Photodynamic Killing Effect Of Methylene Blue On Candida Albicans And Staphylococcus Aureus

Objective To investigate the antibacterial effect and mechanism of methylene blue(MB)-mediated photodynamic therapy(PDT)under a light source suitable for wavelength and light energy,and the antibacterial effect of PDT combination with potassium iodide(KI).Methods The optimal wavelength of the MB-mediated PDT antibacterial effect is determined by measuring the maximum absorption peak of MB solution with spectrophotometer.The first part of the experiment was Candida albicans standard strain DAY286.Firstly,the suspension of Candida albicans prepared at10~7 CFU/mL was divided into the following four groups:blank control group:the fungus suspension without MB or lighting;PDT group:according to the difference of MB concentration,it was divided into 10?mol/L group,50?mol/L group and 100?mol/L group,and was irradiated with 10 J/cm~2,40 J/cm~2 and 80J/cm~2 light after incubation with Candida albicans suspension;the pure light group:the suspension without MB,only light was given,and the light source condition was the same as the PDT group;the simple photosensitizer group:the fungus suspension without light,only MB was given.Determination of MB optimal concentration by different concentrations of MB-mediated PDT against Candida albicans.Secondly,the Candida albicans suspension was divided into three groups:blank control group,PDT group and Illumination group.(1)Blank control group:the fungus suspension without MB or lighting.(2)PDT group:add Candida albicans suspension into MB and incubated for 30 min in the dark,and give different light energies of 0 J/cm~2,10 J/cm~2,20 J/cm~2,40 J/cm~2,80 J/cm~2,160J/cm~2 and 320 J/cm~2 at appropriate wavelengths.(3)Illumination group:the fungus suspension without MB,only light was given,and the light source condition was the same as PDT group.The survival rate of candida albicans after each group was determined by colony forming unit(CFU).In the second part of the experiment,the standard strain of Staphylococcus aureus quality control strain ACCC25923 was selected as the experimental object,and the optical density(OD)was adjusted by a microplate reader to prepare a 10~8 CFU/mL suspension of Staphylococcus aureus.In order to detect the optimal concentration of MB at a light energy of 10 J/cm~2,the experiment was divided into:(1)blank control group:Staphylococcus aureus suspension was not given light or MB.(2)PDT group:It was divided into 0.1?mol/L group,0.2?mol/L group,0.5?mol/L group,1.0?mol/L group and 5.0?mol/L group according to MB concentration.(3)Simple MB group:Staphylococcus aureus suspension is only added to MB,and no light is given.(4)Simple light group:Staphylococcus aureus suspension is not added to MB,only light is given.After determining the optimal concentration of MB through the bactericidal effect of different concentrations,the experiment was divided into the following groups:absolute control group:Staphylococcus aureus suspension was not given light,KI or MB;control group:Staphylococcus aureus suspension was given MB and light without KI;simple KI group:Staphylococcus aureus suspension without MB or light,only KI;PDT(KI)group:Staphylococcus aureus suspension incubate with appropriate concentration of MB for 30 min in the dark,and irradiate with different concentrations of KI after mixing.Light(KI)group:Staphylococcus aureus suspension only added KI,without MB.The same light source as the PDT(KI)group was irradiated;photosensitizer(KI)group:MB and KI were added to the Staphylococcus aureus suspension without any illumination.The survival rate of staphylococcus aureus in each group was determined by CFU.In the third part of the experiment,fluorescent molecules probes SOSG and HPF were used to detect the amount of fluorescence generated by MB under different light energies.The antibacterial mechanism of MB was preliminarily studied.Results(1)The optimal concentration of photosensitizer MB for the killing effect of candida albicans was 100?mol/L.When the light energy is 0J/cm~2 to 320 J/cm~2 under the illumination of a light source with a wavelength of660,the higher the light energy,the stronger the MB-mediated PDT bactericidal effect(P 0.05).At 320 J/cm~2 light energy,MB-mediated PDT had the strongest killing effect against Candida albicans,and the lowest survival rate of Candida albicans was 0.000073%(P 0.05).There was no significant difference between the PDT group and the light group with light energy of 0 J/cm~2 and the blank control group(P 0.05).(2)Selecting MB for the appropriate sterilization of Staphylococcus aureus at a concentration of 0.2?mol/L.MB-mediated PDT combined with KI was used to enhance Staphylococcus aureus killing at light source wavelengths of 660 nm and 10 J/cm~2(P 0.05).There were no significant differences in the number of colonies of Staphylococcus aureus between the KI group,the Light(KI)group and the photosensitizer(KI)group and the absolute control group(P<0.05).(3)The amount of MB fluorescence measured by the fluorescent molecular probe SOSG at the same light energy is higher than that of HPF(P 0.01).Conclusion(1)In the range of 160-320 J/cm~2 light energy,MB-mediated PDT has a good killing effect on Candida albicans.(2)MB-mediated PDT has an enhanced antibacterial effect when combined with KI.(3)The antibacterial effect of MB-mediated PDT is mainly through the type II reaction mechanism.