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Journal of Clinical Microbiology, March 1998, p. 843-844, Vol. 36, No. 3
Institute of Microbial Technology,
Received 15 July 1997/Returned for modification 18 July
1997/Accepted 8 December 1997
We present molecular evidence that a distinct genotype of
Vibrio cholerae O1 which appeared in Calcutta, India, in
September 1993 and which is characterized by a unique ribotype that is
not found in the standardized ribotyping scheme of V. cholerae and that shows a specific pulsed-field gel
electrophoresis profile may have spread to the west African country of
Guinea-Bissau where it was responsible for an epidemic of cholera which
began in October 1994 and continued into 1996.
During the height of the epidemic
caused by Vibrio cholerae O139 in Calcutta, India, the El
Tor biotype of V. cholerae O1 was briefly displaced for a
period of 6 months between January and June 1993 (3). The El
Tor biotype, however, reappeared in July 1993 in Calcutta and
subsequently replaced the O139 serogroup in Calcutta and in most parts
of the Indian subcontinent. Molecular studies using pulsed-field gel
electrophoresis (PFGE) (9), ribotyping, and restriction
fragment length polymorphism of the CTX genetic element (5)
showed that the V. cholerae O1 strains which appeared
after the O139 epidemic in Calcutta showed distinct genotypes compared
with those of O1 strains isolated before the emergence of O139,
indicating that there is continuous genetic reassortment among the El
Tor strains. Genetic changes among O1 strains associated with the
cholera epidemic in South America have recently been reported
(2).
In this report, we present molecular evidence that a distinct genotype
of the O1 strain which appeared in Calcutta in September 1993 and which
is characterized by a unique ribotype (5) that is not found
in the standardized ribotyping scheme of V. cholerae (4) and that shows unique PFGE fragment patterns
(9) may have spread to the west African country of
Guinea-Bissau where it was responsible for an epidemic of cholera which
began in October 1994 and continued into 1996 (1, 7).
Ribotyping was performed by the procedure described by Dalsgaard
et al. (1) in which the restriction enzyme BglI
(Amersham, Arlington Heights, Ill.) was used to digest the
chromosomal DNA, and hybridization was performed with
digoxigenin-labeled 16S and 23S rRNA probes. A 1-kb DNA molecular
weight standard (GIBCO BRL, Gaithersburg, Md.) was used as the weight
marker. PFGE was carried out as described previously (2, 6).
Briefly, DNA was prepared directly in a solid agarose plug for
restriction endonuclease digestion with the enzyme
NotI (Amersham). PFGE was carried out by using a
CHEF-DR III system (Bio-Rad, Richmond, Calif.) with the following
conditions: 6 V/cm at 14°C for 22 h at a field angle of 120°.
The electrophoresis was carried out at switch times of 15 to 25 s
for 3 h and 8 to 25 s for 19 h. Multimeric phage lambda (48.5 kb) DNA (Pharmacia LKB, Uppsala, Sweden) was used as the molecular weight standard. Following electrophoresis, the gels were
stained for 15 min in ethidium bromide (2 µg/ml in water; Sigma),
destained in distilled water for 15 min, and visualized on a UV light
box.
As shown in Fig. 1, the BglI
cleavage pattern of a representative strain of the V. cholerae O1 El Tor biotype (strain CO 840; lane C) isolated after
the O139 epidemic in Calcutta showed eight bands. This ribotype pattern
exactly matches the pattern of a representative V. cholerae O1 El Tor strain recovered from cholera outbreaks in
Guinea-Bissau (strain 9868; lane E). The ribotype of the V. cholerae O1 strain causing cholera in Guinea-Bissau (1)
did not match the ribotypes of the African V. cholerae O1 isolates included in the standardized ribotyping scheme
(4). Although the representative O1 strains isolated in
Calcutta before and after the emergence of O139 only differed by a
single ribotype fragment (Fig. 1, lanes B and C), it was previously
shown that strains isolated before the O139 epidemic harbored two or
more copies of CTX in tandem and a "free" RS1 element located away from CTX, compared with a single copy of CTX and no free RS1 element in
the majority of O1 strains recovered after the emergence of O139
(5). Representative O1 strains from cholera outbreaks in
Guinea-Bissau in 1987 and in 1994 to 1996 showed ribotypes which
differed by four fragments (Fig. 1, lanes D and E). Dalsgaard et al.
