Toluidine Blue O-induced Antimicrobial Photodynamic Therapy For Multidrug Resistant Staphylococcus Aureus In Vitro

Background and Purpose:Staphylococcus aureus（S.aureus）is a Gram-positive bacterium that colonizes burns and scalds wounds,S.aureus strain can be separated in the early course of the disease.Among them,in the burn and scald intensive care unit,methicillin-resistant Staphylococcus aureus（MRSA）accounts for about 40%.Local and systemic infection caused by MRSA can delay wound healing in small burn injuries,and induce sepsis,shock syndrome,and even multiple organ failure in large severe burn injuries,resulting in the death of patients.S.aureus can form biofilms on burn wounds,mucous membranes,medical devices,and dressings.These biofilms are highly organized communities of microbes consisting of bacteria surrounded by extracellular polymeric substances（EPS）.The composition of the EPS varies depending on the organisms within the biofilm,but in general,it includes microbial-derived complex polysaccharides,proteins,lipids,and nucleic acids.The inability of antibiotics and photosensitizers to penetrate through the biofilm is one of the important factors contributing to the capacity of bacterial biofilms in antibiotic tolerance.Biofilms make traditional antibiotic therapy less effective against multidrug-resistant bacteria,and multidrug-resistant bacterial infections in large severe burn injuries often lead to high mortality.The multi-drug efflux system（MES）of S.aureus can efflux a variety of antibiotics and photosensitizers,reducing the drug concentration in the bacteria,and the QS system of biofilms can make the MRSA efflux pump gene highly expressed.The efflux of substrates thus enhances bacterial resistance.Most S.aureus strains secrete several different toxins,including hemolysins,V8protease,and enterotoxins,which can directly damage the immune system cells of patients,stimulate the release of various immune factors,and aggravate infection symptoms,while traditional antibiotics therapies cannot directly target virulence factors.To solve the huge threat of MRSA to burn and scald patients,people are constantly looking for new antibacterial technologies that can be applied to burns and scalds combined with infection wounds at an early stage.Antimicrobial photodynamic therapy（PDT）is a new method of treatment of body surface infections that have emerged in recent years.Its principle is to stimulate a photochemical reaction of a photosensitizer（photosensitizer,PS）by the light of a specific wavelength to produce singlet oxygen（¹O₂）and reactive oxygen species（ROS）such as hydroxyl radicals,hydrogen peroxide,superoxide anions,etc.,can oxidatively damage biological macromolecules such as lipids,proteins or nucleic acids,thereby effectively killing pathogenic microorganisms.This study analyzed the killing effect of toluidine blue O（TBO）as a photosensitizer combined with 635nm red light on multi-drug resistant S.aureus strains cultured in vitro,and explored potassium iodide（KI）and efflux pump inhibitor cyanide m-chlorophenylhydrazone（CCCP）on the efficiency of TBO-mediated PDT to S.aureus.Observe the microscopic appearance of multi-drug resistant S.aureus before and after TBO-PDT by multiple methods.Changes in morphology and detection of changes in multiple virulence factors of multi-drug resistant S.aureus before and after TBO-PDT treatment are used to analyze the mechanism of PDT sterilization and lay a good research foundation for future clinical use.Methods:This study used a multi-drug resistant S.aureus MRSA252 strain.Planktonic experimental group TBO concentration range（0.5μM,0.75μM,1.0μM,1.25μM）and light dose（635±5 nm wavelength;5 J/cm²,10 J/cm²,20 J/cm²）treatment;control groups included a TBO-only group,a light-only group,and a blank control group.The bacterial survival rate was calculated by the colony serial dilution method.The 48 hours mature biofilm of the multidrug-resistant Staphylococcus aureus strain MRSA252 was used.The photodynamic therapy（PDT）group was treated with different concentrations of