Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.
New V. cholerae O1 variants carrying mixed classical and El Tor phenotypes
were first isolated from hospitalized patients with severe watery diarrhoea in Matlab, Bangladesh, in 2002 (3). These isolates could not be allocated into the classical or El Tor biotype using conventional biotyping tests. Genotypically, these were found to carry the El Tor genome backbone including El Tor specific gene clusters: VSP-I and -II and RTX, indicating that these belonged to El Tor lineage. These isolates carried different combinations of alleles of tcpA and CTX prophage repressor gene (rstR) (4). Their classical biotype characteristic was due to the presence of the classical CTX prophage and the deduced amino acids of the nucleotide sequence coding for cholera toxin B subunit belonged to classical biotype. Similar strains were isolated in Mozambique in 2004 (14). Subsequently, V. cholerae O1 El Tor variants have been reported from several Asian countries including China, Japan, Hong Kong, Sri Lanka, and Vietnam and Africa (Zambia) (15). In a retrospective study of V. cholerae strains isolated in Kolkata, India, during a 17 year period (1989-2005), using mis-match amplification mutation assay (MAMA)-PCR for determining ctxB alleles, it was revealed that the El Tor strains carrying ctxB allele of the classical biotype ([ctxB.sup.C]) have emerged since 1991 and co-existed with the prototype El Tor strains until 1995 when these completely replaced the typical El Tor biotype. Arbitrarily, the V. cholerae O1 strains carrying mixed phenotypes of classical and El Tor biotypes [polymyxin B (50 units) susceptibility and positive for chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test] are designated hybrid biotype where as the V. cholerae O1 with typical El Tor phenotypes (resistant to 50 units of polymyxin B, and positive for CCA and VP test) but carrying [ctxB.sup.C] are designated El Tor variant (16). This nomenclature has been followed in this study.
The 7th pandemic cholera arrived in Thailand in 1963, when the El Tor strains completely replaced the classical vibrios and established endemicity (17). The O139 Bengal was first isolated from hospitalized patient with severe watery diarrhoea in Thailand in 1993 (18). The O139 serogroup completely disappeared from Thailand since 1996 (17). Because it is known that classical V. cholerae strains with [ctxB.sup.C] inflicted more severe symptoms than the typical El Tor infection (6,16) and because there had been a resurgence of cases of severe watery diarrhoea that required hospitalization during 1999-2002, it was of interest to make an insight into both phenotypic and genotypic characteristics of V. cholerae O1 isolated from cholera patients in different years 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 strains (4,19) (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.sup.C], [tcpA.sup.E], [hlyA.sup.C] and [hlyA.sup.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 tcpA (19). 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 genes (21-23). 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 III (19). Amplification mixture (25 [micro]l) for ctxB-MAMA-PCR and rstR-PCR composed of 1 [micro]l bacterial genomic DNA template, 2.5 [micro]l 10x PCR buffer, 2 ul each of 2.5 mM deoxynucleotide triphosphate (Fermentas, Vilnius, Lithuania), 2 [micro]l of 25 mM Mg[Cl.sub.2], 2 [micro]l of 10 [micro]M 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 previously (19). 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.sup.E] and [hlyA.sup.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.sup.E]) or combination of the El Tor and classical rstR ([rstR.sup.E/C]) (Table I). MAMA-PCR for cttxB of these isolates revealed that 18 (50%) carried [ctxB.sup.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.sup.E], appeared to be hybrid biotype as they possessed mixed phenotypes (Tables I and IV). There were 11 strains of 1986-1992 (31%) that carried [ctxB.sup.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.sup.E]/C, however, could not be assigned into any of the redefined biotype scheme (16) 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.sup.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.sup.E]/ [rstR.sup.E] i.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.sup.C]/[rstR.sup.E/C] emerged in the country (Table I). It is noteworthy that in 1992 the epidemic V. cholerae O139 strains emerged in Southern India (11). The Fig. shows MAMA-PCR results of representative strains of V. cholerae chronologically isolated in Thailand i.e., [ctxB.sup.C] (Fig. A) and [ctxB.sup.E] (Fig. B).
