Classical ctxB in Vibrio cholerae O1, Kolkata, India

To the Editor: Among the 206 serogroups of Vibrio cholerae, O1 and O139 are associated with epidemic cholera. Serogroup O1 is classified into 2 biotypes, classical and El Tor. Conventionally, the 2 biotypes can be differentiated on the basis of a set of phenotypic traits. Comparative genomic analysis has shown variations in different genes between these biotypes (1). Cholera toxin (CT), the major toxin responsible for the disease cholera, has 2 epitypes or immunologic forms, CT1 and CT2 (2). Another classification recognizes 3 genotypes on the basis of the ctxB gene sequence variation (3). In the past few years, a new emerging form of V. cholerae O1, which possesses traits of both classical and El Tor biotypes, has been isolated in Bangladesh (4,5), Mozambique (6), Vietnam, Hong Kong, Japan, and Zambia (7). These strains were variously labeled as Matlab variants, hybrids, or altered El Tor strains. 
 
Our study analyzed, in chronological order, strains of V. cholerae O1 that were isolated over 17 years (1989–2005). We used strains isolated during diarrhea surveillance conducted at the Infectious Diseases Hospital, Kolkata (Calcutta), to determine precisely when the hybrid strains appeared in this region. A total of 171 strains of V. cholerae O1, which were selected to cover different months of each year, were included in this study, along with 2 reference strains for classical and El Tor biotypes. The V. cholerae strains were confirmed serologically by slide agglutination using a specific polyvalent antiserum to V. cholerae O1. 
 
We focused on the ctxB gene. The strains were examined by mismatch amplification mutation assay (MAMA)–based PCR for detecting the ctxB allele; a common forward primer was used for 2 alleles, FW-Com (5′-ACTATCTTCAGCATATGCACATGG-3′); and 2 allele-specific primers, Re-cla (5′-cctggtacttctacttgaaacg-3′) and Re-elt (5′-CCTGGTACTTCTACTTGAAACA-3′), were used for classical and El Tor biotypes, respectively (8). Results of the MAMA-PCR are summarized in the Table. All of the 123 V. cholerae O1 strains from 1995 through 2005 yielded only the classical type of ctxB, which indicates that since 1995 the classical type has completely replaced the El Tor type ctxB (Table). To reconfirm our PCR-based results, we selected 25 representative strains for DNA sequencing of the ctxB gene. The deduced amino acid sequences were aligned with the CtxB sequences of reference strains N16961 (El Tor) and O395 (classical). The deduced amino acid sequences of all 25 strains were identical to those of the classical reference strain; histidine was at position 39 and threonine was at position 68. Thus, the results from DNA sequencing of the ctxB gene confirmed the MAMA-PCR results. 
 
 
 
Table 
 
Prevalence of different types of ctxB alleles among Vibrio cholerae O1 strains, Kolkata, India, 1989–2005 
 
 
 
Our results highlight a noteworthy event in the evolution of recent V. cholerae strains. Analysis of type ctxB that had been circulating in Kolkata for 17 years (1989–2005) showed that in 1989 only the El Tor allele of ctxB was present. Our results further indicate that classical type ctxB emerged in 1990, although El Tor type ctxB was still present in almost equal numbers during that year. During 1991, a unique event took place when the classical type became predominant, along with strains having both classical and El Tor type ctxB. In 1994, isolation of strains with El Tor ctxB became rare, and the major ctxB allele was of the classical type. V. cholerae O1 strains from 1995 onward were found to carry classical type ctxB, which totally replaced the El Tor type ctxB allele. 
 
Replacement of El Tor type ctxB by the classical allele has been reported in Bangladesh since 2001 (5), but this event seems to have occurred earlier in Kolkata. Perhaps the new type of El Tor strains arose when V. cholerae strains with typical seventh pandemic El Tor genetic background were replaced with strains having the ctxB gene, possibly driven by selective pressure to survive and adapt better in host intestines. Considering the increase in the global prevalence of cholera (9), the origin and spread of these new variants of V. cholerae strains should be tracked in the population by genome analysis. Finally, this study has described a brief period from February 1991 through December 1992 when El Tor strains had CTX prophages of both classical and El Tor types (data not shown), along with the ctxB of both biotypes. Notably, this period coincided with an unprecedented event in the history of cholera—the genesis of the O139 serogroup. After this serogroup reemerged in 1996, it harbored 2 types of CTX prophages, namely, El Tor and Calcutta (10). Furthermore, these strains with ctxB of both biotypes might also have had a pivotal role behind the emergence of El Tor strains with classical ctxB. Further studies are warranted to determine whether any distinct relationship exists between these overlapping events.


