Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand

Material & Methods

Bacterial strains: A total of 330 V. cholerae O1 strains (248 Ogawa, 82 Inaba) isolated from hospitalized patients with cholera in various regions of Thailand from 1986 to 2009 (Table I) were investigated. Nineteen V. cholerae O1 strains collected from Australia, Bangladesh, India, Peru, Romania and Thailand in different years were used as reference strains419 (Table II). Among them, 16 strains were obtained from the collection of the Laboratory Science Division, the International Centre for Diarrhoeal Disease Research of Bangladesh, Dhaka, Bangladesh; two strains (G27875 and SC11) were provided by Dr T. Ramamurthy, the National Centre of Cholera and Enteric Diseases, Kolkata, India; and one strain (295/33) was from the Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. All strains were subjected to conventional biotyping methods (susceptibility to 50 units of polymyxin B, CCA and VP test)20 using strains 569B and N16961 as the classical and El Tor reference strains, respectively.

Conventional- and MAMA-PCRs: All V. cholerae strains were examined for the presence of ctxA, ctxB, zot, ace, toxR, tcpA^C, tcpA^E, hlyA^C and hlyA^E by conventional PCR using strains AR15493 and AR15425 from Bangladesh as positive controls for zot, ace, toxR, and hlyA genes and strain C6706 as positive control for ctxAB and tcpA19. Conventional biotyping methods and a combination of MAMA- and conventional- PCRs were used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotypes, based on their ctxB and rstR genes21–23. Strains MJ1485 from Bangladesh and B33 from Mozambique served as hybrid biotype reference strains while G27875 and SC11 from NICED, India, were El Tor variant reference strains.

Primer sequences used in PCRs are shown in Table III19. Amplification mixture (25 μl) for ctxB-MAMA-PCR and rstR-PCR composed of 1 μl bacterial genomic DNA template, 2.5 μl 10× PCR buffer, 2 μl each of 2.5 mM deoxynucleotide triphosphate (Fermentas, Vilnius, Lithuania), 2 μl of 25 mM MgCl₂, 2 μl of 10 μM of individual forward and reverse primers (Bio Basic Inc., Toronto, Canada), 0.5 units Taq DNA polymerase (Fermentas) and sterile ultra pure distilled water. Amplification of other genes was essentially the same as described previously19. The PCR products were analyzed by using 1.5 per cent agarose (Seakem LE, BMA, Glendate, CA, USA) gel electrophoresis and ethidium bromide staining (Sigma Chemical Co., USA). A Gel Doc 2000 (Bio-Rad, CA, USA) was used for DNA band documentation.

Results & Discussion

All of the 330 V. cholerae O1 Thai clinical strains collected over 24 years (1986-2009) were found to carry ctxA, ctxB, zot, ace, toxR, tcpA^E and hlyA^E which verified genetically their toxin producing capacity and epidemic potential. Two hundred and sixty six strains were prototype El Tor (resistant to the polymyxin B, and positive for CCA and VP test) and the remaining 64 strains were not biotypable (Table I).

Identification of rstR by conventional PCR showed that the 36 strains of 1986-1992 carried either the El Tor rstR (rstR^E/C) or combination of the El Tor and classical rstR (rstR^E/C) (Table I). MAMA-PCR for ctxB of these isolates revealed that 18 (50%) carried ctxB^E. Only 15 of these 18 strains had prototype El Tor phenotype (resistant to 50 units of polymyxin B, and positive for CCA and VP test) indicating that they were typical El Tor biotype. The other 3 strains, although carrying ctxB^E, appeared to be hybrid biotype as they possessed mixed phenotypes (Table I and IV). There were 11 strains of 1986-1992 (31%) that carried ctxB^E/C. Among these only one strain had mixed classical and El Tor phenotypes implying that this was hybrid biotype. The remaining 10 with ctxB^E/C, however, could not be assigned into any of the redefined biotype scheme16 although these showed conventional El Tor phenotype (Tables I and IV. The remaining seven (19%) of the 1986-1992 (all were isolated in 1992) strains carried ctxB ^C; four of these had conventional El Tor phenotypes implying that these were El Tor variant while the other three had mixed phenotypes, and were hybrid (Table I). These data indicate the presence of hybrid biotype of V. cholerae O1 in Thailand since 1986 or even before and these co-existed with the typical El Tor strains. The V. cholerae O1 Thailand strains that carried ctxB^E/rstR^Ei.e., typical El Tor strains, were found for the last time in 1992 in this V. cholerae O1 collection which was the same year when the strains of El Tor variant biotype (strains 30-33) carrying ctxB^C/rstR^E/C emerged in the country (Table I). It is noteworthy that in 1992 the epidemic V. cholerae O139 strains emerged in Southern India11. The Fig. shows MAMA-PCR results of representative strains of V. cholerae chronologically isolated in Thailand i.e., ctxB^C (Fig. A) and ctxB^E (Fig. B).

