| Pseudomonas aeruginoisa（PA）is a gram-negative opportunistic pathogen and exists widely in nature and human skin,respiratory tract and intestine.It is one of the common drug-resistant bacteria in clinical practice that mainly causes ventilation respiratory infection,burn secondary infection,secondary pneumonia and systemic infection in patients with cystic fibrosis.At present,the high separation rate of the multidrug-resistant and extensively drug-resistant,in addition,PA strains cause serious infection,which leads to enormous difficulties in clinical treatment.Many acute and chronic infections were related to the virulence factors of PA.The activity of virulence factors become stronger,and the bacteria will be more rampant,which cause more serious symptoms of infection and more difficult treatment.The virulence factors of PA are regulated by four quorum sensing（Quorum Sensing,QS）systems,namely,las system and rhl system with hyperserine lactone as signal molecule,pqs system with quinolones as signal molecule,and iqs system.The QS network is organized a multi-layered hierarchy mechanisms and interact with each other,which is not independent and complex.The Pseudomonas quinolones signal（PQS）is a multifunctional and crucial signal molecule in the pqs system.It is involved in the regulation of various virulence factors bacterial population density,and nutrient transportion such as Fe3+,antibiotic sensitivity,membrane vesicle formation,and the suppression of the host immune system.This paper investigated the changes of sensitivity of PA under different induced schemes to common antibiotics and the effects of different induced schemes on the expression of virulence factors and QS genes.1.Effects of different induced schemes of exogenous PQS on sensitivity in Pseudomonas aeruginosaObjective:The study aims to investigate the changes of sensitivity of Pseudomonas aeruginosa under different induced schemes to common antibiotics.Methods:（1）Selection and treatment of strains:Collect clinical isolates from blood culture and aseptic body fluids that all sensitive to piperacillin,ceftazidime,amikacin,piperacillin-tazobactam,cefepime,ciprofloxacin and meropenem,after a process of identification by the VITEK-2 compact automatic microbial analysis system,with PAO1 as original strains.Original strains were inoculated in presence of exogenous addition of PQS of different concentrations（10,40,80μM）for various growth time（0,1,2,3,4,5d）,after growth,replace new medium,the strains cultivated for one day were induced strains on day1,simiarly,the strains cultivated for three days and five days were harvested and then saved,all saved strains and original strains were experimental strains.（2）The determination of susceptibility:MICs of CIP were measured for all strains saved by the agar dilution method,antibiotic susceptibility of strains to piperacillin,ceftazidime,amikacin,piperacillin-tazobactam,cefepime,and meropenemwas conducted by disc diffusion method.The results were reported and interpretation was done according to clinical and laboratory standards institute（CLSI）guidelines in 2017.（3）Statistical analysis:Statistical analysis was done using the SPSS version 20.0.The MICs of Pseudomonas aeruginosa under different induced schemes to CIP was analyzed ANOVA for repeated measurement.Results were considered significant when p value was<0.05.Results:（1）Experimental strains chosen:A total of 11 strains of sensitive strains from blood culture and sterile body fluids were collected.Together with PAO1,they were used as original strains.The strains cultivated for one day,three days and five days were harvested and then saved.12 strain series,a total of 120 experimental strains was obtained.（2）The MICs of experimental strains to CIP:The MICs of experimental strains under different induced schemes to CIP showed there was interaction between time and concentrations of PQS（P<0.05）,that is,the MICs showed different trends of variation with extension of exposure duration in the PQS of different levels;MIC of ciprofloxacin changes with different time without consideration of differences in induced concentrations were statistically significant（P<0.001）.（3）The diameters of inhibition zone to other antibiotics:The results showed the changes of the diameter of the inhibitory zone to other antibiotics under different induced schemes were not obvious.（4）The results of susceptibility:The susceptibility rates of all experimental strain to ciprofloxacin,piperacillin,ceftazidime,amikacin,piperacillin-tazobactam,cefepime,and meropenem were 100%.2.Effects of different induced schemes of exogenous PQS on virulence factors in Pseudomonas aeruginosaObjective:To investigate the effect of different induced schemes of exogenous PQS on swimming,pyocyanin,elastase and biofilm in Pseudomonas aeruginosaMethod:（1）The determination of the virulence factors:The swimming ability was determined by the size of the turbid ring,,and the pyocyanin activity is determined by a chloroform-hydrochloric acid two-phase extraction method,the activity of elastase was determined by the enzymatic and staining method,and the formation ability of the biofilm is determined by adopting a crystal violet staining method,and all the experiments were repeated three times independently.