Antimicrobial Effect And Mechanism Of Antimicrobial Photodynamic Therapy In Combination With Potassium Iodide On Streptococcus Mutans And Candida Albicans Dual-species Biofilm

Objective:The aim of this study was to explore the antimicrobial effects,the potentiated mechanism and the anti-biofilm mechanism of potassium iodide（KI）combined with antimicrobial photodynamic therapy（aPDT）on the dual-species biofilm containing Streptococcus mutans（S.mutans）and Candida albicans（C.albicans）.Methods:1)The dual-species biofilm containing S.mutans and C.albicans was established in vitro and confirmed by colony forming unit（CFU）and gram staining.Crystal violet staining,thiazolyl blue tetrazolium bromide（MTT）,p H dropping assay,CFU assay,real-time fluorescent quantitative polymerase chain reaction（RT-q PCR）were performed to compare the biofilm mass,metabolic activity,acid production,acid tolerance,and the expression of virulence genes between monospecies biofilm and dual-species biofilm.The distribution of extracellular polysaccharides（EPS）in dual-species biofilm was observed by fluorescence microscope（FM）.2)In aPDT parameter exploration,a light emitting diode（LED）with a wavelength of 630nm was used as the excitation light source,and Toluidine Blue O（TBO）was used as the photosensitizer.The dependence of KI concentration,TBO concentration,and light dose on the antimicrobial effect of aPDT plus KI was determined by CFU assay.3)Singlet oxygen（¹O₂）probe test and time-resolved ¹O₂ detection,and ¹O₂ quencher experiment were performed to evaluate the role of ¹O₂during TBO-mediated aPDT plus KI.The photobleaching effect in the process of KI combined with TBO-mediated aPDT was recorded by microplate reader.The generation of iodine and hydrogen peroxide（H₂O₂）were analyzed by iodine starch test and Amplex red assay.4)The dual-species biofilm containing S.mutans and C.albicans was cultured on the bovine dentin slices.Scanning electron microscope（SEM）was used to observe the structure of the dual-species biofilm on dentin slices at different periods.The experimental groups were divided into 6 groups,namely,negative control group,0.2%Chlorhexidine（CHX）group,aPDT-L group,aPDT-H group,KI+aPDT-L group,and KI+aPDT-H group.CFU assay,MTT method,crystal violet staining method was used to analyze the effects of different disinfection methods on the number of viable microorganisms,the metabolic activity,and biofilm biomass of the biofilm.SEM and FM were used to observe the microorganism morphology and structure of the dual-species biofilm change in the proportion of dead bacteria.5)Transmission electron microscopy（TEM）was used to observe the effect of KI combined with aPDT on the microbial subcellular structure in the dual-species biofilm.The DCFH-DA kit was used to detect the cellular ROS production changes of KI combined with aPDT in the dual-species biofilm.The flow cytometry was performed to evaluate the effect of KI combined with aPDT on the cell membrane permeability,cell membrane potential,cell apoptosis of the microorganisms in the dual-species biofilm.RT-q PCR was used to detect the expression of related virulence genes in the dual-species biofilm after treating with KI combined with aPDT.Results:1)Two types of colonies of different sizes were visible on the BHI agar plate.Gram staining showed that there were two different morphologies of gram-positive microorganisms,and S.mutans attach tightly to the hyphae of C.albicans.Crystal violet staining,MTT method,CFU assay revealed that the biofilm formation,metabolic activity,and acid tolerance of the dual-species biofilm were higher than either monospecies biofilm（P<0.05）.The p H dropping assay showed that there is no significant difference in acid production between the dual-species biofilm and S.mutans monospecies biofilm,but both of them presented higher acid production ability than C.albicans monospecies biofilm（P<0.05）.RT-q PCR results showed that the biofilm-related adhesion and acid tolerance genes in dual-species biofilm,like gtf B,gtf C,ftf,hwp1,fab M,and phr1 were higher than that of monospecies biofilm（P<0.05）.However,the expression level of the atp D did not change.FM showed observed that EPS of dual-species biofilm increased with time and was distributed among the dual-species biofilm,from the initial sporadic spot-like fluorescence gradually turned into sheet-like fluorescence