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Journal of Clinical Microbiology, December 2002, p. 4779-4781, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4779-4781.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of a Toxigenic Vibrio cholerae O139 Strain Belonging to a New Ribotype and Isolated from a Diarrheal Patient

R. Bhanumathi,1 F. Sabeena,1 Sree Renjini Isac,1 G. Radhakutty,2 and D. V. Singh1*

Rajiv Gandhi Center for Biotechnology, Poojappura, Thiruvananthapuram 695 014, Kerala,1 Department of Microbiology, T. D. Medical College, Alleppey 688 005, Kerala, India2

Received 19 April 2002/ Returned for modification 21 June 2002/ Accepted 21 September 2002


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ABSTRACT
 
We characterized a Vibrio cholerae O139 strain isolated from a diarrheal patient admitted to Taluk Hospital, Cherthala, Alleppey, Kerala, India, on 9 June 2000. The V. cholerae O139 strain possesses the core of the CTX genetic element, colonization toxin-coregulated pilus, the adherence outer membrane protein, and the central regulatory protein encoded by toxR and produces cholera toxin (200 pg/ml). We provide molecular evidence showing that toxigenic V. cholerae O139 strain ALO95 belongs to a distinct genotype characterized by a unique ribotype designated B-VII and has a unique enterobacterial repetitive intergenic consensus sequence PCR fingerprint profile designated E-V.


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TEXT
 
Vibrio cholerae belonging to serogroup O1 biotype El Tor was considered the causative agent of diarrhea until the emergence of V. cholerae O139 Bengal in 1992. This organism caused an explosive epidemic in India and Bangladesh and subsequently in neighboring countries (1, 9, 10). Molecular studies using pulsed-field gel electrophoresis (8), ribotyping (4), and restriction fragment length polymorphism of the CTX genetic element of V. cholerae O139 Bengal have demonstrated the emergence of new clones with temporal changes in phenotypic and genetic properties (2, 4). During the height of the V. cholerae O1 biotype El Tor outbreak in Kerala, India, in the year 2000, we isolated V. cholerae O139 Bengal strain ALO95 from a 66-year-old female diarrheal patient admitted to the Taluk Hospital, Cherthala, Alleppey, Kerala, India, on 9 June 2000.

In this report, we present molecular evidence that toxigenic V. cholerae O139 strain ALO95 belongs to a distinct genotype characterized by a unique ribotype and has a unique enterobacterial repetitive intergenic consensus sequence (ERIC) PCR fingerprint profile.

A V. cholerae strain was isolated from a stool sample from a diarrheal patient and identified by using standard bacteriologic techniques (16). This strain agglutinated with monoclonal O139 antiserum supplied by the World Health Organization Regional Office of Southeast Asia, New Delhi, India, and was confirmed to belong to V. cholerae serogroup O139.

A hexaplex PCR was used to determine the presence of virulence and regulatory genes, including ctxA, zot, ace, tcpA, ompU, and toxR, as described earlier (12). Briefly, the amplification program began with initial denaturation at 94°C for 2 min, followed by 20 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min and 10 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min. A final extension was done at 72°C for 10 min. V. cholerae O1 serotype Inaba biotype classical strain 569B and V. cholerae O1 serotype Ogawa biotype El Tor strain 20 were used as the PCR positive controls for ctxA, zot, ace, tcpA, ompU, and toxR. Aliquots of PCR products were analyzed by agarose (1.8%, wt/vol) gel electrophoresis in 0.5x Tris-borate-EDTA buffer, stained in ethidium bromide, and visualized with a Fluoro-S MultiImager (Bio-Rad, Inc.).

