Mechanism Of Resistance And Biofilm Of Pseudomonas Aeruginosa Induced By Fluoroquinolones In Vitro

Pseudomonas aeruginosa(PA)belongs to non-fermented gram-negative bacilli,carrying huge genetic information to enable it to survive in various environments.PA is the second major cause of nosocomial infections.There are significant morbidity and mortality caused by infections such as pneumonia,wound infections,bloodstream infections,and urinary tract infections.The mechanisms of PA resistance include the formation of biofilms and high expression of active efflux systems.DNA gyrase and topoisomerase IV mutations are the main mechanisms of fluoroquinolone-resistant PA.Fluoroquinolones are commonly used in the treatment of PA infections,especially when?-lactam drug allergies or other causes cannot be used,or as combination therapy,including ciprofloxacin(CIP)and Levofloxacin(LEV)is the most widely used.In this study,CIP and LEV were used as inducing drugs to construct an induced PA model in vitro,which covers a range of inducing concentrations of sub-inhibitory concentrations to high concentrations to simulate different doses of clinical antibacterial agents,aims to from two dimensions of induction concentration and induction time and two levels of phenotype and genotype to analyse the effects on strains induced by CIP and LEV of fluoroquinolone drugs resistance,biofilm formation ability and resistance mechanism with the series induction times and concentrations.1.The effects of Pseudomonas aeruginosa induced by ciprofloxacin and levofloxacin on fluoroquinolones resistance in vitroObjective:To investigate the effect of clinical isolates of sensitive Pseudomonas aeruginosa on fluoroquinolone resistance induced by ciprofloxacin and levofloxacin in vitro.Methods:(1)Preparation of experimental strains:Clinically isolated PA sensitive to both CIP and LEV were collected.CIP and LEV were used as induction drugs with 4 different induction concentrations of 0.5×MIC,1×MIC,2×MIC,and 4×MIC respectively in vitro induced by 5 days.The strains on the Day1,Day3 and Day5 were preserved.The PA strains induced by CIP were defined as CIP group,and the PA strains induced by LEV were defined as LEV group.(2)Effect of drug induction on the minimum inhibitory concentration(MIC):The PA of the original strains and the CIP group and the LEV group were used as the experimental objects.The agar fold dilution method was used to determine MIC values to the ciprofloxacin,levofloxacin,ofloxacinand and moxifloxacin.(3)Statistical analysis was done using the SPSS version 22.0.The influence of induction drugs,concentrations and times was on MIC values was analyzed ANOVA for repeated measurement.Results:(1)A total of 11 strains were sensitive to CIP and LEV.Samples from sterile body fluids and wound secretions.And the PAO1(No.12,ATCC15692)were included in the experiment.After induction by CIP,a total of 135 strains were preserved and 114 strains were stored after induction by LEV.(2)Without consideration the influence of induction concentrations,the effect of induction times on the MIC values of the CIP and LEV strains showed that with the increase of induction times,the MIC values of both groups showed an increasing trends,p<0.001.Without consideration the influence of induction times,the effect of induction concentrations on MIC values showed that with the increase of the inducing concentrations,the MIC values of both groups also showed an increasing trends,p<0.001.(3)The effect of CIP and LEV drug on the MIC values as the induction times increased showed that there was an interaction between the induction drugs and times,p<0.001.The trends of MIC values of the two groups varied with the increase of induction times.On the Day1,the MIC values of the LEV group was higher than CIP group,p<0.05.On the Day3 and Day5,the MIC values of the CIP group were higher than LEV group,p<0.05.(4)The effect of induction concentrations on the MIC values with the increase of induction times showed that there was interactions between concentrations and times for CIP or LEV group,p<0.001.With the increase of induction times,the four concentration trends of the MIC values were different.The MIC values increases as the concentration increases.2.The effects of ciprofloxacin and levofloxacin on biofilm formation of Pseudomonas aeruginosaObjective:To investigate biofilm formation of clinical isolates of Pseudomonas aeruginosa induced by ciprofloxacin and levofloxacin in vitro.Methods:(1)The study subject including original isolates,strains induced by ciprofloxacin and levofloxacin were tested for their ability to form biofilm by crystal violet staining assay.Each strain was carried out three times.After 72h continuous incubation,the optical densities(OD)of stained adherent bacterial biofilms were read at 570nm,then the results were averaged.(2)Statistical analysis was done using the SPSS version 22.0.The influence of induction drugs,concentrations and times was on biofilm formation was analyzed ANOVA for repeated measurement.Results:(1)Without consideration of induction concentrations,the effect of induction times on biofilm of CIP group and LEV group strains showed with extension of induction times,the OD values of two groups changed continuously,p<0.05.Without consideration of induction times,the effect of induction concentration on biofilm of the two groups showed that OD values of the two groups did not differ statistically with the increase of induction concentrations,and p>0.05.(2)The effect of induction drugs on biofilms with extension of induction times showed there was interaction between induction times and drugs,p=0.004.The trends of OD values of two groups varied with the increase of induction times.The biofilm of the CIP group exhibited a process of promoting first and inhibiting during the study,while the LEV group as a whole was an inhibitory process.