Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Novel coordination of lipopolysaccharide modifications in Vibrio cholerae promotes CAMP resistance

In the environment and during infection, the human intestinal pathogen Vibrio cholerae must overcome noxious compounds that damage the bacterial outer membrane. The El Tor and classical biotypes of O1 V. cholerae show striking differences in their resistance to membrane disrupting cationic antimicrobial peptides (CAMPs), such as polymyxins. The classical biotype is susceptible to CAMPs, but current pandemic El Tor biotype isolates gain CAMP resistance by altering the net charge of their cell surface through glycine modification of lipid A. Here we report a second lipid A modification mechanism that only functions in the V. cholerae El Tor biotype. We identify a functional EptA ortholog responsible for the transfer of the amino‐residue phosphoethanolamine (pEtN) to the lipid A of V. cholerae El Tor that is not functional in the classical biotype. We previously reported that mildly acidic growth conditions (pH 5.8) downregulate expression of genes encoding the glycine modification machinery. In this report, growth at pH 5.8 increases expression of eptA with concomitant pEtN modification suggesting coordinated regulation of these LPS modification systems. Similarly, efficient pEtN lipid A substitution is seen in the absence of lipid A glycinylation. We further demonstrate EptA orthologs from non‐cholerae Vibrio species are functional.     

Development of simple and rapid PCR‐fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron

Aims: To develop simple and rapid PCR‐fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. Methods and Results: PCR‐restriction fragment length polymorphism (PCR‐RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat‐amplified fragment length polymorphism (VCR‐AFLP) assay was also developed. In the PCR‐RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR‐AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed‐field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). Conclusions: The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. Significance and Impact of Study: This newly developed method is more discriminatory, simple, rapid and cost‐effective in comparison with PFGE, and thus can be widely applicable.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae

The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non-O1 serotypes.     

Point mutation in the <Emphasis Type="Italic">Virbrio cholerae</Emphasis> O139 <Emphasis Type="Italic">cef</Emphasis> (CHO cell elongating factor) gene alters the substrate specificity of its product

Sequencing of the cef (CHO cell elongating factor) gene of Vibrio cholerae serogroup O139 revealed one nucleotide substitution (T to C at nucleotide 2015) as compared to cef of classical V. cholerae O1 and two substitutions (GT to AC at nucleotides 2014–2015) as compared to cef of V. cholerae O1 El Tor. A comparative bioinformatic analysis showed that the substitution determines a threonine residue in position 672 of the Cef protein, while this position is occupied by an isoleucine residue in the classical strains and a valine residue in the El Tor strains. The latter two amino acids are hydrophobic, while threonine is hydrophilic, having a polar R group. The nonsynonymous substitution affects the predicted secondary and, probably, tertiary structures of the Cef-O139 protein and explained our previous finding that the protein fails to degrade tributyrin, while retaining the tweenase activity spectrum and all other characteristics. It cannot be excluded that the inability of Cef-O139 to cleave triglycerides, along with other genetic specifics, contribute to the fact that the O139 serogroup has been supplanted from a dominating position in etiology of cholera by the El Tor biotype. The nucleotide sequence of the V. cholerae O139 cef gene and the deduced amino acid sequence of its product are reported for the first time and were deposited in GenBank under accession nos. JF499787 and AEC04822.1, respectively.     

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand

Background

Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.

Methods/Findings

A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.

Conclusions

MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.     

Pili of Vibrio cholerae Widely Distributed in Serogroup O1 Strains

The distribution of Vibrio cholerae O1 pili consisting of 16 kDa subunit protein (16K-pili) was examined by Western blotting, using 211 strains from various origins and specific anti-16K-pili sera. The 16 kDa protein was detected in all 211 strains. The pili were purified from 3 El Tor and 3 classical strains, and characterized by hemagglutination and inhibition tests. All purified pili were hemagglutinative. However, the hemagglutinating activity of classical pili disappeared after exposure to 5 M urea and the agglutination induced by the classical pili was inhibited by D -mannose, alpha-methylmannoside, D -glucose and N-acetylglucosamine. On the contrary, El Tor pili were resistant to these sugars and urea.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor

Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double-stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site-specific manner. DNA sequences that cross-hybridize with K139 phage DNA are present in all strains of V. cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139-related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as ‘kappa’. In order to test whether K139 phage is involved in lysogenic conversion of V. cholerae, we constructed a novel mini-transposon, Tn10d-bla, which was designed to produce β-lactamase fusions to phage-encoded, exported proteins. All Tn 10d-bla insertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N-terminal hydrophobic signal sequence. ORF1 was designated the glo gene (G protein-like O RF) because its amino acid sequence shows similarity to eukaryotic Gs(α) protein (34.5% identity over an 81-amino-acid overlap) and its C-terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP-binding proteins. LD₅₀ assays with isogenic Glo⁺ and Glo^? K139 lysogens suggest that glo encodes a secreted virulence determinant of V. cholerae     

