HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Safa, A. Articles by Nair, G. B. Search for Related Content PubMed PubMed Citation Articles by Safa, A. Articles by Nair, G. B. Agricola Articles by Safa, A. Articles by Nair, G. B.

Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes

¹ Laboratory Sciences Division, Enteric Microbiology Laboratory, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Centre for Health and Population Research, GPO Box 128, Dhaka 1000, Bangladesh

² International Medical Research Center of Japan, Toyama, Tokyo, Japan

³ Cine-Science Laboratory, Tokiwadai, Tokyo, Japan

Correspondence
G. Balakrish Nair
gbnair{at}icddrb.org

Received 20 April 2006
Accepted 18 July 2006

Abbreviations: CCA, chicken red cell agglutination; CT, cholera toxin; MSHA, mannose-sensitive haemagglutinin; RTX, repeat toxin; TLC, toxin-linked cryptic; VPI, Vibrio pathogenicity island.

The GenBank/EMBL/DDBJ accession numbers for the ctxB gene sequences of Vibrio cholerae O1 and O139 are DQ523199–DQ523219.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Vibrio cholerae belonging to serogroups O1 and O139 can cause the severe watery diarrhoea known as cholera. To date, more than 200 serogroups of V. cholerae have been recognized, based on variable somatic O antigen composition; O1 was the only known epidemic serogroup of V. cholerae up to 1991. In 1992, serogroup O139 was recognized as the second epidemic serogroup of V. cholerae (Albert et al., 1993; Shimada et al., 1993). There are two well-established biotypes within the V. cholerae O1 serogroup, classical and El Tor, which are distinguished from each other by several properties, including haemolysis of sheep red blood cells, agglutination of chicken red blood cells, Voges–Proskauer reaction, and susceptibility to polymyxin B and to biotype-specific phages (Kaper et al., 1995). The first six cholera pandemics are believed to have been caused by the classical biotype, and in 1961, V. cholerae strains of El Tor biotype initiated the seventh cholera pandemic, which is still continuing.

Different studies have shown that the virulence of V. cholerae O1/O139 involves a number of genes and/or gene clusters. Two large DNA regions, the Vibrio pathogenicity island (VPI)-1 and the CTX prophage, encode the major virulence factors in V. cholerae O1/O139 (Faruque et al., 1998). The tcpA gene of the VPI-1 encodes the major pilin protein which is involved in colonization of V. cholerae in the host intestine and also acts as the receptor for CTX to infect V. cholerae strains. The core part of the CTX is composed of the genes psh, core-encoded pillin (cep), gIII^CTX, ace (accessory cholera enterotoxin), zot (zonula occludens) and ctxAB (Davis & Waldor, 2003). The key virulence factor of V. cholerae, cholera toxin (CT), is encoded by the ctxAB genes. cep is reported to encode a minor coat protein, the function of which is not yet known. Ace and Zot are two CTX-encoded proteins that play a role in phage assembly (Faruque et al., 1998). The gIII^CTX gene plays a role in the processes of phage infection and replication (Heilpern & Waldor, 2003). The core part of CTX is usually flanked by a 2.5 kb RS2 region containing three ORFs, namely rstA, rstB and rstR, which function in the replication, regulation and integration processes, respectively. In El Tor and O139 strains of toxigenic V. cholerae only, CTX is often flanked by an element termed the RS1 element (Waldor et al., 1997; Davis et al., 2000). The RS1 and RS2 regions are similar, except for the rstC gene, which is present in the RS1 element alone (Waldor et al., 1997). Mannose-sensitive haemagglutinin (MSHA) is another virulence factor, and encodes a type IV pilus and consists of six ORFs (Jonson et al., 1991; Faruque et al., 1998). Repeat toxin (RTX) is also reported to be virulence-associated, and encodes a protein that has cytotoxic activity (Lin et al., 1999). This cluster comprises four ORFs, rtxABCD, of which the rtxC gene is seen in the El Tor biotype only. Dziejman et al. (2002) have recently reported two other clusters of genes, namely Vibrio seventh pandemic island (VSP)-I and -II, which are unique to the V. cholerae seventh pandemic El Tor strains. The other genes that may be linked to the pathogenicity of V. cholerae include hlyA, which encodes a haemolysin that is cytolytic for a variety of erythrocytes and mammalian cells in culture and is rapidly lethal for mice (Kaper et al., 1995); toxin-linked cryptic (TLC), a 4.7 kb DNA fragment whose functional relationship to CTX is not yet understood (Rubin et al., 1998); pilE, which encodes a putative fimbrial assembly protein (Heidelberg et al., 2000); and intl4, which encodes a previously unknown integrase that recognizes a family of V. cholerae repeated sequences (VCRs) associated with a ‘gene-VCR’ organization, similar to that of the well-characterized antibiotic-resistance integrons (Mazel et al., 1998; Recchia & Hall, 1997).

