Study On The Mechanisms Of Plasmid-mediated Quinolone Resistance In Enterobacteriaceae

Quinolones are broad-spectrum antibacterial agents,commonly used in clinical medicine.Their extensive use has resulted in bacteria rapidly developing resistance to these agents.The traditional understanding considered that quinolone resistance is acquired only through chromosome mutations and transmitted only vertically.But resently the plasmid-mediated horizontally transferable genes encoding quinolone resistance including qnr genes,a variant aminoglycoside acetyltransferase gene aac(6')-Ib-cr and efflux pump gene qepA have emerged.Both mechanisms provide low-level quinolone resistance that facilitates the emergence of higher-level resistance in the presence of quinolones at therapeutic levels.Their horizontal spread and co-selection with other resistance elements,especially withβ-lactamase genes,indicate that a more cautious approach to quinolone use is needed.Our objective was to characterize the prevalence of plasmid-mediated quinolone resistance determinants qnr,aac(6')-Ib-cr and qepA in extended-spectrumβ-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae,qnrA,qnrB,qnrS, aac(6')-Ib-cr,qepA and ESBL-encoding genes were detected by PCR.MICs of 10 antimicrobial agents were determined by Etest.Pulsed-Field Gel Electrophoresis (PFGE) was used to investigate the clonality of qnr- and aac(6')-Ib-cr-producing isolates.Conjugation and Southern hybridizations were used to confirm whether qnr, aac(6')-Ib-cr or ESBL-encoding genes were located on plasmids.Twenty-nine(8.0%) of 362 isolates were positive for qnr genes,and the qnrA-type,qnrB-type,and qnrS-type genes were detected alone or in combination in 13(3.6%),8(2.2%) and 9 (2.5%),respectively.Sixty-two(17.1%) isolates were positive for aac(6')-Ib,of which 36(9.9%of all) had the -cr variant.Conjugation and Southern hybridization revealed that qnrA,aac(6')-Ib-cr and ESBL-encoding genes were always located on the same plasmids.The plasmids carrying the qnr gene could be transferred by conjugation together with ESBL-encoding genes and aac(6')-Ib-cr.The multiresistance plasmid pKP96 from Klebsiella pneumoniae was sequenced completely and analysed concerning its genetic environment and distributing of antimicrobial resistance genes.The complete sequence of the plasmid was determined using a whole-genome shotgun approach.MICs of 13 antimicrobial agents were performed using Etests.Conjugation experiment was determined in liquid medium. pKP96 was a circularly closed 67,850-bp multiresistance plasmid with an IncN incompatibility group.Seventy putative genes were identified according to the annotation of the finished sequence,including 9 antimicrobial resistance genes.The backbone region of the plasmid,comprising the conjugal transfer and plasmid replication regions,showed 91%identity to the IncN plasmid R46.Several mobile elements were found to be inserted into pKP96 together with antimicrobial resistance genes,including qnrA1,aac(6')-Ib-cr and bla_CTX-M-24.Plasmid pKP96 is a chimera that has acquired its multiple antimicrobial resistance determinants horizontally from different sources.It may have evolved from an ancestor plasmid similar to R46 through the stepwise events of integration or recombination.