A monosialoganglioside (GM1) enzyme-linked immunosorbent assay was used to examine cholera toxin production in V. cholerae O139 strain ALO95 by the method of Svennerholm and Holmgren (14). Briefly, enzyme-linked immunosorbent assay plates were coated with GM1 (Sigma) at a concentration of 2 µg/ml in Tris-HCl buffer (pH 7.5) overnight at 4°C, washed three times with phosphate-buffered saline (PBS)-0.05% Tween 20 (Sigma), blocked with PBS-1.0% bovine serum albumin (BSA; protease free; Sigma) for 2 h at 37°C, and washed again three times. Cell culture supernatant was added, and the plates were incubated for 2 h at 37°C and washed again. Rabbit anti-cholera toxin antiserum (primary antibody; Sigma) diluted 1:10,000 in PBS-0.05% Tween 20-0.5% BSA was added. The plates were incubated for 1 h at 37°C, washed three times again, incubated for 1 h at 37°C with goat anti-rabbit immunoglobulin G-peroxidase conjugate (secondary antibody; Sigma) diluted 1:5,000 in PBS-0.05% Tween 20-0.5% BSA, washed again three times each with PBS-Tween 20 and PBS, and incubated with the TMB substrate system (ready to use; Sigma) for 30 min. The A405 was then read in a model 550 microplate reader (Bio-Rad).

Ribotyping was performed by the procedure described by Faruque et al. (4), in which the restriction enzyme BglI (Amersham Pharmacia Biotech) was used to digest the chromosomal DNA extracted by the cetyltrimethylammonium bromide (Sigma) method described earlier (13) and hybridization was performed with [{alpha}-32P]dCTP (3,000 Ci/mmol; Bhabha Atomic Research Centre, Bombay, India)-labeled 16S and 23S rRNA probes. The ERIC-PCR assay was carried out as described by Rivera et al. (11), with two oligonucleotide primers (5'-ATG TAA GCT CCT GGG GAT TCA C-3' and 5'-AAG TAA GTG ACT GGG GTG AGC G-3'). The amplicons were electrophoresed in 1.8% (wt/vol) agarose at 80 V for 6 h and stained in ethidium bromide. The fingerprint pattern was digitized in a Fluoro-S MultiImager (Bio-Rad, Inc.).

V. cholerae O139 strain ALO95 was tested for antibiotic resistance by the method of Bauer et al. (3) with the antibiotics (Hi-Media Laboratories, Bombay, India) ampicillin (10 µg), chloramphenicol (30 µg), cefotaxime (30 µg), ciprofloxacin (5 µg), cephalexin (30 µg), co-trimoxazole (25 µg), furazolidone (100 µg), gentamicin (10 µg), neomycin (30 µg), norfloxacin (10 µg), polymyxin B (50 U), streptomycin (30 µg), nalidixic acid (30 µg), and tetracycline (30 µg) and the vibriostatic agent pteridine (10 and 150 µg).

With the hexaplex PCR assay, V. cholerae O139 strain ALO95 gave positive results for the ctxA, zot, ace, tcpA, ompU, and toxR genes. The cholera toxin concentration produced by V. cholerae O139 strain ALO95 was 200 pg/ml.

The BglI cleavage patterns of rRNA genes of representative strains of V. cholerae O139 showed four different patterns (I, II, IV, and VII). The restriction patterns consisted of 8 to 10 9- to 2.2-kb bands (Fig. 1). The ribotype of V. cholerae O139 strain ALO95 isolated from a diarrheal patient did not match the ribotypes of the V. cholerae O139 strains of the standardized ribotyping scheme (4). The restriction pattern representing a new ribotype designated B-VII contained a unique 1.8-kb band not present in any of the other restriction patterns. The restriction pattern produced by V. cholerae O139 strain ALO95 has not been reported by us or other investigators (4) who have analyzed a large number of V. cholerae O139 strains. This strain showed resistance to ampicillin, cefotaxime, furazolidone, nalidixic acid, and streptomycin and was sensitive to pteridine.



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  		FIG. 1. Southern hybridization analysis of rRNA genes of V. cholerae O139 strains. Genomic DNA was digested with BglI and probed with a 7.5-kb BamHI fragment of Escherichia coli rRNA clone pKK3535. Restriction patterns corresponding to ribotypes derived from laboratory collection strains are shown in lanes 1 and 2 (B-I), 3 and 4 (B-II), and 5 (B-IV), and the pattern corresponding to a new ribotype, B-VII, derived from V. cholerae O139 strain ALO95, isolated from a diarrheal patient, is shown in lane 6.