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Fig.

Results of MAMA-PCR for amplification of ctxB^C (A) and ctxB^E (B) from representative V. cholerae strains isolated in Thailand during 1986-2009. Lanes 2-6, 1986 strains; lane 7, 1987 strains; lanes 8-9, 1989 strains; lanes 10-22, 1990 strains; lanes 23-26, 1991 strains; lanes 27-36, 1992 strains and lanes 37-56, 1993-2009 strains. Lane M, 100 bp DNA marker. Lane 1 in (A), positive control of ctxB^C (569B); lane 1 in (B), positive control of ctxB^E (N16961).

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The V. cholerae O1 Thailand strains of 1993-2009 (294) were all found to carry ctxBC and either rstR^C or rstR ^E/C. Majority of these strains (237 strains), however, were El Tor variants as their phenotypes were typical El Tor. The minority (57 strains) belonged to hybrid biotype because these had mixed phenotypes of classical and El Tor (Table I). The 1986-2009 Thailand strains with hybrid biotype could be arbitrarily classified into 13 different hybrid groups, 1-13 (Table IV). During 1986-1992, the biotypes of the 36 V. cholerae O1 Thailand strains were 15 prototype El Tor, 7 hybrid (groups 1-5), 4 El Tor variant, and 10 unclassified (unclassified groups 1 and 2) (Tables I and IV). The 294 strains of 1993-2009 belonged to hybrid groups 6-13 (57 strains) and El Tor variants (237 strains) (Tables I and IV).

The V. cholerae O1 of hybrid biotype was isolated from patients in India in 1991 when typical V. cholerae classical and El Tor biotypes co-existed suggesting the horizontal CTX prophage exchange between strains of the two principal biotypes in order for the infecting strains to be more adapted to the host hostile intestinal environment15 which conformed to the more severe cholera symptoms in the afflicted hosts in the recent years32224. It is noteworthy, however, that the classical V. cholerae O1 disappeared from Thailand since 196325 when the 7^th cholera pandemic caused by typical El Tor strains first hit the Kingdom’s population. There has been no report on the period of co-existing classical and El Tor strains during 1986-2009 within Thailand. Our finding that the V. cholerae hybrid biotype could be detected among strains of 1986 suggested that there might be a re-emergence of the classical V. cholerae before or during 1986 or there might be other confounding molecular mechanism(s) in the shifting of the characteristics of V. cholerae bacteria in Thailand. The speculations warrant detail investigation. In 1992, the epidemic O139 strains emerged in India concurrent with the finding of El Tor variant in Thailand for the first time in this series of strain collection (Table I). Between 1992 and 1993, the V. cholerae O1 strains carrying ctxB^C predominated in Kolkata, India15 and Thailand (this study). Thus, there seemed to be incomprehensible event of genetic evolution of the V. cholerae yielding strains of mixed traits/phenotypes of the two authentic biotypes during this period. After 1994, isolates of V. cholerae O1 in Kolkata, India, seemed to carry only ctxB^C; thus these were El Tor variants or hybrids (no phenotypes were given to define the biotype)16. Similarity was found among the Thailand strains of this study, however, two years earlier than the Kolkata’s series. All of the Thai strains after 1992 carried ctxB^C of which 57 (19%) were hybrid biotype and 237 strains (81%) were El Tor variants according to the conventional biotyping method and MAMA- and conventional- PCR determinations. In Punjab and Haryana, northern India, where a re-emergence of classical V. cholerae has not been reported, the V. cholerae hybrid biotype were also found in 2007 (80% of the isolates)26. As has been mentioned earlier, many V. cholerae isolates of several other countries in Asia and Africa were also found to be biotype hybrid/El Tor variant15 indicating that the El Tor V. cholerae bacteria, regardless of the geographical areas, tend to evolve for acquisition of the classical CTX prophage. This phenomenon will have impact, more or less, on the treatment of cholera, public health measures, as well as vaccine development.