uniformly.2)According to CFU assay,25 m M,50 m M,100 m M,and 200 m M KI have no dark or phototoxicity on S.mutans or C.albicans in dual-species suspension.The addition of 200m M KI could highly potentiate TBO-mediated aPDT from eradicating the total microorganisms in plaque suspension.TBO concentration and light dose are positively correlated with the antimicrobial effect of KI combined with aPDT within a certain range.1μg/m L TBO-aPDT combined with 200 m M KI can kill S.mutans and C.albicans completely from dual-species suspension.3)The ¹O₂ fluorescent probe experiment and the time-resolved ¹O₂luminescence signal detection both show that the KI could quench ¹O₂ generated by TBO-mediated aPDT process.TBO presented a photobleaching effect after irradiation,however,KI combined with TBO-aPDT did not exert a photobleaching effect.There was a new peak at 350nm in TBO-mediated aPDT in the presence of KI.Iodine starch assay and Amplex Red assay confirmed that KI combined with TBO-mediated aPDT can produce iodine and hydrogen peroxide（H₂O₂）.And,the addition of 50 m M L-histidine completely abolished the production of free iodine and H₂O₂ generated by TBO-mediated aPDT in combination with KI.When L-histidine was added to the mixture of KI and TBO and then irradiated by LED,its antibacterial effect was similar to that of the negative control group（P>0.05）.4)The CFU assay showed the residual amount of biofilm on the dentin slice in descending order is negative control group,aPDT-L group,aPDT-H group,0.2%CHX group,KI+aPDT-L group,and KI+aPDT-H group.The MTT assay showed that the metabolic activity of biofilms on dentin slice in descending order is negative control group,aPDT-L group,aPDT-H group,0.2%CHX group,KI+aPDT-L group,and KI+aPDT-H group.Crystal violet staining showed the residual biofilm on the surface of dentin slice in descending order is negative control group,aPDT-L group,aPDT-H group,0.2%CHX group,KI+aPDT-L group,and KI+aPDT-H group after different disinfection methods.SEM showed that KI combined with aPDT could significantly reduce microorganism numbers on the surface of dentin slice than aPDT treatment or 0.2%CHX alone,but the microbial morphology did not present any change.The bacterial density and the proportion of dead bacteria showed varying degrees of change after treatments according to FM observation,but the KI combined with aPDT group had the highest proportion of dead bacteria and biofilm dispersion.5)Transmission electron microscopy showed that there were vacuoles inside S.mutans cells,and the cell wall and plasma membrane were partially separated in S.mutans after KI+aPDT treatment.The cell organelle disorganization,chromatin condensation,and increased cell membrane thickness were found in C.albicans after KI+aPDT treatment.The biofilms were subjected to KI+aPDT treatment resulted in increased endogenous ROS levels indicating oxidative damage occurred.The results of flow cytometry confirmed that the increased cell membrane permeability,the depolarized cell membrane,and the cell apoptosis were found in the microbes in dual-species biofilm after KI+aPDT treatment.RT-q PCR showed that biofilm formation-related virulence genes expression of S.mutans and C.albicans were downregulated after KI+aPDT treatment.In addition,oxidative stress-related genes expression of S.mutans were upregulated while the oxidative stress-related genes expression of C.albicans were downregulated.Conclusion:1)The dual-species biofilm of S.mutans and C.albicans showed high cariogenic potential including stronger biofilm formation ability,metabolic activity and acid tolerance when compared to any monospecies biofilm,which can be used as an in vitro caries model.2)The addition of 200 m M KI can potentially enhance the antimicrobial effect of TBO-aPDT on dual-species plaque suspension.3)KI can react with ¹O₂ produced by TBO-aPDT to generate hydrogen peroxide and iodine molecules which are antibacterial.4)The antimicrobial effect of KI+TBO-aPDT is superior to 0.2%CHX,and KI+TBO-aPDT did not produce ant toxicity on dental pulp cells.It is also found that KI+TBO-aPDT may induce changes in microbial cell membrane permeability,cell membrane potential,and even cell apoptosis due to oxidative damage inside the microbes.Meanwhile,it could alter some related virulence genes of S.mutans and C.albicans to exert its antimicrobial effect.