrio anguillarum serovar O2 and Vibrio ordalii

One hundred and twenty-nine strains of Vibrio anguillarum serovar O2 and 14 strains of Vibrio ordalii were ribotyped and examined for plasmid contents. A total of 35 different ribotypes were detected. The V. anguillarum serovar O2 strains were divided into 32 different ribotypes. The V. ordalii strains showed three different ribotypes, clearly distinct from those of the V. anguillarum strains.
Ribotypes were separated into seven clusters, of which one comprised the V. ordalii strains. Clustering of the strains indicated a genetic difference between North European and South European V. anguillarum O2 strains. Sero-subgroups O2a and O2b shared ribotypes; however, three of the clusters did not include O2a strains.
All V. ordalii strains had a plasmid of 32 kb. This plasmid was not detected in any of the V. anguillarum strains. Seventeen different plasmid profiles with 17 different sized plasmids were detected among the V. anguillarum strains. Most of the plasmids were small (< 6 kb) and found in several strains. Except for one South European strain, plasmids were detected only in the North European strains of V. anguillarum O2.     

Natural transformation of Vibrio fischeri requires tfoX and tfoY

Recent evidence has indicated that natural genetic transformation occurs in Vibrio cholerae, and that it requires both induction by chitin oligosaccharides, like chitohexaose, and expression of a putative regulatory gene designated tfoX. Using sequence and phylogenetic analyses we have found two tfoX paralogues in all sequenced genomes of the genus Vibrio. Like V. cholerae, when grown in chitohexaose, cells of V. fischeri are able to take up and incorporate exogenous DNA. Chitohexaose-independent transformation by V. fischeri was observed when tfoX was present in multicopy. The second tfoX paralogue, designated tfoY, is also required for efficient transformation in V. fischeri, but is not functionally identical to tfoX. Natural transformation of V. fischeri facilitates rapid transfer of mutations across strains, and provides a highly useful tool for experimental genetic manipulation in this species. The presence of chitin-induced competence in several vibrios highlights the potential for a conserved mechanism of genetic exchange across this family of environmentally important marine bacteria.     

Use of randomly amplified polymorphic DNA analysis to differentiate isolates of Vibrio parahaemolyticus from cockles (Anadara granosa)

A total of 35 Kanagawa-negative strains of Vibrio parahaemolyticus isolated from cockles (Anadara granosa) were investigated by randomly amplified polymorphic DNA fingerprinting with three primers and their plasmid profiles. Eighteen strains carried small plasmid(s) of 2.4 to 7.3kb that enabled the V. parahaemolyticus to be grouped into eight plasmid patterns. The three primers generated polymorphisms in all 35 strains of V. parahaemolyticus tested, producing bands ranging from 0.25 to 3.9kb. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested, and that cockles in the study area are populated by genetically polymorphic strains of V. parahaemolyticus.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.     

Novel transmissible factors in a non-O1 Vibrio cholerae and a Vibrio sp

Transmissible factors encoding production of lacunae (L factors) were demonstrated in a non-O1 Vibrio cholerae and a Vibrio sp. of recent environmental origin. Lacunae were produced in lawns of non-O1 V. cholerae indicator strains under the same assay conditions as those where lacunae were produced by the well characterized P fertility plasmid of V. cholerae O1 and the V fertility factor found in a non-cholera vibrio strain. The origin of the lacunae produced by strains harbouring the V and L factors was examined. No vibriocin or phage activity was found in culture supernates or in lacunae produced by the strains, suggesting that, as in the case of the P plasmid, the lacunae probably represent sites of active mating. Unlike the P plasmid, neither the Vn or L factor could be detected or isolated by conventional plasmid techniques.     

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.     

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.     

Molecular cloning of the plasmids of Vibrio cholerae 01 and the incidence of related plasmids in clinical isolates and other Vibrio species

Abstract We describe the subcloning of plasmids present in Vibrio cholerae 01 strain V58: the P factor, the large cryptic plasmid (lcp) and small cryptic plasmid (scp). Strains harbouring fragments of the P sex factor and the lcp were examined for plasmid encoded proteins by Coomassie blue staining and analysis in Escherichia coli K-12 minicells.
The distribution of these three plasmids in a variety of Vibrio species has been examined using some of the cloned fragments as DNA probes. Most recent clinical isolates of V. cholerae were found to contain the scp. None of the strains contained the lcp. The P factor was only detected in one clinical isolate in addition to the scp. While plasmids appear to be generally uncommon among V. cholerae , and do not appear to differentiate biovars, the presence of plasmids may be a useful epidemiological adjunct.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage

We identified a 4.7 kb cryptic plasmid in all ctxAB ⁺ Vibrio cholerae strains we tested. An isolate of the V. cholerae classical biotype strain O395 that harbours the cryptic plasmid at high copy number was found. Hybridization analysis demonstrated that sequences highly related or identical to this plasmid exist in all toxigenic strains of V. cholerae but were notably absent in all non-toxigenic environmental isolates that lacked the genes for toxin-co-regulated pili and the filamentous CTX prophage. Accordingly, we have named the cryptic plasmid pTLC for toxin-linked cryptic. The complete nucleotide sequence of pTLC from the high-copy-number isolate was determined. The largest open reading frame in the plasmid is predicted to encode a protein similar to the replication initiation protein (pII) of Escherichia coli F-specific filamentous phages. The nucleotide sequence of pTLC also facilitated the structural characterization of the DNA homologous to pTLC in other strains of V. cholerae . pTLC-related DNA exists in these strains as both low-copy-number, covalently closed circular DNA and tandemly duplicated, chromosomally integrated DNA. Remarkably, the chromosomally integrated form of pTLC is adjacent to the CTX prophage. The strain distribution, chromosomal location and DNA sequence of pTLC suggests that it may be a genetic element that plays some role in the biology of CTXφ, perhaps facilitating either its acquisition or its replication.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Phenotypic characterization and RAPD fingerprinting of Vibrio parahaemolyticus and Vibrio alginolyticus isolated during Tunisian fish farm outbreaks

The genus Vibrio is characterized by a large number of species and some of them are human pathogens causing gastrointestinal and wound infections through the ingestion or manipulation of contaminated fishes and shellfish including Vibrio parahaemolyticus and Vibrio alginolyticus. In this study, we reported the phenotypic and molecular characterization of 9 V. parahaemolyticus and 27 V. alginolyticus strains isolated from outbreaks affecting cultured Gilthead sea bream (Sparus aurata L.) and Sea bass (Dicentrarchus labrax) along the Tunisian coast from 2008 to 2009. All isolates were tested for the presence of DNase, caseinase, protease, lipase, amylase, gelatinase, hemolytic activity and antibacterial resistance to different drugs. Randomly amplified polymorphic DNA was used to examine the genetic relatedness among the V. parahaemolyticus and V. alginolyticus strains.     

Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua)

In 1988, a new plasmid profile was observed for Vibrio salmonicida isolated from cod (Gadus morhua) and Atlantic salmon (Salmo salar) in fish farms in northern Norway. This new plasmid profile, which consisted of plasmids of 61, 21, 3.4, and 2.8 megadaltons, is 1 of 11 plasmid profiles which have so far been observed for V. salmonicida. Plasmid profiling and plasmid DNA hybridization were used in epidemiological studies of cold-water vibriosis. Our results indicate that V. salmonicida was transmitted from Atlantic salmon to cod and vice versa. The 61-megadalton plasmid was found exclusively in V. salmonicida strains originating from northern Norway, which is the only area in which this plasmid has ever been observed. Plasmid DNA hybridization and restriction endonuclease analysis show that the plasmid DNA of V. salmonicida remained stable throughout a 7-year survey.     

Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua).

In 1988, a new plasmid profile was observed for Vibrio salmonicida isolated from cod (Gadus morhua) and Atlantic salmon (Salmo salar) in fish farms in northern Norway. This new plasmid profile, which consisted of plasmids of 61, 21, 3.4, and 2.8 megadaltons, is 1 of 11 plasmid profiles which have so far been observed for V. salmonicida. Plasmid profiling and plasmid DNA hybridization were used in epidemiological studies of cold-water vibriosis. Our results indicate that V. salmonicida was transmitted from Atlantic salmon to cod and vice versa. The 61-megadalton plasmid was found exclusively in V. salmonicida strains originating from northern Norway, which is the only area in which this plasmid has ever been observed. Plasmid DNA hybridization and restriction endonuclease analysis show that the plasmid DNA of V. salmonicida remained stable throughout a 7-year survey.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains

Abstract The structural gene for the haemolysin and two accessory genes from a Vibrio cholerae O1 El Tor strain have previously been cloned in Escherichia coli K-12 to give the plasmid pPM431. This plasmid has been used as a probe with a variety of O1 and non-O1 Vibrio cholerae strains to examine by Southern DNA hybridisations for the presence of homologous DNA. Such experiments show that the DNA homologous to that present in pPM431 is present in all of the 20 strains examined, whether they were haemolytic or non-haemolytic, implying that the genes were present but not expressed in non-haemolytic strains. Using a variety of restriction enzymes to cut the chromosomal DNA of different V. cholerae strains and probing with pPM431, it was possible to distinguish O1 and non-O1 strains, as well as haemolytic or non-haemolytic strains. This variability between hly⁺ and hly⁻ may be indicative of a change in the regulatory region of the haemolysin genes. The results also imply a high degree of homology of the haemolysin of O1 and non-O1 strains.