Several phenotypic and genetic laboratory tests are used to determine whether isolates are classified as classical or El Tor biotypes. Among phenotypic traits distinguishing the 2 biotypes, sensitivity to polymyxin B (50 U) is considered a reliable indicator and stable phenotype for biotyping. Research has shown that the genome of V. cholerae strains is undergoing cryptic changes that influence the strains’ virulence, rapid transmission, and spread (4). Our previous findings showed El Tor strains with few biotype traits of classical strains (5).

Figure

Figure. Isolation profile of polymyxin B–sensitive Vibrio cholerae strains in Kolkata, India, 2003–2014. A) Yearly occurrence of polymyxin B sensitivity and resistance in V. cholerae O1 El Tor variant strains isolated from...

Since the seventh cholera pandemic, which occurred during the 1960s and 1970s and was caused by El Tor strains, the El Tor biotype had been resistant to polymyxin B, a cationic antimicrobial peptide. However, when cholera strains first appeared in patients in Kolkata, India, in June 2012, V. cholerae O1 was found to be sensitive to polymyxin B (6). To determine whether this phenomenon occurred earlier, we tested 255 clinical strains isolated from patients in Kolkata during 2003–2014 and found that, from March 2013, polymyxin B–sensitive El Tor strains had replaced resistant strains (Figure, panel A). The MIC of polymyxin B, determined by Etest (bioMérieux, Marcy l’Etoile, France), confirmed that the El Tor strains were susceptible to this antimicrobial drug (Figure, panel B). In this assay, the El Tor strain (N16961) was highly resistant to polymyxin B (MIC 96 µg/mL), whereas the variant strains in Kolkata showed a drastic reduction in resistance (6,7).

To confirm additional changes in biotype attributes in the variant Kolkata isolates during 2003–2014, we used the Voges-Proskauer test to determine production of acetylmethyl carbinol and found that the tested strains produced acetoin and were positive for chicken erythrocytes agglutination. The rtsC gene encoding the activator protein, which is absent from classical biotype strains but present in El Tor strains, was found in all the tested strains of the El Tor biotype. Biotype-specific CTX prophage repressor rstR was amplified with the El Tor–specific primers, indicating presence of El Tor rstR. The tcpA gene has distinct alleles specific to classical and El Tor biotypes of O1. Our study showed that all strains yielded amplicons with the El Tor-tcpA–specific primers but not with the classical-tcpA–specific primers. However, these strains had a single-base substitution at the 266-nt position of tcpA, also present in variant strains from Haiti. Furthermore, Vibrio seventh pandemic (VSP) gene clusters VSP I and VSP II are unique to El Tor strains of the seventh pandemic. We found presence of VSP I and II encoding genes in all our tested strains, indicating that the strains are El Tor, but with specific classical traits.

We also checked the strains’ sensitivity to many antimicrobial drugs: tetracycline, trimethoprim/sulfamethoxazole, streptomycin, erythromycin, gentamicin, ciprofloxacin, and azithromycin, and all strains were sensitive to all drugs except trimethoprim/sulfamethoxazole and streptomycin. All strains isolated during 2013–2014 were fully resistant to trimethoprim/sulfamethoxazole and streptomycin, but 55% of strains isolated before 2012 were sensitive to these drugs.

Genes encoding lipid IVA acyltrasferase (msbB), biofilm formation, antimicrobial peptide resistance (carR), and 3 aminoacyl lipid modification (almEFG) have been shown to contribute to polymyxin resistance in V. cholerae (6–8). Analysis of these genes from the newly emerged polymyxin-B–sensitive strains may provide additional useful information. We found that these strains contained Haitian variant ctxB (ctxB7) similar to the classical cholera toxin. Our earlier studies identified many new attributes of Haitian V. cholerae variant strains in Kolkata since 2003 (9,10).

We report the emergence of El Tor strains producing classical cholera toxin. These strains have lost an El Tor biotype marker and acquired a vital classical biotype characteristic, a change that has probably altered the regulatory mechanisms of lipid A modification machinery in V. cholerae (6–8). This change is a major event in the history of cholera after 1961, when El Tor strains first appeared. The recent changes in V. cholerae O1 strains should be carefully monitored to determine their clinical and epidemiologic implications.