In our previous studies, we described V. cholerae O1 strains that possess both the classical and El Tor attributes and designated these strains as Matlab types I, II and III, as they were first detected in Matlab, a rural area in Bangladesh (Nair et al., 2002). Different phenotypic traits (haemolysis of sheep erythrocytes, agglutination of chicken erythrocytes, sensitivity to polymyxin B and to specific phages, and reaction to the Voges–Proskauer test) and genotypic traits (ctxA, acfB, tcpA and rstR) examined in the previous study failed to classify them into classical or El Tor biotypes (Nair et al., 2002). Further characterization of the Matlab type strains by PFGE revealed that type I strains had a classical biotype lineage, whereas types II and III were more closely related to the El Tor biotype (Safa et al., 2005). In this study, we employed extensive PCR-based screening and a DNA-sequence-based approach to examine the different virulence genes and/or gene clusters of Matlab variants and other similar strains, and compared the results with those obtained with reference strains of the classical and El Tor biotypes.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains. From the 38 strains of V. cholerae O1 and O139 Matlab variants available in our collection, 17 were included in this study. Of these, five V. cholerae O1 strains were isolated between 1992 and 1994, and were previously designated Matlab type I (MJ1236, MJ1485), II (MG116226) and III (MG116025, MG116926). The remaining 12 strains (10 V. cholerae O1 and two V. cholerae O139) were isolated between 1994 and 1997, and have been characterized previously (Nusrin et al., 2004). Three representative strains from previously characterized strains of V. cholerae O1 El Tor biotype, isolated in 2004 from Beira, Mozambique, were also included in this study (Ansaruzzaman et al., 2004). All strains were grown in Luria–Bertani (LB) broth and stored as frozen stocks in LB broth with 25 %, v/v, glycerol. V. cholerae strain N16961 of El Tor biotype and strain 569B of classical biotype were used as reference strains.

Biotyping. Tests for polymyxin B susceptibility, chicken red cell agglutination (CCA) and phage sensitivity were performed using standard procedures (WHO, 1987).

Genomic DNA isolation and purification. Genomic DNA was extracted and purified from the strains following the method of Sambrook et al. (1989), with some modifications. In brief, cells were harvested by centrifugation from overnight-grown cultures in LB and treated with TES (10 mM Tris, pH 8.0, 10 mM EDTA, 100 mM NaCl) and 10 % SDS at 65 °C for 10–15 min. After proteinase K treatment at 50 °C for 18 h, DNA was extracted with phenol/chloroform–isoamyl alcohol (25 : 24 : 1) and purified by ethanol precipitation, and dried before dissolving in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). RNase treatment was performed at 37 °C for 1–2 h, and final purification was done by ethanol precipitation. The purified DNA was dissolved in TE buffer and stored at –20 °C, and used for PCR analysis. The purity of the DNA was assayed with a spectrophotometer (GeneQuant) by using automatic calculation of the ratio of A₂₆₀ to A₂₈₀.

PCR analysis. PCR was used to assay for 11 virulence-associated genes and/or gene clusters in the genome of the 20 V. cholerae isolates using 31 sets of PCR primers and conditions described previously (Chow et al., 2001; O'Shea et al., 2004; Rivera et al., 2001). Further, using PRIMER3 software (available at http://www.genome.wi.mit.edu/genome_software/other/primer3.html), six sets of primers were designed (Table 2) to amplify regions that are uniquely present in all El Tor strains but absent in classical strains (Dziejman et al., 2002). PCR was performed in a 25 µl reaction mixture as follows: an initial denaturation step at 96 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, 1 min of primer extension at 72 °C and 7 min of final extension at 72 °C for one cycle. Amplicons were separated by agarose gel electrophoresis (1 %) in 0.5x Tris/borate/EDTA buffer. Products were stained with ethidium bromide, destained with distilled water, and visualized under UV light and photographed on a gel-documentation system.

