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