TBO（0.5μM,0.75μM,1.0μM,1.25μM）and different doses of red light（635±5 nm wavelength;30 or 50 J/cm²）.The bacterial survival rate was calculated by the colony serial dilution method.The biofilms viability after PDT were evaluated by crystal violet（CV）staining assay and{2,3-bis（2-methoxy-4-nitro-5-sulfophenyl）-5-[（phenylamino）carbonyl]-2H-tetra-zoliumhydroxide}（XTT）assay.The potentiation effects of inorganic salt（KI）or efflux pump inhibitor（CCCP）on the TBO-PDT in killing biofilm experimented respectively.Confocal laser scanning microscope（CLSM）and scanning electron microscope（SEM）were used to monitor the morphological changes of biofilms.The hemolysis assay and enzyme linked immunosorbent assay（ELISA）were used to determine the virulence of the bacteria.Congo Red assay was used to determine the EPS of biofilms.After PDT,the biofilm was re-cultured for extra 48 h.Its formation viability and virulence were detected again.Results:MRSA planktonic state experiment:1.The killing efficiency of TBO-PDT on the S.aureus strain is positively correlated to TBO concentration and light dose（P<0.05）.2.After TBO-PDT treatment,the planktonic bacteria was observed by SEM,TBO-PDT caused bacteria to shrink,lysis,and size reduction.3.After TBO-PDT treatment,the hemolysis rate and the ELISA results of enterotoxin A showed decreased significantly（P<0.05）.MRSA biofilm state experiment:1.The killing efficiency of TBO-PDT on the S.aureus strain（biofilm state）is positively correlated to TBO concentration and light dose（P<0.05）.2.XTT assays demonstrated the viability of mature MRSA biofilms was reduced after PDT（P<0.05）.The result of crystal violet CV staining,the damage of S.aureus biofilm cells and structure improved with the increase of TBO and light dose（P<0.05）.3.When the TBO concentration was in the range of 0.5μM?1.25μM,the light dose range of 30 J/cm²?50J/cm²,and the KI concentration was 100 m M,the sterilization efficiency can be increased by1 log10?2 log10 on biofilm（P<0.05）.When the TBO concentration was in the range of0.5μM?1.25μM,the light dose was 30 J/cm²?50 J/cm²,and combined with 0.1μM CCCP,it had a significantly higher killing efficiency（2 log10～3 log10）on biofilm compared with TBO-PDT alone（P<0.05）.4.The morphological features of the biofilms showed great changes,such as shrinkage,fissure,fragmentation,and rarefaction after being treated by TBO-PDT and observed by SEM.After TBO-PDT treatment,there was a distinguished increase in the number of cells marked with red fluorescence MRSA biofilms were observed by CLSM.Among them,with the increased of the TBO concentrations,the more dispersion and loss of structure increased showed by three-dimensional images.5.PDT group shows a distinct reduction in RBC hemolysis rate and the concentration of SEA compared to the control groups.（P<0.05）.6.After TBO-PDT treatment,the results of Congo red staining assay showed PDT1（0.5μM TBO,50 J/cm²）group and PDT2（1.25μM TBO,50 J/cm²）group was significantly reduced（P<0.05）.7.After TBO-PDT（1.25μM TBO,50 J/cm²）,the re-formation viability of MRSA cells was inhibited,and its could not formate mature biofilms within 48 h like the control group（C-L-）cells.After TBO-PDT（1.25μM TBO,50 J/cm²）,the hemolysis rate and the enterotoxin A of Staphylococcus aureus biofilm was continuously inhibited within 48 hours（P<0.05）.Conclusions:TBO-PDT has good killing efficiency against S.aureus of both planktonic and mature biofilm states.KI and CCCP can potentiate the killing efficiency of TBO-PDT against S.aureus.In addition,TBO-PDT destroys the structural integrity of the bacterial cell wall,resulting in increased membrane permeability.The results presented in the study indicate that TBO-mediated PDT can inhibit the vitality and function of MRSA,destroy the structure of biofilms and inactivate virulence factors.It also could inhibit the restoration the re-formation and virulence of biofilm after PDT.PDT can be used as a supplement to the treatment of antibiotic-resistant bacterial biofilm infections,and clinical patients could benefit from the combination therapy.