Inhibitory Effect Of N-acetylcysteine On Biofilms Of Pseudomonas Aeruginosa And Its Synergism With Ciprofloxacin

[Objectives]To investigate the inhibitory effect of N-acetylcysteine(NAC) on the bacterial growth of Pseudomonas aeruginosa,on the biofilms of Pseudomonas aeruginosa and mechanisms,and its synergic effect with ciprofloxacin.[Methods]Minimum inhibitory concentrations(MIC) of NAC,piperacillin (PIP),ciprofloxacin(CIP),and amikacin(AMK)against P.aeruginosa were determined by the broth microdilution test.Interactions of NAC and above antibiotics were determined using chequerboard MIC assay.The morphology of biofilms after treatment with NAC,CIP,NAC+CIP was observed using the optical microscope stained by AgNO₃,SEM and CLSM,quantitative analyses of biofilm structures using the image stacks obtained by CLSM were performed using COMSTAT software.The effects of NAC,CIP,NAC+CIP on the biofilm formation and disruption of performed biofilms of P.aeruginosa were assayed by the optical densities stained by crystal violet.Viable counts in biofilms were determined by MTT assay.Glycocalyx production of P.aeruginosa was quantitatively measured by tryptophan assay after treatment with NAC.[Results]A total of 30 strains of P.aeruginosa,83.3%(25/30) strains were inhibited by NAC less than 40mg/ml,the rates of susceptibility to PIP,CIP and AMK were 80.0%,83.3%and 80.0%,respectively.The combination of NAC and PIP demonstrated either synergy(33.3%) or no interaction(66.7%) against P. aeruginosa;50%of the strains showed synergy when NAC was tested in combination with CIP,another 50%strains showed no interaction;NAC displayed no interaction with AMK for 86.7%of the isolates tested,and antagonism was 13.3%.The effects of NAC on mature P.aeruginosa PAO1 biofilms,stained with AgNO₃,SEM and CLSM showed accordant results,bacteria and slime decreased gradually when concentration of NAC(0.5-10mg/ml) increased,biofilms almost disappeared at 10 mg/ml of NAC.The inhibitory effects of CIP(8MIC) on biofilms also enhanced by NAC,and biofilms almost disappeared at CIP(8MIC) combined with 5mg/ml of NAC(observed with AgNO₃ staining and SEM) or with 2.5mg/ml NAC(observed with CLSM).Image stacks were quantitatively analysed with COMSTAT software,bio-volume,substratum coverage,average thickness,maximum thickness and surface area of the biomass in the image stack decreased,surface to bio-volume ratio and roughness coefficient increased gredually when the concentration of NAC increased,and NAC also showed synergism with CIP.There was less effect of small concentration of NAC on synthesis of P.aeruginosa biofilms when assayed by the optical densities stained with crystal violet,but for mature biofilms,reduced more than 50%at 10mg/ml NAC;for 3 day and 6 day old biofilms,CIP(8MIC) combined with 5mg/ml NAC, reduced biofilms to 38.9%±7.7%and 39.0%±9.3%of the control,that was 58.1%±4.9%and 48.9%±11.4%of CIP treatment alone,but there were differences between strains.NAC,CIP,NAC+CIP showed significant kiling on biofilm bacteria at 2.5mg/ml,2MIC and 0.5mg/ml(NAC) combined with 1/2MIC(CIP) (P＜0.01).When 2.5mg/ml NAC combined with CIP(8MIC),bacteria of biofilm decreased to 0.94%of the control,reduced 96.8%and 95.5%as compared with CIP(8MIC) and NAC(2.5mg/ml) treatment.GLX production of P.aeruginosa also decreased 27.64%and 44.59%at treatment with 0.5mg/ml and 1mg/ml NAC respectively.[Conclusions]NAC more than 10mg/ml had inhibitory effects agaist P. aeruginosa,and showed synergism when tested in combination with PIP and CIP to some extent.NAC had disrupted effect on mature P.aeruginosa biofilms and bactericidal effect on biofilm bacteria,also had synergism with CIP;GLX production was inhibited by NAC.