The V. cholerae O1 Thailand strains of 1993-2009 (294) were all found to carry [ctxB.sup.C] and either [rstR.sup.C] or [rstR.sup.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 environment (15) which conformed to the more severe cholera symptoms in the afflicted hosts in the recent years (3,22,24). It is noteworthy, however, that the classical V. cholerae O1 disappeared from Thailand since 1963 (25) when the 7th 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.sup.C] predominated in Kolkata, India (15) 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.sup.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.sup.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 variant (15) 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.
The work was co-supported by the National Research University project of Thailand Office of Higher Education Commission (CHE) through Center for Biopharmaceutical Development and Innovative Therapy, Mahidol University and CHE RG 490329, the Thailand Research Fund (TRF; DPG5380001) and the Japan Health Science Foundation, Japan. P. Srimanote, N. Indrawattana, and N. Sookrung received research support from TRF.
Received February 12, 2010
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M. Na-Ubol, P. Srimanote , M. Chongsa-nguan, N. Indrawattana, N. Sookrung , P. Tapchaisri , S. Yamazaki , L. Bodhidatta , B. Eampokalap , H. Kurazono , H. Hayashi , G.B. Nair , Y. Takeda  & W. Chaicumpa 
Department of Microbiology & Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Graduate Studies, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand,  Office for Research & Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand,  Department of Veterinary Science, Graduate School of Life & Environmental Sciences, Osaka Prefecture University, Osaka, Japan,  Department of Enteric Diseases, Armed Force Research Institute of Medical Science, US Army Component, Bangkok, Thailand;  Bamrasnaradura Institute, Nonthaburi, Thailand,  Obihiro University of Agriculture & Veterinary Medicine, Department of Animal & Food Hygiene, Hokkaido, Japan,  Department of Microbiology & Nutrition, Chugoku-gakuen University, Okayama, Japan,  National Institute of Cholera & Enteric Diseases, Kolkata, India &  Department of Parasitology & Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Reprint requests: Dr Wanpen Chaicumpa, Emeritus Professor, Department of Parasitology, Faculty of Medicine Siriraj Hospital Mahidol University, Bangkok 10700, Thailand e-mail: email@example.com
Table I. V. cholerae O1 strains isolated from Thailand during 1986-2009 Year of Strain no. Serotype Phenotype Genotype isolation (n) PB CCA VP ctxB rstR 1986 (5) 1-2 Inaba R + + E E 3 Inaba R + + E E+C 4 Inaba S - + E E 5 Inaba R + + E+C E 1987 (1) 6 Inaba R + + E E 1989 (2) 7 Inaba R - + E+C E+C 8 Inaba S + + E E+C 1990 (13) 9-12 Inaba R + + E E+C 13-16 Ogawa R + + E E 17-18 Inaba R + + E+C E+C 19-21 Ogawa R + + E+C E 1991 (4) 22 Ogawa R + + E E 23-25 Ogawa R + + E+C E 1992 (11) 26 Inaba R + + E E+C 27 Inaba S + + E E 28 Ogawa R + + E E 29 Ogawa R + + E+C E 30-33 Ogawa R + + C E+C 34-36 Ogawa R - + C E+C 1993(9) 37-38 Inaba R + + C E+C 39-43 Ogawa R + + C E+C 44 Ogawa R + - C E+C 45 Ogawa R + + C C 1994 (7) 46 Inaba R + - C E+C 47-51 