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

Abstract

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata C holera still continues to be an important cause of human infection, especially in developing countries that lack access to safe drinking water and proper sanitation. The recent devastating cholera outbreak in Haiti (13), for the first time in almost a century, placed this ancient disease at the forefront of the global public health agenda. In May 2011, the World Health Assembly recognized the reemergence of cholera as a significant global public health problem and called for the implementation of an integrated and comprehensive global approach to cholera control (17). This dreadful diarrheal disease is caused by the Gram-negative toxigenic bacterium Vibrio cholerae (7). To date, more than 200 serogroups of V. cholerae are known, but only serogroups O1 and O139 cause epidemic and pandemic cholera (7, 16). To date, the world has experienced seven pandemics of cholera. Among these, the first six were caused by the classical biotype strains, whereas the ongoing seventh pandemic has been caused by the El Tor biotype (16). In recent years, the emergence and dissemination of novel pathogenic variants of V. cholerae O1 throughout many Asian and African countries (1,2,3,5,9,10,11,14,15) indicated a cryptic change in cholera epidemiology. Our recent study showed that the El Tor variant strains of V. cholerae O1 have replaced the prototype El Tor biotype strains in Kolkata, India, since 1995 (15). This report, together with the recent massive cholera outbreak in Haiti, caused by V. cholerae organisms with a mutation in the 58th nucleotide of ctxB (3), motivated us to investigate the emergence and dissemination of this new variant of V. cholerae O1 biotype El Tor strains, if any, in Kolkata.In this study, we have developed a double-mismatch-amplification mutation assay (DMAMA) to accurately discriminate the classical, El Tor, and Haitian type ctxB alleles through a rapid and simple PCR-based assay. A total of 142 V. cholerae O1 strains were included in this study. These strains were selected from the repository of the National Institute of Cholera and Enteric Diseases, Kolkata, India, covering different months of each year from 2004 to 2011. V. cholerae O1 strains O395 (serotype Ogawa), N16961 (serotype Inaba), and EL-1786 (Ogawa, El Tor) were used as standard strains for the classical, El Tor, and Haitian type, respectively.Development of the DMAMA-PCR. All 142 tested strains, along with the control strains, were grown in Luria-Bertani broth (Becton Dickinson, Sparks, MD) for 18 h and then streaked on Luria agar (LA) plates. In this study, we focused on ctxB in V. cholerae O1 strains to confirm the strains carrying Haitian, classical, and El Tor alleles in a simple PCR-based assay. Current methods for differentiating the biotype-specific cholera toxin B (CTB) subunit of V. cholerae O1 necessitate MAMA-PCR with ...

CtxB1 outcompetes CtxB7 in Vibrio cholerae O1, Bangladesh

“…This emergence and spread of altered-ET in Asia and Africa had huge epidemiological implications, as ET carrying ctxB1 was associated with more severe disease (Siddique et al, 2010). Further change in the CTX prophage gene (histidine to asparagine at the twentieth position of CtxB) resulting in substitution of ctxB1 with ctxB7 was reported in 2006 from India (Naha et al, 2012). A similar change from ctxB1 to ctxB7 also occurred in V. cholerae O1 responsible for endemic cholera in Dhaka, Bangladesh, but not before 2008 (Rashed et al, 2012), suggesting a possible transmission of V. cholerae carrying the ctxB7 from neighbouring India, which was detected later in Africa and Haiti (Hasan et al, 2012).…”

“…In this study, a significant correlation was observed between the clinical and environmental strains carrying either ctxB1 or ctxB7 (Spearman's correlation coefficient50.973; P50.005). Although the epidemiological significance of the change from ctxB7 to ctxB1, as observed in Bangladesh during 2013-2014, is not well understood, the competition of the ctxB genotypes and their temporal shift in V. cholerae responsible for cholera at different ecological settings provide in situ evidence of CTX prophage-mediated evolution of the bacterium (Naha et al, 2012;Rashed et al, 2013;Alam et al, 2014). The limitation of the present study is that the SNPs of ctxB were monitored, not the genome-wide SNPs that have been proposed to drive positive selection of V. cholerae in Haiti (Azarian et al, 2014); and so, the synergistic role in the observed genotypic shift of SNPs elsewhere in the V. cholerae genome cannot be ruled out.…”

“…The ctxB genotype of V. cholerae O1 was determined by mismatch amplification mutation assay (MAMA)-PCR, and double mismatch amplification mutation assay (DMAMA)-PCR, as described elsewhere (Naha et al, 2012). To complement the MAMA and DMAMA-PCR results, the ctxB of 12 V. cholerae O1 strains showing either ctxB1 or ctxB7 by PCR were subjected to DNA sequencing using primers and conditions as described elsewhere (Olsvik et al, 1993).…”

Genetic characteristics of drug-resistant Vibrio cholerae O1 causing endemic cholera in Dhaka, 2006–2011

“…DMAMA-PCR was recently developed to discriminate the CL (ctxB genotype 1), ET (ctxB gneotype 3) and Haitian types (ctxB genotype 7) of ctxB alleles by focusing on nucleotide positions 58 and 203 of the ctxB gene (Naha et al, 2012). DMAMA-PCR was performed in this study to detect the ctxB genotype using the primers and conditions described elsewhere (Naha et al, 2012). V. cholerae O1 strains O395 (CL), N16961 (ET) and 2010EL-1786 (Haiti variant, genotype 7) were used as control strains for DMAMA-PCR analysis.…”

“…A recent study in India reported that a new CT variant of V. cholerae O1 ET with an amino acid substitution at position 20 caused a large cholera outbreak in Orissa, Eastern India (Kumar et al, 2009). Subsequently, the same CT variant was found to have been associated with cholera in Kolkata, India, since 2006 (Naha et al, 2012). Considering these recent changes, the current study was undertaken in order to understand the phenotypic and genetic traits of contemporary V. cholerae causing endemic cholera in Dhaka, Bangladesh.…”