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

Abstract

Vibrio cholerae O1 biotype El Tor (ET), causing the seventh cholera pandemic, was recently replaced in Bangladesh by an altered ET possessing ctxB of the Classical (CL) biotype, which caused the first six cholera pandemics. In the present study, V. cholerae O1 strains associated with endemic cholera in Dhaka between 2006 and 2011 were analysed for major phenotypic and genetic characteristics. Of 54 representative V. cholerae isolates tested, all were phenotypically ET and showed uniform resistance to trimethoprim/sulfamethoxazole (SXT) and furazolidone (FR). Resistance to tetracycline (TE) and erythromycin (E) showed temporal fluctuation, varying from year to year, while all isolates were susceptible to gentamicin (CN) and ciprofloxacin (CIP). Year-wise data revealed erythromycin resistance to be 33.3 % in 2006 and 11 % in 2011, while tetracycline resistance accounted for 33, 78, 0, 100 and 27 % in 2006, 2007, 2008, 2009 and 2010, respectively; interestingly, all isolates tested were sensitive to TE in 2011, as observed in 2008. All V. cholerae isolates tested possessed genetic elements such as SXT, ctxAB, tcpA(ET), rstR(ET) and rtxC; none had IntlI (Integron I). Double mismatch amplification mutation assay (DMAMA)-PCR followed by DNA sequencing and analysis of the ctxB gene revealed a point mutation at position 58 (C→A), which has resulted in an amino acid substitution from histidine (H) to asparagine (N) at position 20 (genotype 7) since 2008. Although the multi-resistant strains having tetracycline resistance showed minor genetic divergence, V. cholerae strains were clonal, as determined by a PFGE (NotI)-based dendrogram. This study shows 2008-2010 to be the time of transition from ctxB genotype 1 to genotype 7 in V. cholerae ET causing endemic cholera in Dhaka, Bangladesh.

Fig. 1.

Results of DMAMA-PCR performed to determine the ctxB genotype of tested V. cholerae O1 strains (n = 54) from Dhaka, Bangladesh. All representative strains from 2006 and 2007 possessed ctxB genotype 1 only (ctxB1), while ctxB genotype 7 (ctxB7) was predominant during 2008, 2010 and 2011. Although ctxB1 and ctxB7 genotypes co-existed during 2009, none of the strains that were isolated during 2010 and thereafter possessed ctxB1, suggesting that the transition from genotype 1 to genotype 7 took place during 2008–2010.

Fig. 2.

Amino acid sequence alignment of the CTXB subunit of V. cholerae O1 ET strains isolated in Bangladesh (2006–2011) with ET reference (N16961), CL reference (569B) and altered ET (Orissa variant) possessing ctxB genotype 7. Identical amino acid residues are indicated by dots. The amino acid sequences of the CTXB reference and Orissa variant used in the alignment were taken from GenBank.

Fig. 3.

Genomic fingerprinting of drug-resistant V. cholerae O1 ET strains isolated in Dhaka between 2006 and 2011. The dendrogram was constructed by Dice similarity coefficient and UPGMA clustering using PFGE images of NotI-digested genomic DNA. The scale bar at the top left indicates the correlation coefficient (range: 90–100 %). The banding patterns and the similarity coefficient, which is within 90 % for all V. cholerae strains included in the dendrogram, suggest that they all belong to the same clonal lineage. Cluster analysis shows that all V. cholerae strains resistant to SXT and FR, irrespective of their year of isolation, shared a major cluster with an identical banding pattern, while MDR strains having TE resistance showed genetic divergence in their patterns and formed several closely related subclusters.