Characterisation of novel mutations involved in quinolone resistance in Escherichia coli isolated from imported shrimp

Fifty-five nalidixic acid-resistant Escherichia coli strains were isolated from imported shrimp. Purified PCR amplicons of gyrA, gyrB, parC and parE from the template DNA of all isolates were sequenced and analysed for point mutations that confer resistance to nalidixic acid and ciprofloxacin. Point mutations in the quinolone resistance-determining regions (QRDRs) of GyrA at positions 68, 83 and 87 and in ParC at positions 80 and 84 as well as in the non-QRDR of GyrA at positions 112, 127, 128 and 154 along with point mutations in parE at position 476 conferred resistance to these antibiotics. Computational modelling and analysis of the different point mutations and their role in the enhanced resistance to these antibiotics indicated that only mutation at codons 83 (Ser→Ile) and 87 (Asp→Asn) played a vital role in increasing the minimum inhibitory concentration (MIC) to these drugs compared with other mutations. Ethidium bromide experiments indicated higher efflux pump activities in quinolone-resistant E. coli strains compared with their quinolone-sensitive counterparts. Class 1 integrons measuring 0.7-2.3 kb were amplified and sequenced from the template DNA of the isolates. Sequence analysis of the 2.0 kb and 1.7 kb integrons indicated the presence of resistance determinants for trimethoprim (dfrA12 and dfrA17) and aminoglycosides (aadA2 and aadA5). These results indicate that use of nalidixic acid, ciprofloxacin and other antibiotics in shrimp aquaculture ponds may select E. coli resistant to these antibiotics and that imported shrimp is a reservoir of multiple antibiotic-resistant E. coli.