Occurrence of Antibiotic Resistance Gene Cassettes aac(6′)-Ib, dfrA5, dfrA12, and ereA2 in Class I Integrons in Non-O1, Non-O139 Vibrio cholerae Strains in India

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Antimicrobial Agents and Chemotherapy






American Society of Microbiology

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Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6′)-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6′)-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance. Cholera is a serious epidemic disease caused by the gram-negative bacterium Vibrio cholerae. Only V. cholerae strains belonging to the O1 and O139 serogroups are thought to be capable of causing epidemic cholera. Strains belonging to serogroups other than O1 and O139, collectively referred to as non-O1, non-O139 strains, are ubiquitously found in the aquatic environs (19) and are also capable of causing sporadic diarrhea. In 1996, an inexplicable upsurge in the incidence of cholera strains belonging to serogroups other than O1 and O139 occurred in Calcutta, India. After extensive molecular characterization, these strains were found to be devoid of the ctx filamentous phage (CTXφ) (31) and some other virulence genes (27). Based on these findings, it was concluded that some strains of V. cholerae belonging to different serotypes can cause diarrhea clinically indistinguishable from that associated with cholera (5) by a mechanism that could be distinct from that employed by the toxigenic V. cholerae O1 and O139 strains. The nomenclature “enteropathogenic V. cholerae” (EPVC) was proposed to include these serotypes (27). The incidence of EPVC had shown an upward trend from 1997 that continued into 1998. In the months of July and August 1998, the EPVC strains constituted one-third of the V. cholerae strains isolated from hospitalized patients (12). Recently a comparative study of clinical and environmental isolates of non-O1, non-O139 V. cholerae strains belonging to matching serogroups from our laboratories revealed that, despite sharing the same serogroups, the environmental and clinical isolates were genetically heterogeneous and also that the resistance to multiple antibiotics was more common among the clinical isolates (5). Multiple-antibiotic-resistant isolates of non-O1, non-O139 V. choelrae strains were identified in children with diarrhea in Bangkok, Thailand (7). Reports of drug-resistant V. cholerae strains are appearing with increasing frequency (20). Emergence of resistance to multiple drugs is a serious clinical problem in the treatment and containment of the disease, as reflected by the increase in the fatality rate from 1% to 5.3% after the emergence of drug-resistant strains in Guinea-Bissau during the 1996-1997 epidemic of cholera (10). The molecular mechanisms responsible for the emergence of multiple-antibiotic-resistant V. cholerae are not very well worked out. Acquisition of antibiotic resistance genes across genera and species is mediated through horizontal and lateral gene transfer. Plasmids, conjugative transposons, and integrons are all vehicles for the acquisition of resistance genes (13, 14, 24, 29, 32). Integrons are genetic elements capable of integrating and mobilizing individual gene cassettes by a site-specific recombination mechanism involving a DNA integrase intI and an att1 site recognized by the integrase (13). Among the different integron families, class I integrons have been found to be most prevalent among clinical isolates. The 5′ conserved segment (5′-CS) of the class I integrons contains the integrase gene (intI1) and the recombination site attI1 (6). The 3′-CS of class I integrons normally carries the antiseptic resistance gene, qacEΔ1,and the sulfonamide resistance gene, sul1. It has been observed, however, that class I integrons do not always contain the entire 3′-CS (6, 22, 25, 28). Class I integrons are widely present in multidrug-resistant enteropathogens (15-17) and have been identified in V. cholerae O1 strains isolated in Vietnam (8) and Albania and Italy (11), as well as in non-O1, non-O139 strains in Thailand (9). A 150-kb conjugative plasmid was found to contain class I integron in epidemic strains of V. cholerae O1 isolated in Guinea-Bissau (10). So far, there has been no report on the presence of class I integrons in strains of V. cholerae isolated in India. In the present study, 94 strains of V. cholerae (17 of which were environmental isolates) belonging to serogroups other than O1 and O139 isolated in Calcutta, India, during the period 1997 to 1998, were analyzed for the prevalence of class I integrons and the nature of the antibiotic resistance genes carried by them. They were also investigated for the presence of plasmids and the conjugative transposon SXT element (32), which could also contribute to the multidrug resistance observed in these strains.

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