Classical ctxB in Vibrio cholerae 01, 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 E1 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 jot 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 01, which possesses traits of both classical and E1 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 E1 Tot strains.

Our study analyzed, in chronological order, strains of E 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 O 1, which were selected to cover different months of each year, were included in this study, along with 2 reference strains for classical and E1 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 mis match amplification 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-specific primers, Re-cla (5'-CCTGGTACTTCTAC TTGAAACG-3') and Re-elt (5'-CCTGGTACTTCTACTTGAAA CA-3'), were used for classical and E1 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 E1 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 0395 (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.

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 E1 Tor allele of ctxB was present. Our results further indicate that classical type ctxB emerged in 1990, although E1 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 E1 Tor type ctxB. In 1994, isolation of strains with E1 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 E1 Tor type ctxB allele.

Replacement of E1 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 E1 Tor strains arose when V. cholerae strains with typical seventh pandemic E1 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 E1 Tor strains had CTX prophages of both classical and E1 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, E1 Tor and Calcutta (10). Further, 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,

This work was supported in part by the Indian Council of Medical Research, the Government of India, the Japan International Cooperation Agency (JICA/ NICED Project 054-1061-E-O), and the Program of Founding Research Center for Emerging and Reemerging infectious Diseases, Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

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(2.) Finkelstein RA, Burks F, Zupan A, Dallas WS, Jacob CO, Ludwig DS. Epitopes of the cholera family of enterotoxins. Rev Infect Dis. 1987;9:544-61.

(3.) Olsvik O, Wahlberg J, Petterson B, Uhlen M, Popovic T, Wachsmuth IK, et al. 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. 1993;31:22-5.

(4.) Nair GB, Faruque SM, Bhuiyan NA, Kamruzzaman M, Siddique AK, Sack DA. 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. 2002;40:3296-9. DOI: 10.1128/JCM.40.9.3296-3299.2002

(5.) Nair GB, Qadri F, Holmgren J, Svennerholm AM, Safa A, Bhuiyan NA, et al. Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J Clin Microbiol. 2006;44:4211-3. DOI: 10.1128/JCM.01304-06

(6.) Ansaruzzaman M, Bhuiyan NA, Nair GB, Sack DA, Lucas M, Deen JL, et al. Cholera in Mozambique, variant of Vibrio cholerae. Emerg Infect Dis. 2004;10:2057-9.

(7.) Sara A, Sultana J, Cam PD, Mwansa JC, Kong RYC. Classical cholera toxin producing Vibrio cholerae Ol hybrid E1 Tor strains in Asia and Africa. Emerg Infect Dis. 2008;14:987-8.

(8.) Morita M, Ohnishi M, Bhuiyan NA, Nusrin S, Alam M, Siddique AK, et al. Development and validation of a mismatch amplification mutation assay PCR to distinguish between the cholera toxin B subunit of classical and E1 Tor biotypes of Vibrio cholerae O1. Microbiol Immunol. 2008;52:314-7. DOI: 10.1111/j.1348-0421.2008.00041 .x

(9.) World Health Organization. Cholera, 2006. Wkly Epidemiol Rec. 2007;82:273-84.

(10.) Kimsey HH, Nair GB, Ghosh A, Walder MK. Diverse CTXos and evolution of new pathogenic Vibrio cholerae. Lancet. 1998;352:457-8. DOI: 10.1016/S0140-6736(05)79193-5

Amit Raychoudhuri, Tapas Patra, Kausik Ghosh, Thandavarayan Ramamurthy, Ranjan K. Nandy, Yoshifumi Takeda, G. Balakrish Nair, and Asish K. Mukhopadhyay

Author affiliations: National Institute of Cholera and Enteric Diseases, Kolkata, India (A. Raychoudhuri, T. Patra, K. Ghosh, T. Ramamurthy, R.K. Nandy, G.B. Nair, A.K. Mukhopadhyay); and Collaborative Research Center of Okayama University for Infectious Diseases in India, Kolkata (Y. Takeda)

DOI: 10.3201/eid1501.080543

Address for correspondence: Asish K. Mukhopadhyay, National Institute of Cholera and Enteric Diseases, P-33, CIT Rd, Scheme XM, Beliaghata, Kolkata--700010, India; email: [e-mail]

Table. Prevalence of different types of ctxB alleles among
Vibrio cholerae 01 strains, Kolkata, India, 1989-2005

                      No. alleles

             No.
Year        strains   Classical   El Tor   Classical +
isolated    tested      ctxB       ctxB    El Tor ctxB

1989           6          0         6           0
1990           7          4         3           0
1991          10          8         0           2
1992          10          4         5           1
1993           6          4         2           0
1994           9          8         1           0
1995          23         23         0           0
1996          10         10         0           0
1997          10         10         0           0
1998          10         10         0           0
1999          10         10         0           0
2000          10         10         0           0
2001          10         10         0           0
2002          10         10         0           0
2003          10         10         0           0
2004          10         10         0           0
2005          10         10         0           0

* These strains carry the ctxB gene for El Tor,
as well as classical strains.