|Year : 2019 | Volume
| Issue : 4 | Page : 558-560
Detection of Haitian ctxB7 & tcpA alleles in Vibrio cholerae O1 El Tor biotype in Puri, Odisha, India
Anna Salomi Kerketta, Shantanu Kumar Kar, Hemant Kumar Khuntia
Division of Microbiology, ICMR-Regional Medical Research Center, Bhubaneswar 751 023, Odisha, India
|Date of Submission||07-Jul-2017|
|Date of Web Publication||16-Jul-2019|
Hemant Kumar Khuntia
Division of Microbiology, ICMR-Regional Medical Research Center, Bhubaneswar 751 023, Odisha
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Kerketta AS, Kar SK, Khuntia HK. Detection of Haitian ctxB7 & tcpA alleles in Vibrio cholerae O1 El Tor biotype in Puri, Odisha, India. Indian J Med Res 2019;149:558-60
|How to cite this URL:|
Kerketta AS, Kar SK, Khuntia HK. Detection of Haitian ctxB7 & tcpA alleles in Vibrio cholerae O1 El Tor biotype in Puri, Odisha, India. Indian J Med Res [serial online] 2019 [cited 2020 Jul 7];149:558-60. Available from: http://www.ijmr.org.in/text.asp?2019/149/4/558/262870
Vibrio cholerae O1, the causative agent of the majority of cholera outbreaks is classified into two biotypes, classical and El Tor  based on the assays such as chicken cell agglutination (CCA), Voges-Proskauer (VP) reaction, sheep erythrocyte lysis and polymyxin B susceptibility test . Over the decades, three variants of V. cholerae O1 El Tor biotype have been reported globally such as Maltab variants in 2002, Mozambiqe variant in 2004-2005 and the altered El Tor type (El Tor variant) carrying classical cholera toxin since 2002,,.
The clinical manifestation of cholera is caused by cholera toxin, the major virulence factor encoded by ctxAB gene located on the CTX prophage integrated on the V. cholerae chromosome. Classical V. cholerae contains classical type ctxB and El Tor contains El Tor type ctxB. V. cholerae O1 with a typical El Tor phenotypes (resistant to 50 Units of polymyxin B and positive for CCA and VP test) but carrying ctxB classical (ctxB1 genotype) is designated as El Tor variant . On chromosome-1, the single-nucleotide polymorphism (SNP) at codon 19 results in replacement of the classical cholera toxin B histidine with asparagine residue resulting in a new ctxB genotype which is named as ctxB7,.
El Tor variant carrying this new type variant ctxB7 has been reported throughout the world but these were highlighted after the huge epidemic in Haiti in 2010 and designated as Haitian variant . Genomic analysis of Haitian V. cholerae O1 strains revealed mutations in different segments of chromosome including tcpA (toxin co-regulated pilus) allele . Haitian variant gradually spread throughout the world causing cholera outbreaks with predominance ,,. Here, we report the detection of V. cholerae O1 harbouring Haitian variant ctxB and tcpA associated with polymyxin B (pb, 50 U) susceptibility in Puri district, Odisha, India, during 2014-2015. This study was a part of the diarrhoea surveillance programme in Infectious Disease Hospital (IDH) Puri.
During the study, 170 rectal swab samples (72 in 2014 and 98 in 2015) were collected from hospitalized acute diarrhoea patients. The samples were transported to the ICMR-Regional Medical Research Center, Bhubaneswar for isolation of V. cholerae. The samples were incubated for six hours in alkaline peptone water and cultured on thiosulphate-citrate-bile-sucrose agar (TCBS, BD, USA) followed by biochemical analysis and serotyping  with polyvalent O1, O139 and mono-specific Ogawa and Inaba antisera (BD, USA). The study protocol was approved by the Institutional Ethics Committee. Bacteriological analysis of the rectal swabs revealed 20 samples positive for V. cholerae O1 Ogawa, El Tor biotype. All these 20 isolates were found positive for VP test and CCA. Antimicrobial susceptibility test was carried out by Kirby-Bauer disk diffusion method adhering to Clinical and Laboratory Standard Institute guidelines ,. V. cholerae O1 isolates were found sensitive to drugs such as tetracycline, trimethoprim/sulphamethoxazole, chloramphenicol, neomycin, gentamicin, ciprofloxacin, norfloxacin, ofloxacin, doxycycline and azithromycin; and resistant to ampicillin, erythromycin, co-trimoxazole, nalidixic acid and furazolidone. Of the total 20 isolates, 15 (75%) were found sensitive to Pb (50 units). The genetic analysis of isolated V. cholerae isolates included PCR assays for detection of wbe, ctxA and tcpA. Haitian variants ctxB and tcpA were amplified using the primer pairs as described previously ,. PCR analysis revealed that all the 20 isolates were positive for ctxA, tcpA (El Tor) and rfb genes. The presence of ctxA (301 bp)in all V. cholerae isolates confirmed their toxigenic potential, wbe (192 bp) provided the molecular evidence for O1 serogroup and tcpA El Tor (469 bp) determined the El Tor type. Further biotype-specific CTX prophage repressor rstR was amplified with El Tor-specific primers , indicating the presence of El Tor rstR in all these isolates. All V. cholerae isolates were found to carry Haitian variant tcpA allele [(167 bp) [Figure 1]] which confirmed that tcpA had a single base substitution at 266-nt position as was found in strains from Haiti . Double mismatch-amplification-mutation assay (DMAMA) detected Haitian variant ctxB allele (191 bp) in all 20 V. cholerae O1 isolates during the study [Figure 1].
