Alteration in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in Quinolone-resistant Shigella dysenteriae serotype 1 clinical isolates from Kolkata, India.

After the emergence of multidrug-resistant Shigella strains (8, 11), fluoroquinolones, such as ciprofloxacin and norfloxacin, were used in India to treat shigellosis. However, recently, genetically clonal ciprofloxacin-resistant Shigella dysenteriae serotype 1 strains have been isolated from sporadic and epidemic cases of dysentery in southern Asia, including eastern India (1, 9, 12; S. Dutta, A. Ghosh, K. Ghosh, D. Dutta, S. K. Bhattacharya, G. B. Nair, and S.-I. Yoshida, Letter, J. Clin. Microbiol. 41:5833-5834, 2003). Quinolone resistance is linked mainly to mutations located in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) (3, 4, 5). Replacement of residues Ser 80 and Glu 84 in parC commonly supplements gyrA mutations at residues Ser 83 and Asp 87 in members of the Enterobacteriaceae family to develop high fluoroquinolone resistance (13, 14). Besides topoisomerase mutations, overexpression of the energy-dependent multidrug efflux pump AcrAB due to mutations within repressor (AcrR) and multiple target and nontarget gene changes also contribute to fluoroquinolone resistance phenotypes in bacteria (2, 7, 15). In this report, we demonstrate the mutations in the QRDRs of gyrA, gyrB, parC, and parE genes of fluoroquinolone-resistant S. dysenteriae serotype 1 strains isolated in Kolkata, India. The type strain of S. dysenteriae serotype 1 (GTC 786) was procured from the culture collection of the Graduate School of Medicine, Gifu University, Gifu, Japan. Among 12 wild strains of S. dysenteriae serotype 1 included in this study, eight DS strains from sporadic cases (1) and one SKN outbreak strain (9) of dysentery showed resistance to fluoroquinolones, such as nalidixic acid, ciprofloxacin, and norfloxacin. The other three IBM strains were susceptible to all fluoroquinolones (8). These two groups of strains were compared to the type strain to determine any mutations. The QRDRs of the gyrA (648-bp), gyrB (309-bp), parC (249bp), and parE (290-bp) genes for all study strains were amplified with primer pairs designed from the genome sequence of S. flexneri 2457, which is available at the DBGET database. For gyrA, the forward primer 5 TAC ACC GGT CAA CAT TGA GG 3 (nucleotide [nt] 24 to 43) and the reverse primer 5 TTA ATG ATT GCC GCC GTC GG 3 (nt 652 to 671) were used. For gyrB, the forward primer 5 TGA AAT GAC CCG CCG TAA AGG 3 (nt 1170 to 1190) and the reverse primer 5 GCT GTG ATA ACG CAG TTT GTC CGG G 3 (nt 1455 to 1479) were used. For parC, the forward primer 5 GTC TGA ACT GGG CCT GAA TGC 3 (nt 147 to 167) and the reverse primer 5 AGC AGC TCG GAA TAT TTC GAC AA 3 (nt 373 to 395) were used. For parE, the forward primer 5 ATG CGT GCG GCT AAA AAA GTG 3 (nt 1066 to 1086) and the reverse primer 5 TCG TCG CTG TCA GGA TCG ATA C 3 (nt 1334 to 1355) were used. The amplification was performed in a DNA thermal cycler as follows: (i) 30 cycles,