（4）Statistical analysis:The effects of different induced schemes of exogenous PQS on swimming,pyocyanin,elastase and biofilm were analyzed by repeated measurement of variance.Results were considered significant when p value was<0.05.Results:（1）The effect on swimming:On the whole,different induced schemes of exogenous PQS promoted the swimming ability of strains except that 80μM PQS inhibited the swimming mobility of PA stains on day5.When the concentration was 10μM,the swimming ability of strains continued to be promoted with time,when the concentration was 40μM and 80μM,the swimming ability of strains was increased first and then inhibited with the prolongation of time.ANOVA for repeated measurement analysis showed there was an interaction between the induced concentration of PQS and the induced time（P=0.048）,that is,swimming motility varies with exposure time and concentrations of PQS.The results of main effect analysis had no significantly difference.Further single effect showed when the induced concentration was 40μM,the effect of each induced time on the ability of the swimming was statistically different（P=0.006）,the most significant effect was found on day3.The effect of induced concentration on the swimming ability was statistically different on day3（P=0.0011）,the most significant effect was found at 40μM.（2）The effect on pyocyanin:Different induced schemes of exogenous PQS promoted the pyocyanin activity on the whole.When the concentration was 10μM,the activity increased gradually with the prolongation of induced time.When the concentration was 40μM and 80μM,the pyocyanin activity was increased at first and then decreased with time,and the activity of pyocyanin reached the maximum on day3.ANOVA for repeated measurement analysis showed there was no interaction between the induced concentration and induced time（P=0.574）.that is,the activity of pyocyanin showed the same change with the prolongation of induced time under different PQS induced concentration.Without considering the difference of concentration,the effect of different induced time on the activity of pyocyanin was statistically different（P<0.001）,and the activity increased first and then decreased with time.Without considering the difference of induced time,the effect of different induced concentration on the activity of pyocyanin was statistically different（P=0.028）,and the activity of pyocyanin gradually increased with the increase of concentration.Further single effect analysis:when the concentration was 80μM,the effect of different induced time on the activity of pyogenin was statistically different,and the activity of pyocyanin increased at first and then decreased with the prolongation of induced time.The pyocyanin activity was the strongest on day3.The effect of different concentration on the activity of pyocyanin was statistically different on day3（P=0.018）.With the increase of concentration,the activity increased gradually,and the activity of pyocyanin was the strongest when the concentration was80μM.（3）The effect on elastase:the activity of elastase was increased under different induced schemes of exogenous PQS compared with which before induced,and increased at first and then decreased with the prolongation of induced time.Specifically,theactivity of elastase increased with time at the concentration of 10μM,and the strongest activity of elastase was found on day1 when the concentration was 40μM.When the concentration was 80μM,the activity of pyocyanin increased at first and then decreased with the prolongation of time.ANOVA for repeated measurement analysis showed there was no interaction between induced concentration and time（P=0.460）,that is,the activity of elastase showed the same trend with the prolongation of induced time under different PQS concentrations.The effect of different induced time on the formation of elastase was statistically different（P=0.001）,and the activity of elastase increased first and then inhibited with the prolongation of induced time without considering the difference of induced concentration.Further single effect analysis:when the concentration was 10μM,the effect of different time on elastase activity was statistically different,and the activity of elastase increased gradually with the prolongation of induced time.When the concentration was 80μM,the effect of different time on elastase activity was statistically different.With the prolongation of induced time,the elastase activity increased first and then inhibited.