Ogawa R + + C E+C 52 Ogawa S + + C E+C 1995 (11) 53-62 Ogawa R + + C E+C 63 Ogawa R + - C E+C 1996 (3) 64-65 Ogawa R + + C E+C 66 Ogawa S + + C E+C 1997 (3) 67 Ogawa R + + C E+C 68-69 Ogawa R + + C C 1998(2) 70-71 Ogawa R + + C C 1999 (179) 72-78 Inaba R + + C C 79-83 Ogawa R + + C E+C 84-85 Ogawa R + - C E+C 86-115 Ogawa R + - C C 116-247 Ogawa R + + C C 248 Ogawa R - + C C 249 Ogawa R - - C C 250 Ogawa S + + C C 2000 (21) 251-270 Ogawa R + + C C 271 Ogawa R + - C C 2001 (27) 272-294 Inaba R + + C C 295-298 Inaba R + - C C 2002 (13) 299-306 Inaba R + + C C 307 Inaba R + - C C 308-310 Inaba S + + C C 311 Ogawa R + + C C 2003 (8) 312-315 Inaba R + + C C 316 Inaba R + - C C 317 Inaba S + + C C 318 Inaba S + - C C 319 Inaba S - + C C 2004 (9) 320-324 Inaba R + + C C 325-327 Inaba R + - C C 328 Inaba S + - C C 2009 (2) 329-330 Ogawa R + + C C Year of Strain no. Biotype Number of isolation (n) (see also Table IV) strain(s)/total number of strain(s) of the year 1986 (5) 1-2 El Tor 2/5 3 El Tor 1/5 4 Hybrid group 1 1/5 5 Unclassified group 1 1/5 1987 (1) 6 El Tor 1/1 1989 (2) 7 Hybrid group 2 1/2 8 Hybrid group 3 1/2 1990 (13) 9-12 El Tor 4/13 13-16 El Tor 4/13 17-18 Unclassified group 2 2/13 19-21 Unclassified group 1 3/13 1991 (4) 22 El Tor 1/4 23-25 Unclassified group 1 3/4 1992 (11) 26 El Tor 1/11 27 Hybrid group 4 1/11 28 El Tor 1/11 29 Unclassified group 1 1/11 30-33 El Tor variant 4/11 34-36 Hybrid group 5 3/11 1993(9) 37-38 El Tor variant 2/9 39-43 El Tor variant 5/9 44 Hybrid group 6 1/9 45 El Tor variant 1/9 1994 (7) 46 Hybrid group 6 1/7 47-51 El Tor variant 5/7 52 Hybrid group 7 1/7 1995 (11) 53-62 El Tor variant 10/11 63 Hybrid group 6 1/11 1996 (3) 64-65 El Tor variant 2/3 66 Hybrid group 7 1/3 1997 (3) 67 El Tor variant 1/3 68-69 El Tor variant 2/3 1998(2) 70-71 El Tor variant 2/2 1999 (179) 72-78 El Tor variant 7/179 79-83 El Tor variant 5/179 84-85 Hybrid group 6 2/179 86-115 Hybrid group 8 30/179 116-247 El Tor variant 132/179 248 Hybrid group 9 1/179 249 Hybrid group 10 1/179 250 Hybrid group 11 1/179 2000 (21) 251-270 El Tor variant 20/21 271 Hybrid group 8 1/21 2001 (27) 272-294 El Tor variant 23/27 295-298 Hybrid group 8 4/27 2002 (13) 299-306 El Tor variant 8/13 307 Hybrid group 8 1/13 308-310 Hybrid group 11 3/13 311 El Tor variant 1/13 2003 (8) 312-315 El Tor variant 4/8 316 Hybrid group 8 1/8 317 Hybrid group 11 1/8 318 Hybrid group 12 1/8 319 Hybrid group 13 1/8 2004 (9) 320-324 El Tor variant 5/9 325-327 Hybrid group 8 3/9 328 Hybrid group 12 1/9 2009 (2) 329-330 El Tor variant 2/2 n, total number of strain(s) of the indicated year; PB, susceptibility to 50 units of polymyxin B; CCA, chicken red blood cell agglutination; VP, Voges-Proskauer test; MAMA, mismatch amplification mutation assay; R, resistant; S, sensitive; +, positive; -, negative; C, classical; E, El Tor Table II. V. cholerae O1 reference strains isolated from various countries No. Name of Year of Country of Serotype Phenotype isolate isolation origin (n = 19) PB CCA VP 1 569B 1948 India Inaba S - - 2 GP71 1971 India Ogawa R + + 3 N16961 1975 Bangladesh Inaba R + + 4 2463-78 1978 Australia Inaba R - - 5 GP156 1979 Australia Ogawa R + - 6 2164-88 1988 United states Inaba R + + 7 295/33 1990 Thailand Ogawa R - + 8 C6706 1991 Peru