The ERIC-PCR fingerprint profile results supported those obtained by ribotyping because V. cholerae O139 strain ALO95 showed a distinct PCR fingerprint profile. The representative V. cholerae O139 strains showed five ERIC-PCR fingerprint profiles (I through V) consisting of 4 to 15 0.31- to 3.5-kb bands (Fig. 2). The fingerprint profile of V. cholerae O139 strain ALO95 isolated from a diarrheal patient did not match the ERIC-PCR fingerprint profiles previously noted by us (unpublished observation). The ERIC-PCR profiles representing a new ERIC genotype designated E-V lack a 0.85-kb band and possess 0.71-, 1.02-, 1.13-, and 1.64-kb bands (not present in E-I). Thus, results obtained with two different typing methods strongly indicate that toxigenic V. cholerae O139 strain ALO95 belongs to ribotype B-VII and that ERIC genotype E-V represents a new clone.



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  		FIG. 2. DNA fragments of V. cholerae O139 strains generated by ERIC-PCR amplification. Fingerprints corresponding to ERIC genotypes E-I through E-IV derived from laboratory collection strains are shown in lanes 1 through 4, and the fingerprint corresponding to a new ERIC genotype, E-V, derived from V. cholerae O139 strain ALO95, isolated from a diarrheal patient, is shown in lane 5.

Epidemiological surveillance of cholera caused by V. cholerae O139 demonstrated the emergence of new clones of V. cholerae O139. Seven ribotypes, B-I through B-VI and NB-1 (derived from a nontoxigenic strain isolated in Argentina in 1993), have been described recently among V. cholerae O139 strains (4). During the initial outbreak of O139 vibrios in 1992 and 1993, only two ribotypes, designated B-I and B-II, were involved. Ribotype B-III was reported recently and found to be associated with an outbreak of cholera in two north central districts of Bangladesh (5). It is interesting that a single strain (MDO2) belonging to this ribotype was isolated in the southern part of India (Madurai) as far back as 1993 but was not associated with an epidemic outbreak (4).

Ribotyping has been successfully used to study clonal diversity among V. cholerae O139 strains, and the enzyme BglI is more useful for discrimination of strains than are other enzymes used for ribotyping (15). The results of this study indicate that toxigenic V. cholerae O139 strain ALO95 belonging to a new ribotype, B-VII, and ERIC genotype E-V carries the virulence-associated genes necessary for an epidemic strain (7). It is known that V. cholerae O139 can exist in the environment in association with plankton in a viable but nonculturable state (6). It may be possible that the new clone of V. cholerae O139, like V. cholerae O139 strain MDO2, belonging to ribotype B-III, may be present in the aquatic environment in a viable but nonculturable state and, under hitherto unknown favorable environmental conditions, may grow selectively to cause an explosive epidemic. To elucidate this hypothesis, further ecological, epidemiological, and bacteriological investigations are required.


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ACKNOWLEDGMENTS
 
This research was supported by funds contributed by the Department of Biotechnology, New Delhi, India, to the Rajiv Gandhi Centre for Biotechnology. Junior research fellowships awarded by the Council of Scientific and Industrial Research, New Delhi, India, to R. Bhanumathi and Sree Renjini Isac are gratefully acknowledged.

We thank D. Karunagaran for critically reading the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Rajiv Gandhi Center for Biotechnology, Thycaud P.O., Poojappura, Thiruvananthapuram 695 014, Kerala, India. Phone: 91 471 345 899. Fax: 91 472 348 096. E-mail: durg-singh{at}mailcity.com. Back


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Journal of Clinical Microbiology, December 2002, p. 4779-4781, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4779-4781.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mohapatra, S. S., Ramachandran, D., Mantri, C. K., Singh, D. V. (2007). Characterization of the genetic background of Vibrio cholerae O1 biotype El Tor serotype Inaba strains isolated in Trivandrum, southern India. J Med Microbiol 56: 260-265 [Abstract] [Full Text]  
  • Singh, D. V., Bhanumathi, R., Colwell, R. R. (2004). Characterization of a Cholera Toxin Gene-Negative Clinical Strain of Vibrio cholerae O139 Bengal. J. Clin. Microbiol. 42: 1381-1381 [Full Text]  

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