|Figure 1: PCR analysis showing Haitian variant ctxB and tcpA allele in V. cholerae O1 isolates. Lane M, 100 bp size ladder; lane1, Haitian control 2010EL-1786; lane 2, classical control, O395; from lane 3 through lane 16, V. cholerae O1 isolates.|
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A surveillance study conducted in three hospitals including IDH during 2004-2006 revealed the incidence rate of V. cholerae as 17.3 per cent where all V. cholerae O1 were El Tor biotype . During 2011-2013, following a study on operational feasibility of an oral cholera vaccine in Satybadi Block of Puri district , the post-vaccination diarrhoea surveillance demonstrated the incidence rate of V. cholerae O1 as 3.4 per cent (unpublished data) and bio-typing of isolates revealed V. cholerae O1 Hybrid and El Tor variants . In the present study, 11.8 per cent V. cholerae isolates were found to be Haitian variants.
The emergence of Haitian variant, tempted researchers to investigate its incidence and spread causing cholera outbreaks. Two cholera outbreaks in western and southern Odisha during 2013 and 2016 respectively presented the ctxB7 genotype (unpublished data). Our previous study depicted that a large cholera outbreak occurred in western Odisha during 2014 was due to V. cholerae O1 with ctxB7 and retrospective analysis of laboratory strains revealed that the origin of Haitian variant was since 1999 in Odisha . Earlier studies in Kolkata reported V. cholerae with tcpA of Haitian allele, ctxB7 allele and Pb susceptibility since 2003, 2006 and 2012, respectively ,,. In north India, including Delhi, Haryana and Uttar Pradesh, the combination of Haitian ctxB (ctxB7), classical ctxB (ctxB1) and tcpA of Haitian allele has been reported since 2008. A cholera outbreak in Maharashtra during 2012 was due to V. cholerae O1 carrying Haitian variant ctxB. The present study showed the combination of 100 per cent Haitian variants ctxB and tcpA associated with high (75%) susceptibility to Pb without the detection of ctxB1 allele. This suggested that Haitian strains might have replaced the El Tor variant in this region as has been reported for El Tor being replaced by El Tor variant strains during 2008-2009 in Odisha . Similar to the Kolkata strains , the high percentage (75%) of Pb susceptibility in our isolates was a unique phenotypic change contrast to the El Tor biotype of earlier decades and Haitian variants in Kerala, south India .
The cryptic change in the genetic backbone is the current evolutionary outcome in V. cholerae O1 that influences its virulence, transmission and spread. The presence of hypervirulent Haitian variant in Puri has significance in connection to the spread of cholera outbreaks. An effective surveillance acts as a resource to develop early warning to implement prevention and preparedness for disease outbreak. This report of Haitian variant in coastal Odisha warrants constant surveillance in other parts of Odisha and India.
Financial support & sponsorship: Authors acknowledge the Indian Council of Medical Research, New Delhi, for financial support.
Conflicts of Interest: None.
| References|| |
Baumann P, Furniss AL, Lee JV. Genus I. Vibrio pacini
1854. In: Kreig NR, Holt JG, editors. Bergey's manual of systematic bacteriology
, Vol. 1. Baltimore: Williams and Wilkins; 1984. p. 518-38.
Kaper JB, Morris JG Jr., Levine MM. Cholera. Clin Microbiol Rev
Safa A, Bhuyian NA, Nusrin S, Ansaruzzaman M, Alam M, Hamabata T, et al
. Genetic characteristics of Matlab variants of Vibrio cholerae
O1 that are hybrids between classical and El Tor biotypes. J Med Microbiol
(Pt 11) : 1563-9.
Ansaruzzaman M, Bhuiyan NA, Nair GB, Sack DA, Lucas M, Deen JL, et al
. Cholera in Mozambique, variant of Vibrio cholerae
. Emerg Infect Dis
Nair GB, Qadri F, Holmgren J, Svennerholm AM, Safa A, Bhuiyan NA, et al
. Cholera due to altered El Tor strains of Vibrio cholerae
O1 in Bangladesh. J Clin Microbiol
Raychoudhuri A, Mukhopadhyay AK, Ramamurthy T, Nandy RK, Takeda Y, Nair GB, et al.
