Potassium Iodine Potentiate Antimicrobial Photodynamic Therapy Of P.aeruginosa Mediated By Methylene Blue

Part 1 the Mechanism of Enhancement of antimicrobial Photodynamic Therapy Effect of Methylene blue on P.aeruginosa by Potassium iodineObjective Study the mechanism of enhancement of potassium iodine（KI）of antimicrobial photodynamic therapy（aPDT）effects mediated by methylene blue（MB）on P.aeruginosa（P.aeruginosa）.Methods P.aeruginosa was prepared into 10⁸CFU/ml.After the treatment,the bacterial suspension was diluted.The number of bacterial colonies was counted by plate count method.（1）The effect of KI on aPDT mediated by MB.The experiments were divided into aPDT group,control group,photosensitizer control group and light control group.APDT group:MB and bacteria was mixed with（or without）KI.After incubation for 30min,the compound was irradiated by red light with the energy density of 20J/cm.Control group:without photosensitizer or light.Photosensitizer control group:bacteria was not mixed with any MB nor KI.After incubation,the compound was irradiated by the light.Light control group:MB（with or without KI）was mixed with bacteria.After incubation,the compound was not irradiated by the light.（2）KI was mixed with MB and bacterial suspension.After incubation in the dark,the compound was irradiated by the light with energy density 20J/cm.（3）On the basis of the above experiments,the order of the addition of KI was changed.Then divided the experiment into 4 groups.Control group was without any MB nor light.After group:the compound mixed by KI and MB was irradiated by light then added into bacteria suspension.In group:KI,MB and bacteria mixed then irradiated by light after incubation.Washed group:the compound which was mixed by bacteria and MB was incubation and centrifugation,then added into the KI.（4）MB was added（or not added）with KI,mixed with the bacterial suspension.The compound incubated then lighted.The bacterial suspension was collected.Then made bacterial sections.The morphological and structural changes of the bacteria were observed by electron microscopy.Results（1）In this study,aPDT mediated by KI and MB resulted in 6.21㏒₁₀units reduction compared to aPDT mediated by MB.（2）P.aeruginosa was vulnerable to aPDT at concentrations of MB（10μmol/L）and concentrations of KI（7.5mmol/L and 10mmol/L）.（3）There were more than 6 units reduction in the“in group”,while only 1 log of killing in the“washed group”and“after group”.（4）With electron microscope,the integrity of cell membrane of P.aeruginosa was destroyed by aPDT which mediated by KI and MB.And the cytoplasmic structure was filamentous and reticular.Conclusions KI potentiates the killing effect of aPDT mediated by MB on P.aeruginosa.Within certain concentration of MB and light conditions,the elimination of aPDT mediated by KI and MB is positively correlated with KI concentrations.Short-lived substances generated during the aPDT process play major role in eliminating bacteria.The action sites of KI-MB-aPDT include the bacterial cell membrane and cytoplasm,which are non-specific.Part 2 Effects of Photodynamic Factors on aPDT Mediated by KI and MBObjective To study the effect of photodynamic factors on aPDT mediated by MB and KI to killing P.aeruginosa.Methods P.aeruginosa was prepared into10⁸CFU/ml.（1）KI and MB were mixed with the bacterial suspension and incubated against light for 30min.Red light with an energy density of 20J/cm²was used for repeated aPDT irradiation.（2）MB and bacteria mixed with（or without）KI.After incubation for 30min,aPDT was performed by changed the light energy density.（3）MB and bacterial suspension mixed with（or without）KI.Varied the incubation time,the compounds were lighted with an energy density of 16J/cm².Results（1）In this study,aPDT mediated by KI and MB resulted in3㏒₁₀units reduction.（2）With given concentrations of MB and KI,changed the light energy density（0 to 16 J/cm²）,the reduction of bacterial colony respectively raised to 2㏒₁₀units colony by MB-aPDT and to 5.4㏒₁₀units colony by KI-MB-aPDT.With certain concentrations of MB and KI,after incubation time varied from 5 min to 20 min,the reduction of bacterial colony raised to 5.4㏒₁₀units colony by KI-MB-aPDT.Conclusion Repeated application of aPDT does not result in drug resistance of P.aeruginosa.With certain concentrations of KI and MB and incubation time,the effect of aPDT killing P.aeruginosa was enhanced with the increase of light energy density.The effect of aPDT on killing was enhanced with the extension of incubation time with certain KI and MB concentrations and light energy density.Part 3 The Effect of efflux pump inhibitors on aPDT mediated by KI and MBObjective To study the efflux pump inhibitor phenylalanine-arginine-Β-naphthylamine（PAβN）and the order of addition of aPDT mediated by KI and MB.Methods P.aeruginosa was prepared into 10⁸CFU/ml.（1）The experiment was divided into four groups.PAβN-aPDT group:KI of different concentrations were mixed with MB,PAβN and bacteria,respectively,and incubated for 30min in dark.Then,red light irradiation with energy density of 20J/cm was used.aPDT group:only MB and KI,which are the same as PAβN-aPDT group,were added to photodynamic reaction.PAβN group:only PAβN was incubated with the bacteria without irradiation.Control group:no MB,KI and PA,no irradiation.（2）According to the order of adding of PAβN,the experiment was divided into four groups:PAβN-first group,PAβN-after group,PAβN-in group and Control group.PAβN-first group:the bacterial suspension was added to PAβN for incubation.After centrifuged,the compound was re-suspended in PBS.At the same time,MB was added for incubation.Then centrifuged,the compound was re-suspend in KI of different concentrations and red light irradiation with an energy density of 20J/cm was given.PAβN-after group:the bacterial suspension was added to MB for dark incubation,after centrifuged to remove supernatant,resuspended in PBS and added PAβN for dark incubation.The compounds centrifuged to remove the supernatant,then resuspended in KI of different concentration and PBS for irradiation.PAβN-in group:the bacterial suspension was added to PAβN and MB for incubation.Then the supernatant was centrifuged,and resuspended in PBS and KI of different concentraion for irradiation.Control group:no MB,KI and PA,no irradiation.Results（1）PAβN enhanced the effect of KI-MB-aPDT on P.aeruginosa.（2）the effect of adding PAβN before MB and KI was better than adding after mb-ki and pathogen incubation and the clearance of aPDT mediated by co-incubation of MB-MB and PAβN.The elimination of P.aeruginosa was proportional to KI concentration.Conclusion PAβN can further enhance the scavenging effect of KI-MB-aPDT on P.aeruginosa.The mechanism may be to reduce the bacterial excretion of MB by inhibiting the active efflux pump.