Impact Of OqxAB-Positiveplasmids On The Development Of Fluoroquinolone Resistance And Bacterial Fitness In Escherichiacoli

Plasmid-mediated quinolone resistance(PMQR)determinants can confer low-level resistance to fluoroquinolones and their worldwide spread among Enterobacteriaceae may facilitate the emergence of fluoroquinolone resistance.oqx AB,as one of the most popular PMQR determinants in China,mediates multidrug efflux pump which confers resistance to multiple agents,including quinolones and quinoxalines.In this study,inducing experiments and bacterial fitness will be conducted to investigate the role of plasmid-borne oqx AB on the process of quinolone resistance development.Fifteen oqx AB-positive E.coli strains obtained from food animals,companion animals,healthy volunteers,and retail meat in 2010–2011 were randomly selected andsubjected to transformation experiments.Single oqx AB-carrying plasmid from E.coli strain GAC59 and FKD436 F was successfully transferred to the E.coli recipient strain TOP10,designated as pHNGC59(~75-kb)and pHNFD436(~50-kb),respectively.Plasmid pHNGC59 belonged to the IncF type,but the replicon type for pHNFD436 could not be determined with the PBRT method.Compared with the E.coli recipient strain TOP10,theoqx ABcarrying plasmids contributed to a 4-8 fold increase of the ciprofloxacin MIC and also increased the ciprofloxacin MPC by8-16-fold.Furthermore,p HNGC59 and p HNFD436 remained stable without selective pressure and their acquisition rarely affected the growth of E.coli TOP10.Two transformants harboring the OqxAB-encoding plasmids and recipient strain TOP10 were selected to subjected to serial passages with 0.5×MIC of ciprofloxacin as to investigate the development of fluoroquinolone resistance with/without plasmid-borne oqx AB.The results showed that the MIC of ciprofloxacin for the two transformants increased faster than that of E.coli TOP10 by serial passaging.Furthermore,TOP10:p HNGC59 and TOP10:pHNFD436 developed resistance to ciprofloxacin after 16 or 12 passages(MIC >1mg/L),respectively,whereas TOP10 was still susceptible after 20 passages.Additionally,novel mutations in gyr B(A468P or F458V)were observed in two transformants.Mutations in gyrA were distributed at codons 83(novel mutation ?S83)and 87(D87Y or D87N)in the two transformants,whereas mutation A119 E in gyrA dominated in the TOP10 mutants.The two oqx AB-bearing plasmids caused a decrease in fitness in vitro(13% and 24%,respectively),but their fitness increased when combined with more than one chromosomal mutation in vitro,and biological benefits were observed in vivo.The mutations in gyr B were associated with a fitness cost,which could be compensated for by additional mutations.The novel mutation gyrA?S83,only appeared after 5 and 6 passages for two transformants,significantly reduced biological fitness both in vitro and in vivo,and was thus quickly replaced by more beneficial mutations in the population.Similarly,the gyrA mutation D87 Y had a fitness advantage over D87 N and may be responsible for the disappearance of the gyrA D87 N mutation after 12 passages.Furthermore,the mutant TOP10-5D1(gyrA A119E)had significantly increased fitness compared with the mutant TOP10-5D5 without a gyrA mutation.Interestingly,the mutant TOP10-16D1(gyrA A119E)displayed the highest fitness value(2.034)with a 64-fold increase in the MIC of ciprofloxacin,suggesting that other undetected mutations may contribute to the increased MIC and bacterial fitness.In conclusion,although both oqx AB-carrying plasmids cause a fitness cost,they can be stably maintained without selective pressure.Additionally,the presence of plasmid-borne oqx AB facilitate the rapid development of fluoroquinolone resistance,and the biological cost could be compensated by two or more additional chromosomal mutations.