Study Of Qnr Gene To Mutant Selection Window And The Effects Of Fitness Cost On Bacterial Quinolone Resistance

Objectives In this study,it is proposed to reconstruct experimental strains contain qnr gene.Through detected the minimum inhibitory concentration,mutant prevention concentration and the fitness cost of reconstruct experimental strains.Explore the effect of qnr gene to mutant selection window and the effects of fitness cost on bacterial quinolone resistance.Methods 1.Using TA cloning and other molecular biology methods to reconstruct experimental strains containing qnr gene and through Blue-White screening as experiment subjects.2.The agar dilution method was carried out on Mueller-Hinton agar containing quinolones according to CLSI guidelines to determine minimum inhibitory concentration(MIC)of nalidixic acid,levofloxacin,ciprofloxacin,norfloxacin and moxifloxacin against reconstruct experimental strains.Mutant prevention concentration(MPC)was determined by broth enrichment method according to MIC,and calculate mutant selection window(MSW)and mutant prevention index(MPI).3.Measure the growth curve of the experimental strains by broth culture method and comprehend the growth characteristics of the experimental strains at different stages.4.In vitro competition experiments were conducted on the three groups(E.coli TOP10 qnr A/E.coli TOP10 p GMT group,E.coli TOP10 qnr B/E.coli TOP10 p GMT group and E.coli TOP10 qnr S/E.coli TOP10 p GMT group).In vitro competition and cross inhibition experiment between three E.coli TOP10 qnr strains using broth co-culture method,analyze the growth performance and fitness cost between two strains in the same growth environment.5.The in vitro bactericidal effect of quinolones(nalidixic acid,levofloxacin,ciprofloxacin,norfloxacin and moxifloxacin)on E.coli TOP10 qnr strains was implemented by broth culture method,the in vitro bactericidal effect of quinolones was analyzed and describe the time-kill curves.Results 1.In this study,the minimum inhibitory concentration(MIC)range of nalidixic acid to E.coli TOP10 qnr was 4-8 μg/ml,MIC of levofloxacin was 0.125 μg/ml,ciprofloxacin was 0.0625-0.125 μg/ml,norfloxacin and moxifloxacin was 0.125-0.25 μg/ml.2.The mutant prevention concentration(MPC)range of nalidixic acid to E.coli TOP10 qnr was 32-64 μg/ml,MPC of levofloxacin was 0.5-1 μg/ml,ciprofloxacin was 0.25-0.5 μg/ml,norfloxacin was 0.5-2 μg/ml and moxifloxacin was 0.25-0.1 μg/ml.The range of mutation prevention index(MPI)of quinolones to E.coli TOP10 qnr was 2-8.3.From the growth curve,each strain entered the logarithmic growth phase was about 3 h and each strain entered the plateau phase about 11 h.With the increase of cultivation time,the number of bacteria increases with the extension of the culture time,number of bacteria has also gradually increased,no significant difference in growth(P>0.05).4.The fitness cost of the strain in vitro competition experiment between E.coli TOP10 qnr A with E.coli TOP10 p GMT was 1.030,E.coli TOP10 qnr B with E.coli TOP10 p GMT was 1.056,E.coli TOP10 qnr S with E.coli TOP10 p GMT was 1.016,no statistically significant in competition experiment(P>0.05).5.The fitness cost of the strain in vitro competition experiment between E.coli TOP10 qnr A with E.coli TOP10 qnr B was 1.002,E.coli TOP10 qnr B with E.coli TOP10 qnr S was 1.064,E.coli TOP10 qnr A with E.coli TOP10 qnr S was 0.939(P<0.05),differences were statistically significant.6.In vitro cross competition experiment,probably produce some cross-inhibitory effect between E.coli TOP10 qnr A with E.coli TOP10 qnr S strain.7.When the quinolones concentration was 0.5× MIC or 1× MIC,the time-kill effect is not very significant,more obvious bactericidal effect in 2× MIC.With the increase of drug concentration,the bactericidal capacity gradually increased.Conclusion 1.qnr gene can reduce the susceptibility of bacteria to quinolones and have different sensitivity to fluoroquinolones,which is mediated low level quinolone resistance.2.qnr gene can increase the MPC of bacteria relatively and broaden the range of mutation selection windows,there is no significant difference in the range of mutant selection window of three kinds of qnr genes to increase bacterial quinolone resistance.3.qnr gene may have no obvious effect on the growth of bacteria,the qnr gene has no fitness cost to bacteria,the low fitness costs between the qnr A strain and qnr S strain indicate that the two may have a weaker competitive relationship,and advantages of growth and competition may lead to the epidemic clonal of qnr S strains in the same environment.