Classical ctxB in Vibrio cholerae O1, Kolkata, India
To the Editor: Among the 206 serogroups of Vibrio cholerae, O1 and O139 are associated with epidemic cholera. Serogroup O1 is classifi ed into 2 biotypes, classical and El Tor. Conventionally, the 2 biotypes can be differentiated on the basis of a set of phenotypic traits. Comparative genomic analysis has shown variations in different genes between these biotypes (1). Cholera toxin (CT), the ma-jor toxin responsible for the disease cholera, has 2 epitypes or immunologic forms, CT1 and CT2 (2). Another classifi cation recognizes 3 genotypes on the basis of the ctxB gene sequence variation (3). In the past few years, a new emerging form of V. cholerae O1, which possesses traits of both classical and El Tor biotypes, has been isolated in Bangladesh (4,5), Mozambique (6), Vietnam, Hong Kong, Japan, and Zambia (7). These strains were variously labeled as Matlab variants, hybrids, or altered El Tor strains.
We focused on the ctxB gene. The strains were examined by mis-match amplifi cation mutation assay (MAMA)-based PCR for detecting the ctxB allele; a common forward primer was used for 2 alleles, FW-Com (5′-ACTATCTTCAGCATATGCACAT-GG-3′); and 2 allele-specifi c primers, Re-cla (5′-CCTGGTACTTCTAC TTGAAACG-3′) and Re-elt (5′-CCTGGTACTTCTACTTGAAA CA-3′), were used for classical and El Tor biotypes, respectively (8). Results of the MAMA-PCR are summarized in the Table. All of the 123 V. cholerae O1 strains from 1995 through 2005 yielded only the classical type of ctxB, which indicates that since 1995 the classical type has completely replaced the El Tor type ctxB (Table). To reconfi rm our PCR-based results, we selected 25 representative strains for DNA sequencing of the ctxB gene. The deduced amino acid sequences were aligned with the CtxB sequences of reference strains N16961 (El Tor) and O395 (classical). The deduced amino acid sequences of all 25 strains were identical to those of the classical reference strain; histidine was at position 39 and threonine was at position 68. Thus, the results from DNA sequencing of the ctxB gene confi rmed the MAMA-PCR results.

LETTERS
Our results highlight a noteworthy event in the evolution of recent V. cholerae strains. Analysis of type ctxB that had been circulating in Kolkata for 17 years (1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005) showed that in 1989 only the El Tor allele of ctxB was present. Our results further indicate that classical type ctxB emerged in 1990, although El Tor type ctxB was still present in almost equal numbers during that year. During 1991, a unique event took place when the classical type became predominant, along with strains having both classical and El Tor type ctxB. In 1994, isolation of strains with El Tor ctxB became rare, and the major ctxB allele was of the classical type. V. cholerae O1 strains from 1995 onward were found to carry classical type ctxB, which totally replaced the El Tor type ctxB allele.
Replacement of El Tor type ctxB by the classical allele has been reported in Bangladesh since 2001 (5), but this event seems to have occurred earlier in Kolkata. Perhaps the new type of El Tor strains arose when V. cholerae strains with typical seventh pandemic El Tor genetic background were replaced with strains having the ctxB gene, possibly driven by selective pressure to survive and adapt better in host intestines. Considering the increase in the global prevalence of cholera (9), the origin and spread of these new variants of V. cholerae strains should be tracked in the population by genome analysis. Finally, this study has described a brief period from February 1991 through December 1992 when El Tor strains had CTX prophages of both classical and El Tor types (data not shown), along with the ctxB of both biotypes. Notably, this period coincided with an unprecedented event in the history of cholera-the genesis of the O139 serogroup. After this serogroup reemerged in 1996, it harbored 2 types of CTX prophages, namely, El Tor and Calcutta (10). Further, these strains with ctxB of both biotypes might also have had a pivotal role behind the emergence of El

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