DNA and protein sequence analysis. The chromatogram sequencing files were inspected using Chromas 2.23 (Technelysium). Nucleotide sequences of the test isolates were compared with the corresponding sequences of the N16961 El Tor reference strain (NC_002505), the 569B classical reference strain (U25679), the O139 reference strain (X76391), and the B33 (AY648939) and B65 (AY648940) strains, retrieved from GenBank using Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997). Multiple sequence alignments were developed using CLUSTALX 1.81.13, and DNA sequences were translated using GeneDoc version 2.6.002 alignment editor.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
All strains tested were resistant to polymyxin B and positive for CCA. Phage typing of seven of the 10 V. cholerae O1 strains tested showed resistance to phage IV and susceptibility to phage 5. The remaining strains were resistant to both phages (Table 1). We examined for the presence of the three important virulence-associated genes, namely tcpA, toxT and acfB, of the VPI-1 cluster. Fifteen strains, including three previously characterized strains from Mozambique, showed the presence of tcpA of El Tor type, and the remaining five Matlab reference strains (MT-I, MT-II and MT-III) showed tcpA of the classical type (Table 1). The remaining two genes were positive for all strains. All strains, including the reference strains, were positive for rstA, gIII^CTX and zot located in the CTX prophage region. Seven strains, including the El Tor N16961 reference strain, were positive for the rstC gene, indicating the presence of the RS1 element, while the remaining strains, including the classical reference strain 569B, were negative for rstC (Table 1). PCR analysis of all strains tested showed the presence of the MSHA gene cluster. All test strains, including El Tor N16961, showed a positive amplification for the rtxC gene of the RTX gene cluster, whereas only classical 569B reference strain was negative for the same gene. Another gene in this cluster, rtxA, was present in all the test strains, including classical and El Tor reference strains. Genes in the VSP-I and -II gene clusters were present in all the strains, including the El Tor reference strain, but were absent from the classical reference strain. Four pairs of primers were used to examine four individual loci, namely hlyA, pilE, tlc and intl4. Of these, pilE and intl4 were present in all strains tested, including the classical and El Tor reference strains, hlyA was present in all except two strains, whereas tlc was absent in all strains that were negative for rstC (Table 1). PCR analysis of all the six loci that are unique to El Tor strains but absent in classical strains showed the expected size of PCR product in all Matlab type reference strains (MT-I, MT-II and MT-III), including the El Tor reference strain (Fig. 1), but none in the classical reference strain. The deduced amino acid sequence of the CtxB subunit of the single representative strain of Matlab type II and one of two Matlab type III representative strains showed 100 % homology to the N16961 El Tor reference strain of genotype 3, which has tyrosine at position 39, phenylalanine at position 46 and isoleucine at position 68 (Table 1). In contrast, the amino acid sequence of the CT-B subunit of the remaining 12 strains, including two O139 strains, was identical to that of the 569B classical reference strain of genotype 1 (histidine at position 39, phenylalanine at position 46 and threonine at position 68) (Table 1).