Biotyping of Vibrio cholerae
O1: Time to redefine the scheme. Indian J Med Res
Reimer AR, Van Domselaar G, Stroika S, Walker M, Kent H, Tarr C, et al.
Comparative genomics of Vibrio cholerae
from Haiti, Asia, and Africa. Emerg Infect Dis
Goel AK, Jain M, Kumar P, Bhadauria S, Kmboj DV, Singh L, et al.
A new variant of Vibrio cholerae
O1 El Tor causing cholera in India. J Infect
Piarroux R, Barrais R, Faucher B, Haus R, Piarroux M, Gaudart J, et al
. Understanding the cholera epidemic, Haiti. Emerg Infect Dis
Ghosh P, Naha A, Basak S, Ghosh S, Ramamurthy T, Koley H, et al.
Haitian variant tcpA
in Vibrio cholerae
O1 El Tor strains in Kolkata, India. J Clin Microbiol
Díaz-Quiñonez A, Hernández-Monroy I, Montes-Colima N, Moreno-Pérez A, Galicia-Nicolás A, Martínez-Rojano H, et al.
Outbreak of Vibrio cholerae
serogroup O1, serotype Ogawa, biotype El Tor strain – la Huasteca Region, Mexico, 2013. MMWR Morb Mortal Wkly Rep
Marin MA, Vicente ACP. Variants of Vibrio cholerae
O1 El Tor from Zambia showed new genotypes of ctxB. Epidemiol Infect
Marin MA, Thompson CC, Freitas FS, Fonseca EL, Aboderin AO, Zailani SB, et al.
Cholera outbreaks in Nigeria are associated with multidrug resistant atypical El Tor and non-O1/non-O139 Vibrio cholerae
. PLoS Negl Trop Dis
World Health Organization. Manual for laboratory investigations of acute enteric infections
. WHO documents CDD/83.3/Rev.1.113. Geneva: WHO; 1987.
Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by standardized single disk method. Am J Clin Pathol
Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disk
, approved standard M2-A10
. Wayne, PA, USA: CLSI; 2007.
Naha A, Pazhani GP, Ganguly M, Ghosh S, Ramamurthy T, Nandy RK, et al.
Development and evaluation of a PCR assay for tracking the emergence and dissemination of Haitian variant ctxB
in Vibrio cholerae
O1 strains isolated from Kolkata, India. J Clin Microbiol
Chatterjee S, Patra T, Ghosh K, Raychoudhuri A, Pazhani GP, Das M, et al
. Vibrio cholerae
O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant. J Med Microbiol
(Pt 2) : 239-47.
Chin CS, Sorenson J, Harris JB, Robins WP, Charles RC, Jean- Charles,et al
. The origin of the Haitian cholera outbreak strains. N. Engl J Med
Samal SK, Khuntia HK, Nanda PK, Satapathy CS, Nayak SR, Sarangi AK, et al.
Incidence of bacterial enteropathogens among hospitalized diarrhoea patients from Orissa, India. Jpn J Infect Dis
Kar SK, Sah B, Patnaik B, Kim YH, Kerketta AS, Shin S, et al.
Mass vaccination with a new, less expensive oral cholera vaccine using public health infrastructure in India: The Odisha model. PLoS Negl Trop Dis
Wierzba TF, Kar SK, Mogasale VV, Kerketta AS, You YA, Baral P, et al.
Effectiveness of an oral cholera vaccine campaign to prevent clinically-significant cholera in Odisha state, India. Vaccine
Pal BB, Khuntia HK, Nayak SR, Mohanty A, Biswal B. Vibrio cholerae
O1 Ogawa strains carrying the ctxB7
allele caused a large cholera outbreak during 2014 in the tribal areas of Odisha, India. Jpn J Infect Dis
Samanta P, Ghosh P, Chowdhury G, Ramamurthy T, Mukhopadhyay AK. Sensitivity to polymyxin B in El Tor Vibrio cholerae
O1 strain, Kolkata, India. Emerg Infect Dis
Kumar D, Sharma NC, Ramamurthy T, Gupta SK, Seth DK. Haitian variant tcpA
in Vibrio cholerae
O1 El Tor strains in National Capital Region (India). Indian J Med Res
Kumar P, Mishra DK, Deshmukh DG, Jain M, Zade AM, Ingole KV, et al.
Haitian variant ctxB
producing Vibrio cholerae
O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Moharastra, India after causing a cholera outbreak. Clin Microbiol Infect
Khuntia HK, Pal BB, Samal SK, Kar SK. Rapid spread of Vibrio cholerae
O1 El Tor variant in Odisha, Eastern India, in 2008 and 2009. J Clin Microbiol
Divya MP, Sivakumar KC, Sarada Devi KL, Remadevi S, Thomas S. Novel multiple mutations in the topoisomerase gene of Haitian variant Vibrio cholerae
O1. Antimicrob Agents Chemother