Effects Of Antimicrobial Peptide CRAMP Combined With Antibacterial Drugs On The Formation Of Pseudomonas Aeruginosa Biofilm

Pseudomonas aeruginosa(P.a)is a common conditional pathogen and wide distribution,causing chronic repeated infection in low immunity organisms.P.a is readily resistant to a variety of familiar antibacterial drugs,and drug resistance mechanism is relatively complicated,which is also one of the main reasons for biofilm formation.Futhermore,biofilm formation,the release of diverse virulence factors and multi-drug resistance are the primary reasons that generate P.aeruginosa refractory infections.The quorum sensing system(QS)in P.aeruginosa can regulate numerous pathogenic factors and play a critical role in the infection process.The virulence factors secreted by p.a(pyocyanin,rhamnolipid,alginate,etc.)and the formation of biofilm are regulated by QS.The virulence factors play a crucial role in biofilm formation and bacterial resistance.P.aeruginosa mainly contains two separate quorum sensing systems,the lasI/ lasR and rhlI / rhlR systems,activating other related gene regulation to jointly promote the generation of bacterial biofilms.Therefore,if the expression of related genes in the QS system can be effective in reducing or blocking,and thus decreasing the production of virulence factors and finding reliable QS blocker may be inhibiting the formation of p.a biofilm.This is likewise a promising subject in biofilm infection treatment.Additionally,the antimicrobial peptides exhibit broad-spectrum antimicrobial activity.As a type of antibacterial agents that is not prone to drug resistance,they are widely regarded as potential alternatives to antibiotics,and providing a new approach for controlling bacterial infection of biofilm and solving drug resistance of P.aeruginosa.In this study,the wild-type standard strain PAO1 of Pseudomonas aeruginosa was used as the test strain,establishing the biofilm in vitro.The effects of the antimicrobial peptide CRAMP combined with antibacterial drugs on P.aeruginosa biofilm formation were discussed.In addition,mRNA expression levels of correlative regulatory genes of lasR/lasI?rhlR/rhlI in Pseudomonas aeruginosa biofilm quorum sensing system and the inhibitory effect on the release of main virulence factors of PAO1 biofilms were investigated.Part 1The effects of combination of antimicrobial peptide CRAMP and antibacterial drugs on P.aeruginosa biofilm formation Objective: To screen out the optimum compatibility of CRAMP combined with antibacterial drugs to inhibit biofilm formation.Methods: Establishment of biofilm on 96-well cell culture plate to detect PAO1 biofilm biomass.The effect of the compatibility of the preliminary screening in sub-MIC on the formation of PAO1 biofilm was investigated.The biofilm biomass and viable bacteria counting were measured by 96-well cell culture plate crystal violet staining,as well as the calculation of the synergy coefficient then ultimately elected the optimal compatibility and intervention concentration and other conditions.Finally,PAO1 biofilm biomass and the biofilm bacteria counting were detected by 6-well cell culture plate under the ideal compatibility,and subsequently CLSM microscopy was determined to observe the morphological changes to further verify its effect.Results: 96-well plate crystal violet assayed PAO1 biofilm biomass,and therefore,tobramycin(TOB)and colistin sulfate(COL)were preliminary screened as the ideal combination drugs.The results of crystal violet and viable counting showed that1/2MIC CRAMP significantly affected planktonic bacteria in the stage of biofilm modeling,while 1/4MIC CRAMP had no significant difference compared with the blank well.The synergy coefficient calculated that tobramycin synergism was superior to colistin sulfate,but tobramycin would significantly affect the planktonic bacteria stage of biofilm modeling at 1/2?1/8 MIC concentrations and colistin sulfate showed no