View larger version (90K): [in this window] [in a new window]   Fig. 1. Gel image showing amplified genes indicated by the annotation numbers given in Table 2. Lane M, molecular size marker (100 bp ladder); lanes 1–5, 8–11, 14–18, 21–25, 28–32 and 34–38 represent Matlab type strains MJ1236 (MT-I), MJ1485 (MT-I), MG116226 (MT-II), MG116025 (MT-III) and MG116926 (MT-III), respectively; lanes 6–7, 12–13, 19–20, 26–27, 33–34 and 39–40 represent V. cholerae El Tor N16961 and the classical 569B reference strain.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The VPI-1 is a large DNA region that is composed of two gene clusters, namely toxin-coregulated pilus (TCP) and accessory colonization factor (ACF) (Herrington et al., 1988; Kovach et al., 1996). The tcpA gene of the TCP cluster has alleles specific for classical and El Tor biotypes of O1. In this study, the tcpA gene analysis showed variable results, and either a single allele or both the alleles were found among the strains tested (Table 1). Another important gene of this cluster is toxT, which encodes a second positive regulatory protein that directly activates a number of virulence genes (DiRita et al., 1996). This gene was present in all the strains tested and indicated the presence of an active virulence-gene regulatory system. Immediately adjacent to and downstream of the TCP cluster the ACF gene cluster is located. This cluster comprises four ORFs (ABCD), and the presence of acfB, which encodes a potential colonization factor, in all the strains is significant from the viewpoint of virulence potential. The genes of the MSHA cluster are expressed by strains of the El Tor biotype, but are only rarely expressed by strains of the classical biotype (Jonson et al., 1991). However, all strains in this study were positive for the MSHA gene cluster, including the classical reference strain. The hlyA gene is reported to be present in all classical, El Tor and non-O1 strains of V. cholerae (Brown & Manning, 1985). In this study, two strains were negative by PCR for this gene, and a further examination can indicate whether the gene is deleted or is present in a mutated form in these two strains. The presence of all these genes, including pilE and intl4, further indicated the pathogenic potential of these strains.

CT genes were previously identified in all strains examined here (Ansaruzzaman et al., 2004; Nair et al., 2002; Nusrin et al., 2004). However, PCR identification of other neighbouring genes (rstA, gIII^CTX and zot) indicated the presence of an intact core region of the CTX prophage genome. rstR is a regulatory gene and present in the RS2 part of the CTX prophage. This gene has four variants: rstR^classical, rstR^{El Tor}, rstR^Calcutta and rstR^{environmental}, and our previous studies (Ansaruzzaman et al., 2004; Nair et al., 2002; Nusrin et al., 2004) have shown that either a single rstR variant or an assortment of rstR variants is present in all the strains tested. Kimsey et al. (1998) first reported the presence of different types of rstR in a single V. cholerae strain, but the implication of this assortment is not yet known. Faruque et al. (2002) have recently shown that RS1 is a self-transducing phage, and that the rstC gene acts as an antirepressor in the phage replication process (Davis & Waldor, 2003). This gene is unique to the strains of the El Tor biotype and is absent in classical strains. Thus, we checked all the strains for the rstC gene and found that 10 strains, including Mozambique strains, lacked the rstC gene (Table 1). In other words, the absence of the rstC gene classified these strains as classical type, which is contrary to the earlier findings with the tcpA gene, but matches the rstR result obtained in our previous studies (Ansaruzzaman et al., 2004; Nusrin et al., 2004). Notably, all the rstC-negative strains were also negative for the TLC (Table 1), and require further examination to understand the relationship.

Recent studies have reported that rtxC, which encodes the activator protein, is absent from strains of classical biotype and present in the El Tor biotype only (Dziejman et al., 2002; Lin et al., 1999; O'Shea et al., 2004). The presence of the rtxC gene grouped all the strains as El Tor biotype. The VSP-I gene cluster encompasses a 16 kb region from VC0175 to VC0185, and most of the genes encode hypothetical or conserved proteins with no known function. On the other hand, the VSP-II region is a 27 kb region that encompasses VC0490 to VC0516 (O'Shea et al., 2004). As these two clusters are unique to the El Tor strains of the seventh pandemic, finally we checked for the presence of VSP-I and -II in the genome of these strains. All strains examined were positive for VSP-I and -II, pointing to the seventh-pandemic El Tor ancestry of these strains. The presence of VSP-I and -II also indicated that these strains have the potential to initiate pandemic spread, as these two regions are thought to be associated with the pandemicity of the El Tor biotype. Dziejman et al. (2002) also identified six genetic loci that are unique to strains of the El Tor biotype but absent from the classical biotype (Fig. 1). All these loci encode either hypothetical proteins or a putative acetyl transferase. All the six loci were present in all strains tested, except the classical reference strain, which further underscores the El Tor lineage of these strains.

