Bacterial Strains
In this study, a set of 172 V. cholerae O1 strains were isolated from clinical samples collected in Dhaka, Bangladesh, between 2015 and 2021, as a part of an epidemiological surveillance conducted by the International Centre for Diarrheal Disease Research, Bangladesh (icddr,b). The strains were isolated from the stool of suspected cholera patients seeking treatment at icddr,b Dhaka Hospital. Informed consent was obtained from the patients or legal guardians for minors’ patients prior to the collection of stool samples. Isolation and identification of V. cholerae were performed according to standard cultural and molecular methods as described previously (14).
Serogrouping
The serogroup of the V. cholerae isolates was confirmed by slide agglutination test, using V. cholerae O1 and O139 specific polyvalent antisera and were tested further using serotype specific monoclonal antibodies, Inaba and Ogawa as described previously (5). For molecular confirmation of serogroups, and detection of cholera toxin, multiplex PCR targeting O1-(wbe), O139-(wbf), and ctxA genes were performed (10).
Antibiotic Susceptibility Assay
Bacterial susceptibility to antimicrobials was determined by standard disc diffusion test on Muller-Hinton agar (BD, USA) according to Clinical and Laboratory Standards Institute guidelines (Bauer et al., 1966; CLSI, 2010). All strains of V. cholerae were tested for susceptibility to ampicillin (AMP, 10 µg), ceftriaxone (CRO, 30 µg), ciprofloxacin (CIP, 5 µg), mecillinam (MEL, 25µg), erythromycin (E, 15 µg), nalidixic acid (NA, 30 µg), imipenem (IMP, 10 µg), sulfamethoxazole/trimethoprim (SXT, 25 µg), streptomycin (S, 10 µg), levofloxacin (LEV, 5 µg), cephalothin (KF, 30 µg), cefixime (CFM, 5 µg), cefepime (FEP, 30 µg), tetracycline (TE, 30 µg), aztreonam (ATM, 30 µg), azithromycin (AZM, 15 µg), chloramphenicol (C, 30 µg), and gentamicin (CN, 10 µg) using commercially available discs (BD BBL SensiDisc). The minimum inhibitory concentration (MIC) was determined by E-test (Biomeuriex). Suspected Extended Spectrum Beta Lactamase (ESBL) producing V. cholerae isolates were screened for the production of ESBLs by using a double disk synergy test (DDST) (15).
Polymerase Chain Reaction (Pcr) Assay
PCR assays were performed to detect genes encoding zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA); (16), SXT-related integrase (intSXT)(17) and biotype-specific (El Tor and Classical) phage encoded repressor (rstR; Kimsey et al., 1998)); toxin coregulated pilus (tcpA) (16), and repeat in toxin (rtxC)(18) using primers and conditions described previously. All antibiotic-resistant V. cholerae O1 strains were tested for gene encoding the class 1 and class 2 integron using primers and procedures described elsewhere (19). Double mismatch amplification mutation assay (DMAMA)-PCR was performed to determine three genotypes of the cholera toxin gene, Classical (ctxB genotype 1, ctxB1), El Tor (ctxB genotype 3, ctxB3), and Haitian type (ctxB genotype 7, ctxB7) based on nucleotide substitution at position 58, 115, and 203 (20). V. cholerae O1 strains O395 (Classical), N16961 (El Tor), and EL-1786 (Haitian variant, ctxB7) were used as control for the PCR.
PCR was also performed to detect genes for ESBL, AmpC, carbapenemase, and quinolone resistance (21–25). Specifically, multiplex PCR (6 sets) was performed to detect ESBL genes blaTEM/blaSHV/blaOXA-1-like genes, blaCTX-M including phylogenetic groups 1, 2 and 9, 142 carbapenemase genes blaVEB/blaGES/blaPER, blaVIM/blaIMP/blaKPC, blaGES/blaOXA-48- like genes, and the AmpC group of genes blaMOX/blaFOX/blaEBC/blaCIT/blaDHA/blaACC. Simplex PCR was used to detect the following genes: ESBL (blaCTX-M-8/-25) (21); carbapenemase (blaAIM, blaGIM, blaSIM, and blaDIM) (22); blaNDM (25), quinolone resistance related qnrVC (23), efflux pump encoding qepA, acetylase encoding aac(6’)-Ib-cr (24).
Sequencing Of Antibiotic Resistant Genes
PCR amplified antibiotic resistant genes were sequenced using an ABI Big-Dye Terminator v.3.1 Cycle Sequencing Reaction kit (Applied Biosystems) on an ABI PRISM 3500 XL genetic analyzer (AppliedBiosystems, Foster City, USA). The BLASTN program (www.ncbi.nlm.nih.gov/BLAST) was used for homology searching. The partial sequences of the genes were submitted to GenBank (Accession Numbers: MK992813, MK992814, MK992815).