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目的分析新型光敏剂钌化合物(Ru complex)介导的光动力治疗(Photodynamic therapy,PDT)对铜绿假单胞菌PAO1浮游菌和生物被膜内细菌活性的影响,观察PDT对其生物被膜的清除作用。方法实验对象为细菌悬液和生物被膜模型,均分为四组。细菌悬液组的光敏剂浓度为0.1、0.25、1、2.5、10和25μM;生物被膜组的光敏剂浓度为1、2.5、10和25μM。(1)PDT组:加入不同浓度的光敏剂后,孵育时间30 min,采用激光照射,功率密度为40 mW/cm2,照射时间10 min。(2)单纯光敏剂组:光敏剂孵育时间30 min,不予激光照射。(3)单纯照光组:仅激光照射,功率密度40 mW/cm2,照射时间10 min。(4)空白对照组:不加入光敏剂,不予激光照射。用稀释平板法和MTT法分别测定各组处理后浮游菌存活率和生物被膜内细菌活性。生物被膜经过冲洗、固定、结晶紫染色后,用光镜观察其结构改变。结果在本实验浓度范围内,PDT杀伤铜绿假单胞菌的作用呈光敏剂浓度依赖性。以10和25μM Ru-PDT处理PAO1浮游菌和生物被膜均能够显著杀伤,浮游菌杀伤分别达到99.99%和99.9999%以上,生物被膜内菌的杀伤分别为60.1%和66.0%,进一步提高光敏剂浓度至50μM,光动力作用没有显著增强。单纯照光或光敏剂孵育对细菌存活无影响。光镜观察PDT处理组生物被膜发现其结构完整性遭到破坏,整体稀疏分散。结论 Ru-PDT在体外对铜绿假单胞菌浮游菌和生物被膜内细菌都具有很好的杀伤作用;浮游菌相比生物被膜内的细菌对PDT作用更加敏感。PDT不仅可以使生物被膜内细菌失去活性,还可以破坏其结构,具有清除作用。
OBJECTIVE: To analyze the effect of photodynamic therapy (PDT) mediated by a new photosensitizer Ru complex on the bacterial activity of Pseudomonas aeruginosa PAO1 in planktonic bacteria and biofilm, and observe the scavenging effect of PDT on its biofilm . Methods The experimental subjects were bacterial suspension and biofilm model, all divided into four groups. The photosensitizer concentration of the bacterial suspension group was 0.1, 0.25, 1, 2.5, 10 and 25 μM; the biofilm group had photosensitizer concentrations of 1, 2.5, 10 and 25 μM. (1) PDT group: After adding different concentrations of photosensitizer, the incubation time was 30 min, laser irradiation, the power density of 40 mW / cm2, irradiation time of 10 min. (2) pure photosensitizer group: photosensitizer incubation time 30 min, no laser irradiation. (3) Simple illumination group: Only laser irradiation, power density 40 mW / cm2, irradiation time 10 min. (4) blank control group: no photosensitizer, no laser irradiation. The survival rate of the planktonic bacteria and the bacterial activity in the biofilm were measured by the dilution plate method and the MTT method respectively. Biofilm after washing, fixed, crystal violet staining, with a light microscope to observe the structural changes. Results The concentration of PDT killed Pseudomonas aeruginosa was photosensitizer concentration-dependent. Both 10 and 25μM Ru-PDT treatment PAO1 planktonic bacteria and biofilm were able to kill significantly, the killing of bacteria were 99.99% and 99.9999%, respectively, biofilm killing bacteria were 60.1% and 66.0%, and further increase the concentration of photosensitizer At 50 μM, there was no significant photodynamic enhancement. Pure light or photosensitizer incubation had no effect on bacterial survival. Light microscopy PDT treatment group biofilm found that its structural integrity has been destroyed, the overall sparse dispersion. CONCLUSION Ru-PDT has a good killing effect on Pseudomonas aeruginosa in vitro and bacteria in biofilm. Bacterioplankton is more sensitive to PDT than bacteria in biofilm. PDT can not only deactivate the bacteria in the biofilm, but also destroy its structure and have a scavenging effect.