RESUMO
Objective To investigate the mechanism of the different levels of ciprofloxacin resistance in qnrA-containing transconjugants.Methods E. coli J53AzR as the recipient,4 qnrA-containing transconiugants were constructed by conjugation from 4 qnrA-carrying clinical isolates.MICs of the transconjugants were measured by E test.aac(6')-Ib-cr was detected by PCR,and qnrA mRNA expression level was determined by real-time RT-PCR.The promoter sequences of qnrA were amplified by PCR from qnrA-bearing plasmids and cloned into plasmid pKK232-8,then transformed into HB101.All promoter fragments were sequenced.Resuits The MICs of ciprofloxacin against 4 transconjugants demonstrated a 10-fold difference from 0.094 μg/ml to 1.000 μg/m1.Of 4 qnrA-bearing plasmids in E.coli J53,ciprofloxacin MICs of pHS4 and pHS5 were 0.094 μg/ml and 0.125 μg/ml,respectively;pHS3,which contained the aac(6')-Ib-cr gene as well,MIC was 0.25μg/ml;and pHS5,which had a high expression level of qnrA and the aac(6')-Ib-cr gene,MIC was 1.00μg/ml.The relative expression levels of qnrA mRNA in J53 pHS6 was 32.5,much higher than the other 3 transconjugants(from 1.0 to 2.5).The promoter in plasmid pHS6 was 12-fold stronger than that in the other 3 plasmids.Compared with pHS3,there was 7 bp(GTTAGCA)deletion between the transcription initiation site and the start of qnrA in pHS6.Conclusion Co-existence of qnrA and aac(6')-Ib-cr in a single plasmid and high level of qnrA expression can account for the different levels of ciprofloxacin resistance in transconjugants.