The In Vitro Effects Of Photodynamic Therapy Against MRSA In Planktonic And Biofilm Forms

Objective To investigate the effects of photodynamic therapy (PDT) mediated by the cationic benzylidene cyclopentanone photosensitizers P2 against MRSA in planktonic and biofilm forms in vitro.Method(1) In the PDT group, Bacterial suspension (2?108 CFU/ml) was mixed with equal volume of P2 solution at different final concentrations (2.5?5?10? 25 ?M). After incubation for 30 min, irradiate the mixtures with 532-nm laser(irradiance:40 mW/cm2, exposure time:10 min, energy density:24 J/cm2). Blank control, photosensitizer group and light group were also included. Each group was carried out in triplicate with three samples for each time. The antimicrobial effects against planktonic MRSA in vitro were evaluated via serial dilutions and subsequent colony counting.(2) In the PDT group, biofilms of MRSAO were cultivated and treated with PDT mediated by P2 at a concentration of 10 ?M. The parameters were the same as above. After the treatment, SYTO9 and PI fluorescence probe were added to the wells, then confocal laser scanning microscopy (CLSM) was used to observe the survival of biofilm-embedded bacteria. The SYTO9 stained the surviving bacteria, showing green fluorescence. The PI stained dead bacteria, showing red fluorescence. After that, biofilms of MRSAO were cultivated and treated with PDT mediated by P2 at concentrations of 10 ?M and 25 ?M. XTT assay was used to detect the survival rates of bacteria in biofilms. Blank control, photosensitizer group and light group were also included. Each group was carried out in triplicate with six samples for each time.(3) Biofilms of MRSA were cultivated and treated with PDT mediated by P2 at different concentrations(10?M and 25 ?M). The density of the biofilms, the change of colony forms and biofilm structures were observed with microscope after crystal violet staining. Damage of the biofilms was quantified subsequently with the plate reader. After incubation with the FITC-Cona fluorescent probes, which can bind with polysaccharides specifically, the biofilms were observed with CLSM for the change of the contents of polysaccharides. Microporous membrane filter tube method was used to established to test the MRSA biofilm permeability. After PDT treatment mediated by P2 at 10 ?M,400-?l levofloxacin (LVFX) solution (10 ?g/ml) was added to the tube followed by 6-hour incubation. High performance liquid chromatography (HPLC) was used to detect the concentrations of LVFX that had permeated the biofilms. Blank control group was also included. Each group included 6 specimens.(4) The combination effects of P2-PDT and LVFX were tested in 96-well plates. MRSA biofilms were treated with PDT mediated by 10-?M P2. Then 10 ?g/ml LVFX solution were added to the wells. After 24 h, the survival fraction of bacteria embedded in the biofilms was determined by XTT assay. Control group, LVFX group and PDT group were included with 6 replicates for each group.Results(1) The antimicrobial effects of PDT against the three MRSA strains were P2 concentration-dependent. At a concentration of 5 ?M,> 3-log1o decrease of bacteria survival was obtained for all of the three strains, whereas the maximum killing was obtained at P2 concentration of 10 ?M.(2) The images of CLSM showed that the average intensity (299.84 ± 53.39 a.u.) of green fluorescence (surviving bacteria) in the PDT group is obviously weaker than that (485.44 ± 120.21 a.u.) in the control group (P< 0.01). The ratio of the green fluorescence to the red fluorescence is weaker for the PDT group (31.87% ± 6.25%)than the blank control (61.67%±11.07%)(P< 0.01), which indicates of a lot of bacterial death. The results of XTT assay showed the decreased bacterial survival rates in the PDT group. And a better inactivation was achieved at a higher P2 concentration. For the PDT group with lower P2 concentration, the bacterial survival rate was 15.77 ± 6.86%, while in the PDT group with higher P2 concentration, the bacterial survival rate was 6.25 ±3.36%.(3)The destruction of biofilm structures was verified by the microscopy. The colonies became less dense with a loss of complexity in the stereostructures of the biofilms. More serious damage was observed for the PDT group with higher P2 concentration. After crystal violet staining, the OD value for the PDT group is significantly lower than that of the control group. And the OD value for the PDT group at 25 ?M (1.046±0.157) was significantly lower than the PDT group at 10 ?M (1.283±0.280) (P< 0.01). Through the CLSM images, the intensity of the green fluorescence (183.41±0.31 a.u.) of FITC-Cona in the PDT group was obviously weaker than that (474.57±117.26 a.u.) in the control group (P< 0.01), indicating a significant reduction of the contents of polysaccharide in biofihns. After PDT treatment, the concentration of LVFX through MRSA biofilm for the PDT group and control group were 2.02±0.2 ?g/ml and 1.60 ± 0.2 ?g/ml, respectively, showing significant difference (P< 0.01).(4) The survival rate of group A, B, C and D were 100% 86.49 ±3.5%,57.42 ± 0.9% and 0.76 ± 0.6%, respectively. The survival rate of group D was lower than those of group B and C, and there were significant differences among them (P< 0.01).Conclusion1. PDT mediated by P2 is effective in inactivating the three strains of MRSA that we used in planktonic form.2. PDT mediated by P2 is effective in inactivating the bacteria of MRSAO embedded in biofilms, but higher concentrations of P2 were required to achieve the similar killing as to the planktonic cultures.3. PDT mediated by P2 can destroy the structure of MRSAO biofilms, thus can increase the permeability of LVFX through MRSAO biofilm.4. PDT combined with the LVFX can enhance the inactivation of the bacteria in the MRSA biofilm.