Study On The Effect Of Ciprofloxacin On The Pseudomonas Aeruginosa Regulation Of Quorum Sensing Genes In Vitro

Objective:The study is to investigate the effect of different ciprofloxacin?CIP?concentrations and induction times on the Pseudomonas aeruginosa?PA?resistance,virulence factors and quorum sensing?QS?genes,after the in vitro induction of sensitive PA by different concentrations of CIP.Further to determine whether CIP participated in the QS regulation to virulence factors during the induction process,providing a basis for clinical rational use of quinolones.??Effect of CIP on resistance to PA in vitroMethods:1.Selection of experimental strains:40 PA non-repetitive clinical isolates from2012 to 2013 were collected early in the research group.The strains were re-identified by VITEK-2.Strains that were sensitive to CIP were screened by determining the minimum inhibitory concentrations?MICs?to CIP.Finally selected the strains derived from sterile body fluids and wound exudates to make up the experimental strains together with PAO1.2.In vitro induction of CIP:According to the MICs value of each experimental strain to CIP,they were inoculated to the corresponding agar plates with the CIP concentrations of 0.5×MIC,1×MIC,2×MIC,and 4×MIC.The inoculation was subcultured each 24 hours,and not stop until 5 days.The strains on the 1st day of induction were defined as 1st generation strains,2nd day were defined as 2nd generation strains,and so on.The 1,3,and 5 generations of strains were stored separately.3.Determination of the resistance and statistical analysis:The MICs of all the stored strains to CIP were determined by the agar dilution method,and the change of resistance rates at different induction concentrations and times was calculated.After the lg transformation,MICs were analyzed by repeated measures analysis of variance to investigate the effect of different CIP concentrations on the MICs.P<0.05 was statistically significant.Results:1.Selection of experimental strains:The 40 isolates of non-repetitive PA collected in the early research group mainly consisted of 26 strains?65%?of sputum specimens,followed by 5?12.5%?wound secretions and 3?7.5%?urine strains and blood2 strains?5%?and so on.Isolates were obtained from clinical inpatient departments.Among them,the most isolated were ICU 7?17.5%?and respiratory 7?7.5%?,followed by 6?15%?in hematology,5?12.5%?in neurology and orthopedics 5?12.5%?.Among the 40 strains,31 strains were sensitive to CIP?77.5%?,among which 11 strains were from sterile body fluids and wound secretions.The experimental strains consisted the 11strains PAO1.2.In vitro induction of CIP:The 12 strains were induced for 5 generations at four concentrations:0.5×MIC,1×MIC,2×MIC,and 4×MIC.Two of the strains at 4×MIC concentration and one strain at 2×MIC concentration were discarded because they did not grow always continuous culturing for 48 hours.Therefore,a total of 9 strains were discarded,and 135 strains were successfully induced and stored.3.Determination of the resistance and statistical analysis:The MICs to CIP were detected in all the successful strains.The results showed that the resistance rate increased gradually with the induction of each concentration.By the fifth generation,the resistance rate increased to 58.33%¹00.00%.After repeated measurement analysis of variance,the interaction between induction time and concentration was significant?P<0.001?,ie,with the prolongation of induction time,the MICs in different inducing concentrations showed different trends.The fastest increasing concentrations in 1,3,and5 generations were respectively 4×MIC,4×MIC and 2×MIC.There was a significant difference between the main effect induction time?P<0.001?,that is,the MICs gradually increased during the induction process and the growth rate gradually decreased.The difference between the concentrations was significant?P=0.001?,ie,the ability to induce MICs increase was:4×MIC>2×MIC>0.5×MIC>1×MIC.??Effect of CIP on virulence factors in vitroMethods:1.Swimming:A single colony was picked with an inoculum needle and inserted into a tryptic plate.The culture was incubated at 32?for 16 hours.The diameter of the turbid circle was measured.All experiments were repeated three times.2.Pyocyanin:The activity of pyocyanin was determined by two-way extraction methhod?chloroform and hydrochloric acid?.OD520/OD600 was used to indicate pyocyanin activity.All experiments were repeated three times.3.Statistical Analysis:The effects of different CIP concentrations on the virulence factors?motility,pyocyanin?were studied using repeated measures analysis of variance.The single factor analysis method was used the t-test or Kruskal-Wallis rank sum test based on the normality of the data.Results:1.Swimming:After repeated measurement analysis of variance,there was no significant difference between the induction time and the induction concentration?P=0.157?.That is,with the prolongation of induction time,the variation trend of the swimming ability of each concentration were same.There was a significant difference in the main effects induction time?P<0.001?,ie,with the induction time prolonged,the swimming was firstly suppressed then promoted.There was no significant difference between the induced concentrations?P=0.910?,but the single-effect analysis showed that the inhibitory effect of CIP at 0.5×MIC and 1×MIC decreased with time,and the promoting effect gradually increased;while 2×MIC and 4×MIC only has a promoting effect and does