The amplified fragments of int and attP were directly sequenced on both strands using the same PCR primers. The sequencing
reactions were repeated once to generate a consensus sequence and to eliminate the possibility of errors due to amplification by Taq polymerase (Promega). The sequencing was performed by the ABI Prism Big Dye Terminator Kit using an ABI PRISM 3100 DNA sequencer (Applied Biosystems). The nucleotide and deduced protein sequences were analysed using bioedit software (version 7.0.9.0.) (Hall, 1999) and blast network available at NCBI. The serological analysis confirmed that the strain investigated belong to the Ogawa serogroup. The antibiotic susceptibility pattern of V. cholerae, MCV09 revealed that it exhibited resistance to ampicillin, polymixin B, MLN0128 cell line co-trimoxazole, trimethoprim, streptomycin, spectinomycin, furazolidone, tetracycline, ciprofloxacin and nalidixic acid and intermediate resistance to norfloxacin, neomycin and ofloxacin, while it showed susceptibility to gentamycin, chloramphenicol and cephotaxime only. The MIC values for ciprofloxacin, nalidixic acid, tetracycline and trimethorim were found to be 1, 64, 8 and >32 μg mL−1, respectively. The PCR analysis revealed the presence of SXT and drug resistance genes viz dfrA1, strB and sul2 Ferroptosis inhibitor drugs (Fig.
1). The results of PCR correlated with antibiotic resistance phenotypes. The sequencing and blast analysis of the int gene of MCV09 indicated that Idoxuridine it is 1242 bp (GenBank accession no. GQ495075) in size and 96% similar to int of MO10. The comparison of the deduced amino acid sequence (413 residues) showed substitution of Ser-148-Ala, Ser-198-Gly, Ser-333-Gly and Val-334-Ile. The last three substitutions were similar to the variant of SXT reported from Vibrio fluvialis. The PCR analysis of the attP attachment sites of MO10 and MCV09 indicated a difference in the amplicon size. The MCV09 yielded a 641-bp product in contrast to 785 bp of MO10 (Fig. 2). The sequencing and blast analysis of this product (GenBank accession no. GQ495076) revealed that it is similar to the attP site of V. fluvialis
(GenBank accession no. AB125369) rather than V. cholerae. Similar results were obtained when attP attachment sites were amplified and sequenced from MCV08 (GenBank accession no. GQ495077) and A880 (GenBank accession no. GQ495078) isolated recently from Kerala (Fig. 2). Interestingly, sequencing results also showed a single base substitution (C to T) in 17-bp attP core sequences in MCV09 as well as in all recently isolated O1 strains of clinical and environmental origin (Fig. 3). Collectively, these results confirm the presence of a variant of an SXT in MCV09 as well as recently isolated O1 strains characterized in the present investigation. The conjugation experiment of MCV09 with E. coli revealed that the variant SXT element could transfer all drug resistance genes to recipient E. coli.
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