Molecular Evidence that a Distinct Vibrio cholerae O1 Biotype El Tor Strain in Calcutta May Have Spread to the African Continent

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 enzymeNotI (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. choleraeO1 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.

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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.

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).

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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. choleraeO1, 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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