not change significantly with time.The study also found that each concentration had a weak inhibitory effect on the swimming ability at the 1st induction day;at the 3rd day each concentration had a weak promoting effect;by the 5th day,there was a different promotion effect of each induced concentration,the difference is that0.5×MIC is the strongest and 4×MIC is the weakest.2.Pyocyanin:After repeated measures analysis of variance,there was no significant difference in the interaction between induction time and induction concentration?P=0.528?.That is,with the prolongation of induction time,the changes of pyocyanin were the same in all concentrations.There was no significant difference in the main effects induction time?P=0.404?,and there was no significant difference between the induced concentrations?P=0.901?.That is,it is promoted by a similar degree inducing by the different CIP concentration for 5 days.However,it is worth noting that the 4×MIC concentration can inhibit the pyocyanin,and the inhibitory effect gradually increases with the induction time.??The effect of CIP on the QS-related genes expressionMethods:1.Screening of strains:Excluded the incompletely induced strains of 12 strains.Random data sheets were used to randomly screen 3 strains.The 3 strains,PAO1 and their respective inducing strains were formed the experimental strains.2.Quantitative analysis of QS-related gene expression:Extracted the total RNA of the experimental strains,cDNA was reversely synthesized.Designed the PA QS-related genes primers of lasR,lasI,rhlI,rhlR,and pqsR.GAPDH was used as an internal reference gene.Then amplified by RT-PCR.Amplification curves were plotted,and the gene expression level was determined by 2^-??CT.3.Statistical analysis:The effect of different CIP concentrations on the QS-related genes expression was studied by repeated measures analysis of variance.The single factor analysis method was used the t-test or Kruskal-Wallis rank sum test based on the normality of the data.The correlation between QS gene and MICs,between QS gene and virulence factor?swimming and pyocyanin?,between all the QS genes were analyzed by the Pearson correlation coefficient.Results:1.Screening of strains:9 strains?including PAO1?were obtained after excluding the incompletely inducing strains in 12 strains.3 strains were randomly selected from the8 strains except PAO1 using a random data table method.The 3 strain,PAO1 and the respective induced strains were made up the experimental strains including totally 52strains.2.QS-related gene expression:There was no interaction between the induction time and concentration?P>0.05?after repeated measurement of variance analysis,that is,with the prolongation of induction time,the expression of QS related genes in all CIP concentrations were down-regulated that gradually weakened,and up-regulated that gradually enhanced.The upregulation intensity was:rhlR>lasI>lasR>pqsR>rhlI.The main factor of induction time:There was no difference in the times of lasR and pqsR genes?all P>0.05?,that is,CIP induced with the similar up-regulation degree of lasR and pqsR genes at different times;The difference in times of lasI,rhlI,and rhlR genes was significant?P<0.05?,that is,the las I and rhlI genes were first down-regulated and then up-regulated,while the rhlR genes were gradually enhanced over time.There was no difference in the concentration of each gene?P>0.05?,that is,the induction of different concentrations increased the expression of lasI,lasR,rhlI,rhlR and pqsR genes to a similar degree.3.Relationship between QS-related genes and phenotypes:The statistical results show that the expression of lasI,lasR,and rhlI genes during the induction of CIP is positively related to the gradual increase of MICs?P<0.05,r>0?.The rhlR gene had a low positive correlation with the first-inhibition and post-promotion of the swimming ability?P<0.05,r=0.417?.There was no significant correlation between the expression of each gene and the promotion of pyocyanin?P>0.05?.4.Correlation between the QS-related genes:There was a positive correlation between the expression levels of lasI,lasR,rhlI,rhlR and pqsR genes?P<0.05,r>0?.This suggests that the QS system in Pseudomonas aeruginosa is complex and interrelated.So it is difficult to determine that the expression of a virulence factor is independently regulated by a single gene.The correlation degree results showed that lasI and lasR both had three high correlations?r?0.8?and one moderate correlation?0.5?r<0.8?with other genes,and had a greater degree of correlation than other genes.It is suggested that the las system may be the main regulation system in the QS system.Conclusion:1.Induction of CIP caused P.aeruginosa MICs gradually increased and the increasing rate gradually decreased.The induction ability was:4×MIC>2×MIC>0.5×MIC>1×MIC.2.The low concentration of CIP induced the swimming ability of P.aeruginosa firstly be inhibited and then be promoted,while the high concentration promoted the similar degree with the time prolonged.3.The induction of CIP at different concentrations promoted the similar degree of P.aeruginosa pyocyanin.4.The induction of CIP caused P.aeruginosa lasI and rhlI genes to be down-regulated and then up-regulated with the time prolonged,while the rhlR gene was continuously up-regulated.5.The expression of P.aeruginosa las I,lasR and rhlI genes had certain correlations with the change of MICs;the expression of rhlR gene had a low correlation with the ability of swimming.