A naturally occurring point mutation in the 13-mer R repeat affects the <Emphasis Type="Italic">oriC</Emphasis> function of the large chromosome of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1 classical biotype

The genome of Vibrio cholerae consists of two circular chromosomes of different sizes. Here, a comparative analysis of the replication origins of the large chromosomes (oriCI_VC) of classical and El Tor biotypes of the pathogen is reported. Extensive nucleotide sequence analyses revealed that the oriCI_VC region has six DnaA boxes instead of the five found in Escherichia coli oriC. The additional DnaA box, designated R_V, was unique in V. cholerae as well as in other members of the family Vibrionaceae. However, R_V was not found to be essential for the autonomous replication function of the 307-bp oriCI_VC minimal region. In contrast to El Tor and the recently evolved V. cholerae O139 strains, the oriCI_VC region of the classical biotype showed only a single base transition (TG) in a highly conserved AT-rich 13-mer R repeat region. From the minichromosome copy number and its transformational efficiency analyses, it appears that the single base substitution in the oriCI_VC of the classical biotype has a significant effect on its replication initiation.     

Molecular diversity of CTX prophage in Vibrio cholerae

Aims: The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6‐year period (2004–2009) in Iran. Methods and Results: The results of mismatch amplification mutation assay (MAMA)‐PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA‐PCR indicative of presence of two copies of CTX phages with different genotypes (rstR^ET ctxB^class and rstR^ET ctxB^ET) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstR^ET ctx^class) in 70·6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. Conclusions: Enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and ribosomal gene spacer‐PCR (RS‐PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction‐based methods. Significance and Impact of the Study: Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.     

Analysis of Vibrio seventh pandemic island II and novel genomic islands in relation to attachment sequences among a wide variety of Vibrio cholerae strains

Vibrio cholerae O1 El Tor, the pathogen responsible for the current cholera pandemic, became pathogenic by acquiring virulent factors including Vibrio seventh pandemic islands (VSP)‐I and ?II. Diversity of VSP‐II is well recognized; however, studies addressing attachment sequence left (attL) sequences of VSP‐II are few. In this report, a wide variety of V. cholerae strains were analyzed for the structure and distribution of VSP‐II in relation to their attachment sequences. Of 188 V. cholerae strains analyzed, 81% (153/188) strains carried VSP‐II; of these, typical VSP‐II, and a short variant was found in 36% (55/153), and 63% (96/153), respectively. A novel VSP‐II was found in two V. cholerae non‐O1/non‐O139 strains. In addition to the typical 14‐bp attL, six new attL‐like sequences were identified. The 14‐bp attL was associated with VSP‐II in 91% (139/153), whereas the remaining six types were found in 9.2% (14/153) of V. cholerae strains. Of note, six distinct types of the attL‐like sequence were found in the seventh pandemic wave 1 strains; however, only one or two types were found in the wave 2 or 3 strains. Interestingly, 86% (24/28) of V. cholerae seventh pandemic strains harboring a 13‐bp attL‐like sequence were devoid of VSP‐II. Six novel genomic islands using two unique insertion sites to those of VSP‐II were identified in 11 V. cholerae strains in this study. Four of those shared similar gene clusters with VSP‐II, except integrase gene.

     

Molecular phylogenetic analysis ofVibrio cholerae O1 El Tor strains isolated before, during and after the O139 outbreak based on the intergenomic heterogeneity of the 16S-23S rRNA intergenic spacer regions

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eightrrn operons (rrna-rrnh) ofVibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O139 outbreak. ISR classes ‘a’ and ‘g’ were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O139 serogroup and post-O139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.     

Transfer of cholera toxin genes from O1 to non‐O1/O139 strains by vibriophages from California coastal waters

Aims: Vibrio cholerae is an important bacterial pathogen that causes global cholera epidemic. Although they are commonly found in coastal waters around the world, most environmental isolates do not contain cholera toxin genes. This study investigates vibriophages in southern California coastal waters and their ability to transfer cholera toxin genes. Methods and Results: Lytic phages infecting V. cholerae were isolated from Newport Bay, California, between May and November, while none was found in winter. Some of the phage isolates can infect multiple environmental V. cholerae strains and El Tor strains. All phages contained double‐stranded DNA. Transduction experiments using kanamycin‐resistant gene marked CTXΦ demonstrated that some environmental vibriophages can transfer CTXΦ genes from O1 El Tor strain to environmental non‐O1/O139 V. cholerae via generalized transduction. Conclusions: Vibriophages are important components of the natural aquatic ecosystem. They play an important role in influencing the dynamics and evolution of V. cholerae in the environment. Significance and Impact of the Study: This study demonstrates the significance of vibriophages in the coastal environment and transduction as one of the mechanisms of pathogenicity evolution among environmental V. cholerae.