（4）The effect on biofilm:Exogenous PQS had a weak inhibitory effect on biofilm formation of strains compared with that before induction on the whole.The analysis of variance showed that there was no interaction between the induction concentration of PQS and the induction time（P=0.864）,that is,the biofilm formation ability showed the same trend with the prolongation of induced time under different induced concentrations.The results of main effect analysis and single effect analysis were not statistically significant.3.Effects of different induced schemes of exogenous PQS on the expression of QS genes of Pseudomonas aeruginosaObjective:To explore the effect of different induced schemes of exogenous PQS on the expression of QS genes of Pseudomonas aeruginosa,and further explore the relationship between virulence factors and the QS genes.Method:（1）Strain Selection:Three series of strains were randomly selected by drawing lots from No.1-11 strains,together with PAO1 series used as totally 48 experimental strains（2）Measurement of QS gene expression:the primers of lasI,lasR,rhlI,rhlR and pqsR were designed,extract the total RNA of the strain,and the cDNA was synthesized by reverse transcription.The GAPDH was used as the internal reference gene and amplified by the real-time fluorescent quantitative PCR.The relative quantitative 2-△△CTmethod was used to analyze the RT-PCR data.（3）Statistical Analysis:The effect of different PQS induced schemes on the expression of QS genes was analyzed by analysis of variance of repeated measures.The correlation between QS gene expression and virulence phenotype was analyzed by Pearson product difference.Results were considered significant when p value was<0.05.Results:（1）Strain Selection:Finally,the number of 0,1,5 and 10 series of strains were used to carried out QS gene amplification experiments.（2）Expression of QS genes:On the whole,PQS up-regulated the expression of lasI,rhlI and pqsR genes and slightly down-regulated the expression of lasR and rhlR.ANOVA for repeated measurement analysis showed there was interaction between induced time and concentration on lasI,rhlI rhlR and pqsR（P<0.05）,that is,with the prolongation of induced time,the variation trend of gene expression in different concentrations was different.For lasR,there was no interaction between induced time and concentration,that is,with the prolongation of induced time,the variation trend of relative gene expression was the same for each concentration.Without consideration of induced time,the relative expression of pqsR was significantly affected by induced concentrations,and the up-regulation was enhanced with the increase of concentration.The effect of different induced time on the relative expression of each gene was statistically significant without consideration of induced concentration（P<0 05）.With the prolongation of induced time,the relative expression of lasI was upregulated,and the trend of upregulation increased first and then weakened.Both lasR and rhlR were down-regulated in varying degrees;rhlI was first up-regulated and then decreased,;pqsR was up-regulated first and then slightly down-regulated,and the upregulation increased first and then decreased gradually.（3）Correlation between QS genes expression and virulence phenotype:The rhlR gene was highly negatively correlated with the first promotion and then inhibition of swimming ability induced by different concentrations of PQS for different time（P=0.001,r=-0.921）,lasI was lowly correlated with swimming（P=0.011,r=0.449）,the results suggest that PQS may enhance the ability of swimming by down-regulating the expression of rhlR gene and up-regulating the expression of pqsR.The pqsR gene lowly correlated with the promotion of pyocyanin under different concentrations for different time（P=0.405,r=0.318）,PQS may enhance the activity of pyocyanin by up-regulating the expression of pqsR gene.There is a moderate positive correlation between pqsR gene and elastase with the first promotion and then inhibition induced by different concentrations of PQS for different time（P<0.05,r=0.652）,lasI and rhlR were lowly correlated with elastase（r=0.328,P=0.389;r=-0.444,P=0.231）,PQS may enhance the activity of elastase by up-regulating the expression of pqsR and lasI genes,and down-regulating the expression of rhlR.rhlR gene was moderately correlated with the inhibition of biofilm induced by different concentrations of PQS for different time（P<0.05,r=0.576）,lasR was lowly correlated with biofilm（P=0.221,r=0.453）,PQS may have a weak inhibitory effect on biofilm formation by down-regulating the expression of rhlR and lasR gene.Conclusions:1.Different induction schemes of exogenous PQS did not change the susceptibility of PA to common antibiotics.2.The lower concentration（10μM）of PQS had a single effect on the virulence phenotype,while the higher concentration（40μM,80μM）of PQS had a dual regulatory effect on the virulence phenotype.3.PQS may enhance the ability of swimming of PA by down-regulating the expression of rhlR gene and up-regulating the expression of lasI,it’s likely the activity of pyocyanin was enhanced by up-regulating the expression of pqsR gene,and enhance the elastase activity by up-regulating the expression of pqsR and lasI genes and down-regulating the expression of rhlR..PQS weakly inhibit the formation of biofilm by down-regulating the expression of rhlR and lasR genes. |
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