On the basis of DNA and protein sequencing, heterogeneity within the B subunit was first reported in the early 1990s (Brickman et al., 1990; Kaper et al., 1995), and since then ctxB typing has been used as a tool for differentiating V. cholerae strains. To date, three ctxB genotypes of V. cholerae O1 strains of both biotypes and serotypes have been identified globally (Mekalanos et al., 1993; Olsvik et al., 1993). Based on the amino acid residue substitution at the three positions 39, 46 and 68, all classical and El Tor strains from the Gulf Coast of the USA are categorized as ctxB of genotype 1, El Tor strains associated with the Australian environmental reservoir are genotype 2, and El Tor strains of the seventh pandemic and the recent Latin American epidemic are classified as ctxB genotype 3. In this study, all except two strains examined possessed the classical type of ctxB DNA sequence or genotype 1, which is in contrast to the results obtained by PCR from different virulence-associated genetic regions. Notably, the PFGE results from our previous study (Safa et al., 2005) that clustered reference strains (MT-I, MT-II and MT-III) of Matlab variants with the El Tor biotype also disagree with the ctxB typing result.

The overall analysis showed that in all these Matlab variants most of the genetic traits tested are El Tor-like, indicating that these have an El Tor genome backbone. In contrast, the classical biotype attributes in these strains are mainly confined to the CTX prophage region, which is typically the classical CTX prophage. Lee et al. (2006) have recently shown that the Mozambique strains of El Tor biotype carry a tandem array of the classical prophages. Furthermore, in 1961, with the beginning of the seventh pandemic, the classical strains were gradually replaced by the El Tor biotype, and in Bangladesh the replacement started from 1968 and ended by 1973. However, the classical biotype reappeared in 1982 (Samadi et al., 1983) in Bangladesh and caused disease until 1993. Interestingly, the Matlab variants were isolated during a period when the classical biotype was disappearing from Bangladesh for the second time. The hybrid make-up of these strains thus appears to be built on the El Tor backbone, in which classical biotype attributes might have been acquired through horizontal gene transfer from classical strains that were on the verge of extinction. The similarity of PCR results and identical DNA sequence of the ctxB gene of these Matlab variants to those of the strains isolated during a recent outbreak in Mozambique are also key observations of this study which underscore the epidemiological significance of these variants.

	ACKNOWLEDGEMENTS

 
The Japan Health Science Foundation funded this study. ICDDR,B acknowledges with gratitude the commitment of the Japan Health Science Foundation to the Center's research efforts.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Albert, M. J., Siddique, A. K., Islam, M. S., Faruque, A. S. G., Ansaruzzaman, M., Faruque, S. M. & Sack, R. B. (1993). Large outbreak of clinical cholera due to Vibrio cholerae non-O1 in Bangladesh. Lancet 341, 704.[Medline]

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[Abstract/Free Full Text]

Ansaruzzaman, M., Bhuiyan, N. A., Nair, G. B., Sack, D. A., Lucas, M., Deen, J. L., Ampuero, J., Chaignat, C. L. & The Mozambique Cholera Vaccine Demonstration Project Coordination Group (2004). Cholera in Mozambique, variant of Vibrio cholerae. Emerg Infect Dis 10, 2057–2059.[Medline]

Barrett, T. J. & Blake, P. A. (1981). Epidemiological usefulness of changes in hemolytic activity of Vibrio cholerae biotype El Tor during the seventh pandemic. J Clin Microbiol 13, 126–129.[Abstract/Free Full Text]

Brickman, T. J., Boesman-Finkelstein, M., Finkelstein, R. A. & McIntosh, M. A. (1990). Molecular cloning and nucleotide sequence analysis of cholera toxin genes of the CtxA^– Vibrio cholerae strains Texas Star-SR. Infect Immun 58, 4142–4144.[Abstract/Free Full Text]

Brown, M. H. & Manning, P. A. (1985). Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains. FEMS Microbiol Lett 30, 197–201.[CrossRef]

Chow, K. H., Ng, T. K., Yuen, K. Y. & Yam, W. C. (2001). Detection of RTX toxin gene in Vibrio cholerae by PCR. J Clin Microbiol 39, 2594–2597.[Abstract/Free Full Text]