Inaba R + + 9 C7754 1991 Romania Ogawa R + - 10 MJ1485 1994 Bangladesh Inaba R - + 11 B33 2004 Mozambique Ogawa R + - 12 AR15493 Unknown Bangladesh Inaba R + + 13 AR15425 Unknown Bangladesh Inaba R + + 14 G27875 Unknown India (NICED) Ogawa R + + 15 SC11 Unknown India (NICED) Ogawa R + + 16 GP12 Unknown India Ogawa R + - 17 AS230 Unknown India Ogawa R + + 18 AS231 Unknown India Ogawa R + + 19 AS233 Unknown India Ogawa R - + No. Genotype Biotype Originally identified ctxB rstR biotype 1 C C Classical Classical 2 C E El Tor variant El Tor 3 E E El Tor El Tor 4 C C Hybrid El Tor 5 C E Hybrid El Tor 6 C C El Tor variant El Tor 7 E+C E Hybrid El Tor 8 E+C E Hybrid El Tor 9 C E+C Hybrid El Tor 10 C C Hybrid El Tor 11 C C Hybrid El Tor 12 C E El Tor variant El Tor 13 C E El Tor variant El Tor 14 C E El Tor variant El Tor 15 C E El Tor variant El Tor 16 C E Hybrid El Tor 17 C E El Tor variant El Tor 18 C E El Tor variant El Tor 19 C E Hybrid El Tor PB, susceptibility to 50 units of polymyxin B; CCA, chicken red blood cell agglutination; VP, Voges-Proskauer test; R, resistant; S, sensitive; +, positive; -, negative; C, classical; E, El Tor Table III. PCR primers for the study of V. cholerae O1 genes Gene (s) Primer sequence Size of PCR amplicon (bp) Simple PCR rstRE Forward: GCACCATGATTTAAGATGCTC 501 Reverse: TCGAGTTGTAATTCATCAAGAGTG (El Tor) rstRC Forward: CTTCTCATCAGCAAAGCCTCCATC 474 Reverse: TCGAGTTGTAATTCATCAAGAGTG (Classical) MAMA-PCR ctxB Forward: ACTATCTTCAGCATATGCACATGG Reverse for El Tor: CTGGTACTTCTACTTGAAACA Reverse for classical: CTGGTACTTCTACTTGAAACG Gene (s) PCR condition Initial Denaturation Annealing denaturation Simple PCR rstRE 94[degrees]C, 94[degrees]C, 58[degrees]C, 5 min 60 s 60 s rstRC 94[degrees]C, 94[degrees]C, 64[degrees]C, 5 min 60 s 60 s MAMA-PCR ctxB 96[degrees]C, 96[degrees]C, 55[degrees]C, 2 min l0 s l0 s Gene (s) PCR condition Reference Extension Final No. of extension cycles Simple PCR rstRE 72[degrees]C, 72[degrees]C, 30 22 90 s 7 min rstRC 72[degrees]C, 72[degrees]C, 30 22 90 s 7 min MAMA-PCR ctxB 72[degrees]C, 72[degrees]C, 25 2l 30 s 2 min MAMA-PCR, mismatch amplification mutation assay-PCR Table IV. Biotypes of the 330 V. cholerae Thailand clinical strains Biotype Genotype Phenotype ctxB rstR PB CCA VP Classical C C S - - El Tor E E R + + El Tor E E+C R + + Hybrid group 1 E E S - + Hybrid group 2 E+C E+C R - + Hybrid group 3 E E+C S + + Hybrid group 4 E E S + + Hybrid group 5 C E+C R - + Hybrid group 6 C E+C R + - Hybrid group 7 C E+C S + + Hybrid group 8 C C R + - Hybrid group 9 C C R - + Hybrid group 10 C C R - - Hybrid group 11 C C S + + Hybrid group 12 C C S + - Hybrid group 13 C C S - + El Tor variant C C R + + El Tor variant C E+C R + + Unclassified group 1 E+C E R + + Unclassified group 2 E+C E+C R + + PB, susceptibility to 50 units of polymyxin B; CCA, chicken red blood cell agglutination; VP, Voges-Proskauer test; R, resistant; S, sensitive; +, positive; -, negative; C, classical; E, El Tor
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|Author:||Na-Ubol, M.; Srimanote, P.; Chongsa-nguan, M.; Indrawattana, N.; Sookrung, N.; Tapchaisri, P.; Yamaz|
|Publication:||Indian Journal of Medical Research|
|Date:||Apr 1, 2011|
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