significant difference.Therefore,the final selection of 1/4MIC CRAMP was combined with the different sub-inhibitory concentrations of colistin sulfate(1/4?1/8?1/16MIC)for use.The determination of biofilm biomass was used crystal violet method in 6-well plate.The results declared that 1/4MIC CRAMP+1/4MIC COL and 1/4 MIC CRAMP+1/8MIC COL drastically reduced the biofilm biomass compared with the blank well(P<0.001).Nevertheless,the consequences of biofilm viable counting showed that only 1/4MIC CRAMP+1/4MIC COL could effectively lessen the biofilm viable count.Eventually,the compatible combination of 1/4MIC CRAMP+1/4MIC COL was selected.Laser confocal microscopy(CLSM)results indicated that 1/4MIC CRAMP+1/4MIC COL could availably decrease the biofilm biomass and have obvious synergistic effect on biofilm formation,and the effect was better than that the control group and the single drug group.Conclusion: According to biofilm biomass detection,biofilm viable counting,and CLSM morphology observations screen and verify that 1/4MIC CRAMP + 1/4MIC COL has obvious synergistic effect on PAO1 biofilm formation.Part 2 Effects of CRAMP combined with colistin sulfate on common virulence factors of PAO1 biofilm Objective: To observe the inhibitory effect of CRAMP combined with colistin sulfate on P.a biofilm-related virulence factors.Methods: Constructed the biofilm in vitro with 6-well cell culture plate,and at the same time,dividing them into control group,single drug group(1/4MIC CRAMP and1/4MIC COL)and the combined drug group(1/4MIC CRAMP+1/4MIC COL)were set up to detect the contents of alginate and rhamnolipid.The Las B elastolytic activity in bacteria was measured by employing Elastin-Congo red.Protease activity in bacteria was determined by using Azo-casein.Chloroform extraction method was used for assaying the activity in the pyocyanin on 14 h co-culture.Results: Contrasted to the control group,the pyocyanin production of colistin group(1/4MIC COL)and combined drug group(1/4MIC CRAMP+1/4MIC COL)were effectively lower(P < 0.05),and even the combined drug group(1/4MIC CRAMP+1/4MIC COL)significantly reduced alginate content(P<0.01).In colistin group,1/4MIC COL was effective in reducing rhamnolipid content(P<0.05),but drug combination(1/4MIC CRAMP+1/4MIC COL)decreased more(P<0.01).However,the elastase and proteolytic enzyme activities were obviously increased in both the single drug group and the combined drug group.Conclusion: The colistin group(1/4MIC COL)and the combined drug group(1/4MIC CRAMP+1/4MIC COL)can suppress the release of multiple virulence factors of P.a.The inhibitory effect of alginate,rhamnolipid in the combined group is preceded the single drug group.Part 3 Effects of CRAMP combined with colistin sulfate on mRNA levels of QS system related genes in PAO1 biofilm Objective: The major research is the effect of CRAMP combined with colistin sulfate on mRNA levels of the QS system related genes(lasI/lasR and rhlI/rhlR)in PAO1 biofilm.Methods: Constructed the biofilm in vitro with 6-well cell culture plate,then extracted the total RNA of the biofilm,and subsequently,c DNA was reversely synthesized.TB Green fluorescence embedding method was used to detect the changes in the expressions of lasR,lasI,rhlR and rhlI genes of biofilm bacteria and rpsL was used as an internal reference gene.Results: The drug combination of 1/4MIC CRAMP+1/4MIC COL could be remarkably down-regulated the mRNA levels of PAO1 biofilm QS related genes lasI,lasR,rhlR and rhlI(P<0.05 or 0.01).Only 1/4MIC COL significantly down-regulated the mRNA levels of rhlR in the single drug group(P<0.01).Conclusion: The combination of colistin in sub-bacteriostatic concentration and the antimicrobial peptide CRAMP(1/4MIC CRAMP+1/4MIC COL)can significantly reduce the formation of P.a biofilm and the mRNA expression levels of QS system related genes(lasR,lasI,rhlR,rhlI)and its inhibitory effect is significantly better than that of the single drug group.