Davis, B. M. & Waldor, M. K. (2003). Filamentous phages linked to virulence of Vibrio cholerae. Curr Opin Microbiol 6, 35–42.[CrossRef][Medline]

Davis, M. B., Moyer, K. E., Boyd, E. F. & Waldor, M. K. (2000). CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes. J Bacteriol 182, 6992–6998.[Abstract/Free Full Text]

DiRita, V. J., Neely, M., Taylor, R. K. & Bruss, P. M. (1996). Differential expression of ToxR regulon in classical and El Tor biotypes of Vibrio cholerae is due to biotype-specific control over ToxT expression. Proc Natl Acad Sci U S A 93, 7991–7995.[Abstract/Free Full Text]

Dziejman, M., Balon, E., Boyd, D., Fraser, C. M., Heidelberg, J. F. & Mekalanos, J. J. (2002). Comparative genomic analysis of Vibrio cholerae: genes that correlate with cholera endemic and pandemic disease. Proc Natl Acad Sci U S A 99, 1556–1561.[Abstract/Free Full Text]

Faruque, S. M., Albert, M. J. & Mekalanos, J. J. (1998). Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiol Mol Biol Rev 62, 1301–1314.[Abstract/Free Full Text]

Faruque, S. M., Asadulghani Kamruzzaman, M., Nandi, R. K., Ghosh, A. N., Nair, G. B., Mekalanos, J. J. & Sack, D. A. (2002). RS1 element of Vibrio cholerae can propagate horizontally as a filamentous phage exploiting the morphogenesis genes of CTX. Infect Immun 70, 163–170.[Abstract/Free Full Text]

Heidelberg, J. F., Eisen, J. A., Nelson, W. C. & 29 other authors (2000). DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477–483.[CrossRef][Medline]

Heilpern, A. J. & Waldor, M. K. (2003). pIII^CTX, a predicted CTX minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae. J Bacteriol 185, 1037–1044.[Abstract/Free Full Text]

Herrington, D. A., Hall, R. H., Losonsky, G., Mekalanos, J. J., Taylor, R. K. & Levine, M. M. (1988). Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med 168, 1487–1492.

Jonson, G., Holmgren, J. & Svennerholm, A. M. (1991). Identification of a mannose-binding pilus on Vibrio cholerae El Tor. Microb Pathog 11, 433–441.[CrossRef][Medline]

Kaper, J. B., Bradford, H. B., Roberts, N. C. & Falkow, S. (1982). Molecular epidemiology of Vibrio cholerae in the U. S. Gulf Coast. J Clin Microbiol 16, 129–134.[Abstract/Free Full Text]

Kaper, J. B., Morris, J. G., Jr & Levine, M. M. (1995). Cholera. Clin Microbiol Rev 8, 48–86.[Abstract]

Karalois, D. K. S., Lan, R. & Reeves, P. R. (1995). The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-O1 Vibrio cholerae. J Bacteriol 177, 3191–3198.[Abstract/Free Full Text]

Kimsey, H. H., Nair, G. B., Ghosh, A. & Waldor, M. K. (1998). Diverse CTX and evolution of new pathogenic Vibrio cholerae. Lancet 352, 457–458.[Medline]

Kovach, M. E., Shaffer, M. D. & Peterson, K. M. (1996). A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae. Microbiology 142, 2165–2174.[Abstract]

Lee, J. H., Han, K. H., Choi, S. Y. & 11 other authors (2006). Multilocus sequence typing (MLST) analysis of Vibrio cholerae O1 El Tor isolates from Mozambique that harbour the classical CTX prophage. J Med Microbiol 55, 165–170.[Abstract/Free Full Text]

Lin, W., Fullner, K. J., Clayton, R., Sexton, J. A., Rogers, M. B., Calia, K. E., Calderwood, S. B., Fraser, C. & Mekalanos, J. J. (1999). Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci U S A 96, 1071–1076.[Abstract/Free Full Text]

Mazel, D., Dychinco, B., Webb, V. A. & Davies, J. (1998). A distinctive class of integron in the Vibrio cholerae genome. Science 280, 605–608.[Abstract/Free Full Text]

Mekalanos, J. J., Swartz, D. J., Person, G. D. N., Harford, N., Groyne, F. & deWilde, M. (1993). Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature 306, 551–557.