(3)The effect of agents concentrations on biofilms with extension of induction time showed there was no interaction between agents concentrations and induction time.The trend of OD values of four concentrations of two agents were coincident,in other words,it was no statistically significant(p>0.05).Biofilm formation was advanced at first and then inhibited on four concentrations of CIP group while inhibited at first and then advanced on four concentrations of LEV group.3.Mutations of DNA gyrase and topoisomerase IV genes of fluoroquinolones-resistant Pseudomonas aeruginosa which induced by ciprofloxacinObjective:The aim of this study was to examine mutations in the quinolone-resistance-determining region(QRDR)of gyrA,gyr B,parC and parE genes in fluoroquinolones-resistant Pseudomonas aeruginosa isolates which induced by ciprofloxacin.Methods:(1)In the CIP group,the strain number induced by three induction times were selected,and resistant PA against four fluoroquinolones were further selected.The primers of quinolone resistance determining region(QRDR)of gyr A,gyrB,parC and parE genes were designed and amplified by polymerase chain reaction(PCR).The product fragment size was determined by agarose gel electrophoresis and DNA products were directly sequenced.(2)Snapgene software was used to compare the base sequence and amino acid between experimental strains and PAO1 sequence of Gene Bank.The effect of induction concentrations and times on gene mutation was analyzed.Results:(1)A total of 4 original strains and 23 strains induced by CIP were selected for PCR amplification experiments.(2)The QRDR sequence of the gyrA,gyrB,parC and parE genes of the original No.12(PAO1)strain was identical to the Gene Bank PAO1 sequence.(3)The QRDR of gyrA gene sequence alignment results:Only No.1 original strains had 1 base change(283 from C to T).Induced strains which 2×MIC(No.1),0.5×MIC(No.10),4×MIC(No.11),0.5×MIC(No.12)on Day5 and 4×MIC(No.12)on Day3 showed amino acid mutations,respectively were 54 from glutamic acid to lysine(Glu?Lys),83from threonine to isoleucine(Thr?Ile),128 from alanine to the threonine(Ala?Thr),83from threonine to isoleucine(Thr?Ile)and 139 from glutamic acid to histidine(Glu?His).In addition to the high concentration induction of PAO1 strain,the amino acid mutation occurred in Day3,and the other strains on Day5 whether the concentration of sub-inhibition or high concentration.Compared with the original strain,inducted strains with the increase of induction times and concentrations,there were also an increase in the unmeaning base mutations.(4)The QRDR of gyrB gene sequence alignment results:The No.10 and No.11original strains showed 2 base mutations(150 G?A,177 A?G).2×MIC(No.10)on Day5,4×MIC(No.10)on Day1 and Day3,4×MIC(No.11)on Day3 and 2×MIC(No.12)on Day5compared with original strain:not only the number of base mutation increased,but also 4amino acid mutations per strain,that were 372 amino acids from alanine to leucine(Ala?Leu),424 from isoleucine to leucine(Ile?Leu),464 from leucine to isoleucine(Leu?Ile),and 483 from glutamic acid to aspartic acid(Glu?Asp).(5)The QRDR of parC and parE gene sequence alignment results:The parC gene of No.1,No.10,No.11 original and induced strains and 12(PAO1)induced strains showed a base mutation(283 from T to G).The parE gene of No.1 original and induced strains showed a base mutation(474 C?T).Only No.12(PAO1)induced strain were occurred five bases mutations:371 G?A,392 T?C,398 T?C,407 C?T,545 G?T.The parC and parE genes did not increase the new base and amino acid mutations.4.Study on expression of active efflux pump gene of Pseudomonas aeruginosa induced by ciprofloxacinObjective:To investigate how the drug concentration and induction time affect expression level of MexA and MexE active efflux pump genes of Pseudomonas aeruginosa.Methods:(1)Screened No.1,No.10,No.11,No.12 and 48 strains inducted by CIP.Antibiotic susceptibility tests of PA to amikacin,piperacillin,piperacillin/tazobactam,ceftazidime,cefepime and meropenem were determined by disk-diffusion method.(2)The target genes were amplified by real-time PCR.PCR Primer designed against target gene include mexA and mexE,meanwhile the GADPH was used as reference gene.Futhermore,amplification curve were observed and relative expression of initial and inducted strains were calculated based on C_T value.(3)Statistical analysis was done using the SPSS version 22.0.The influence of induction concentrations and times on the expression level of genes was analyzed ANOVA for repeated measurement.Results:(1)No.10 strain were resistant to meropenem under concentration of 1×MIC,2×MIC,4×MIC on Day3,and under concentration of 1×MIC on day 5.Other strains after incubation were sensitive to above six drugs.(3)Without consideration of induction concentrations,the effect of induction time on relative expression of mexA and mexE showed with extension of induction times,relative expression of two genes changed,p<0.05.During the study period,relative expression of two genes were lower than 1,but the expression of mexA showed declined,and mexE showed declined then increased on Day3.Without consideration of induction times,the effect of induction concentration showed that relative expression of mex A and mexE did not differ statistically with the increase of induction concentrations,and p>0.05.(3)The effect of incubation concentrations on relative expression of mex A and mex E genes:as incubation time increased,relative expression of tow genes showed similar trend under any concentration,p>0.05.Mex A gene showed rising tendency and mexE gene showed first decline then increase.Conclusion:1.Within 24 hours,PA strains induced by LEV were more resistant to fluoroquinolone than CIP,but by CIP resistance was stronger than LEV with increasing induction time.2.In the CIP group,the ability of BF formation was promoted on the Day1 and the inhibitory effect was enhanced with time.With the increase of induction concentrations in LEV group,the inhibitory capacity of BF gradually increased,showing a dose-dependent manner.3.The fluoroquinolone-resistant strains induced by CIP,the drug resistance targets of the main mechanism exist in the QRDR region of DNA gyrase of gyr A and gyrB genes.Most strains of the gyrA gene have amino acid mutations on Day5.The time of amino acid mutation of gyrA gene in PAO1 strain induced by high concentration of CIP drug was advanced.4.After CIP induction,the relative expression levels of Mex A and MexE active efflux pump genes in PA were inhibited.