(1) previously reported that strains from the two outbreaks in Guinea-Bissau also differed in CTX genotypes and antibiotic susceptibility patterns.
The results of the PFGE typing supported the results obtained by
ribotyping since identical PFGE typing patterns were shown by strains
CO 840 from Calcutta and 9868 from Guinea-Bissau (Fig. 2). Thus, the use of two different typing
methods strongly indicates that the two representative strains belong
to the same clone. The representative O1 strains isolated in Calcutta
before and after the emergence of O139 showed PFGE types which differed
by more than five fragments (Fig. 2).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Evidence that a Distinct Vibrio
cholerae O1 Biotype El Tor Strain in Calcutta May Have Spread to
the African Continent
ABSTRACT
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FIG. 1.
Ribotypes of V. cholerae O1 strains
associated with disease outbreaks in Calcutta and Guinea-Bissau. Lanes
(including, where applicable, strain designation, place of isolation,
and year of isolation): A, 1-kb molecular mass standard; B, VC 20, Calcutta, 1992; C, CO 840, Calcutta, 1995; D, 1407, Guinea-Bissau,
1987; E, 9868, Guinea-Bissau, 1996.
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FIG. 2.
PFGE fragment patterns of NotI-digested total
cellular DNAs from representative V. cholerae O1
strains isolated in Calcutta, India, and in Guinea-Bissau. Lanes
(including, where applicable, strain designation, place of isolation,
and year of isolation): A and F, multimers of phage lambda DNA (48.5 kb) as molecular size markers; B, VC 20, Calcutta, 1992; C, CO 840, Calcutta, 1995; D, 1407, Guinea-Bissau, 1987; E, 9868, Guinea-Bissau,
1996.
Chronologically, the El Tor biotype of V. cholerae O1 with this hitherto unreported ribotype and PFGE type appeared in Calcutta around September 1993 (5) when strains showing this genotype were sporadically associated with hospitalized patients with cholera. By March 1994, the incidence of this distinct V. cholerae O1 strain exceeded the incidence of the O139 serogroup in Calcutta, and its genotype subsequently became the dominant genotype in India. The distinct strain of V. cholerae O1 was possibly introduced into Guinea-Bissau in October 1994 where it was associated with an epidemic of cholera (1, 7). Therefore, it appears that the distinct O1 strain emerged in Calcutta around September 1993 and may subsequently have been introduced into Guinea-Bissau. Further studies should confirm the clonal relationship of strains associated with cholera in Calcutta and Guinea-Bissau. However, it appears that while it took nearly a decade for the 1961 El Tor clone to spread from the initial focus of the Celebes Islands to the African continent, the distinct O1 strain has spread in a matter of months from Calcutta to the west African coast. Strangely, even in 1970 when the seventh pandemic of cholera entered Africa, the epidemic started in Guinea and was caused by V. cholerae O1, biotype El Tor, serotype Ogawa, the source of which remained unknown (8). This report further underscores the usefulness of ribotyping and PFGE typing in tracing the pandemic spread of clones of V. cholerae.
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ACKNOWLEDGMENTS |
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Anders Dalsgaard was supported by the Danish Council for Development Research, Danish International Development Assistance (DANIDA), fellowship no. 90810.
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FOOTNOTES |
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* Corresponding author. Mailing address: National Institute of Cholera and Enteric Diseases, P-33, CIT Rd., Scheme XM, Beliaghata, Calcutta 700 010, India. Phone: 91-33-350-1176. Fax: 91-33-350-5066. E-mail: krishgb{at}giascl01.vsnl.net.in.
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REFERENCES |
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Journal of Clinical Microbiology, March 1998, p. 843-844, Vol. 36, No. 3
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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