Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003-2011

Abstract

Cholera, caused by Vibrio cholerae, results in significant morbidity and mortality worldwide, including Thailand. Representative V. cholerae strains associated with endemic cholera (n = 32), including strains (n = 3) from surface water sources, in Khon Kaen, Thailand (2003-2011), were subjected to microbiological, molecular and phylogenetic analyses. According to phenotypic and related genetic data, all tested V. cholerae strains belonged to serogroup O1, biotype El Tor (ET), Inaba (IN) or Ogawa (OG). All of the strains were sensitive to gentamicin and ciprofloxacin, while multidrug-resistant (MDR) strains showing resistance to erythromycin, tetracycline, trimethoprim/sulfamethoxazole and ampicillin were predominant in 2007. V. cholerae strains isolated before and after 2007 were non-MDR. All except six diarrhoeal strains possessed ctxA and ctxB genes and were toxigenic altered ET, confirmed by MAMA-PCR and DNA sequencing. Year-wise data revealed that V. cholerae INET strains isolated between 2003 and 2004, plus one strain isolated in 2007, lacked the RS1 sequence (rstC) and toxin-linked cryptic plasmid (TLC)-specific genetic marker, but possessed CTX(CL) prophage genes ctxB(CL) and rstR(CL). A sharp genetic transition was noted, namely the majority of V. cholerae strains in 2007 and all in 2010 and 2011 were not repressor genotype rstR(CL) but instead were rstR(ET), and all ctx(+) strains possessed RS1 and TLC-specific genetic markers. DNA sequencing data revealed that strains isolated since 2007 had a mutation in the tcpA gene at amino acid position 64 (N→S). Four clonal types, mostly of environmental origin, including subtypes, reflected genetic diversity, while distinct signatures were observed for clonally related, altered ET from Thailand, Vietnam and Bangladesh, confirmed by distinct subclustering patterns observed in the PFGE (NotI)-based dendrogram, suggesting that endemic cholera is caused by V. cholerae indigenous to Khon Kaen.

Fig. 1.

clustal w alignment of amino acids encoded by the tcpA gene of the test V. cholerae O1 strains with the sequence from the ET reference strain N16961. Dots indicate identical amino acids. The amino acid sequences of TcpA of V. cholerae O1 strains isolated in Thailand between 2003 and 2004 showed 100 % identity with those of TcpA of reference strain N16961. Strains isolated in 2007 and thereafter showed a mutation at amino acid position 64 (N→S) of the tcpA gene.

Fig. 2.

Genomic fingerprinting patterns of V. cholerae O1 strains isolated from diarrhoea and environmental sources in Khon Kaen, Thailand (2003–2011). The dendrogram was prepared by Dice similarity coefficient and UPGMA clustering using PFGE images of the NotI-digested genomic DNA. The scale-bar at the top left indicates similarity coefficient (%). The PFGE types (A–D) of the V. cholerae O1 strains are shown. The major cluster, A, comprised most of the Thai V. cholerae O1 strains (2003–2011) and all of the representative Bangladeshi (2009–2010) and Vietnamese (2008–2010) V. cholerae O1 strains showing clonal relatedness. The seven different PFGE pattern-based subclusters within cluster A are marked with grey bars. Distinct signatures for clonally related altered ET in Thailand, Vietnam, and Bangladesh, as confirmed by subclustering patterns in the dendrogram, suggest endemic cholera to be caused by V. cholerae population indigenous to Khon Kaen, Thailand. INET, Inaba El Tor; OGET, Ogawa El Tor; Clin, clinical; Env, environmental.