Mitra, S. N., Mukhopadhay, R., Ghosh, A. N. & Ghosh, R. K. (2000). Conversion of Vibrio El Tor MAK757 to classical biotype: role of phage PS166. Virology 273, 36–43.[CrossRef][Medline]

Nair, G. B., Faruque, S. M., Bhuiyan, N. A., Kamruzzaman, M., Siddique, A. K. & Sack, D. A. (2002). New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. J Clin Microbiol 40, 3296–3299.[Abstract/Free Full Text]

Nusrin, S., Khan, G. Y., Bhuiyan, N. A. & 9 other authors (2004). Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh. J Clin Microbiol 42, 5854–5856.[Abstract/Free Full Text]

Olsvik, Ø., Wahlberg, J., Petterson, B., Uhlen, M., Popovic, T., Wachsmuth, I. K. & Fields, P. I. (1993). Use of automated sequencing of polymerase chain reaction-generated amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae O1 strains. J Clin Microbiol 31, 22–25.[Abstract/Free Full Text]

O'Shea, A. Y., Reen, J. F., Quirke, A. M. & Boyd, E. F. (2004). Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles. J Clin Microbiol 42, 4657–4671.[Abstract/Free Full Text]

Recchia, G. D. & Hall, M. R. (1997). Origins of the mobile gene cassettes found in integrons. Trends Microbiol 5, 389–394.[CrossRef][Medline]

Rivera, I. N., Chun, J., Huq, A., Sack, R. B. & Colwell, R. R. (2001). Genotypes associated with virulence in environmental isolates of Vibrio cholerae. Appl Environ Microbiol 67, 2421–2429.[Abstract/Free Full Text]

Rubin, E. J., Lin, W., Mekalanos, J. J. & Waldor, M. K. (1998). Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage. Mol Microbiol 28, 1247–1254.[CrossRef][Medline]

Safa, A., Bhuiyan, N. A., Alam, M., Sack, D. A. & Nair, G. B. (2005). Genomic relatedness of the new Matlab variants of Vibrio cholerae O1 to the classical and El Tor biotypes as determined by pulsed-field gel electrophoresis. J Clin Microbiol 43, 1401–1404.[Abstract/Free Full Text]

Samadi, A. R., Shahid, N., Eusuf, A., Yunus, M., Huq, M. I., Khan, M. U., Rahman, A. S. M. M. & Faruque, A. S. G. (1983). Classical Vibrio cholerae biotype displaces El Tor in Bangladesh. Lancet 1, 805–807.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Shimada, T., Nair, G. B., Deb, B. C., Albert, M. J., Sack, R. B. & Takeda, Y. (1993). Outbreak of Vibrio cholerae non-O1 in India and Bangladesh. Lancet 341, 1347.[CrossRef][Medline]

Waldor, M. K., Rubin, E. J., Pearson, G. D. N., Kimsey, H. & Mekalanos, J. J. (1997). Regulation, replication, and integration functions of the Vibrio cholerae CTX are encoded by region RS2. Mol Microbiol 24, 917–926.[CrossRef][Medline]

WHO (1987). Manual for Laboratory Investigations of Acute Enteric Infections. Geneva: World Health Organization.

This article has been cited by other articles:

				D. M. Adin, K. L. Visick, and E. V. Stabb Identification of a Cellobiose Utilization Gene Cluster with Cryptic {beta}-Galactosidase Activity in Vibrio fischeri Appl. Envir. Microbiol., July 1, 2008; 74(13): 4059 - 4069. [Abstract] [Full Text] [PDF]

				N. Gonzalez-Escalona, B. Whitney, L.-A. Jaykus, and A. DePaola Comparison of Direct Genome Restriction Enzyme Analysis and Pulsed-Field Gel Electrophoresis for Typing of Vibrio vulnificus and Their Correspondence with Multilocus Sequence Typing Data Appl. Envir. Microbiol., November 15, 2007; 73(22): 7494 - 7500. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Safa, A. Articles by Nair, G. B. Search for Related Content PubMed PubMed Citation Articles by Safa, A. Articles by Nair, G. B. Agricola Articles by Safa, A. Articles by Nair, G. B.