光动力对铜绿假单胞菌PAO1浮游菌与生物被膜的体外清除作用  

Photodynamic Antibacterial Activityon Pseudomonas Aeruginosain Planktonic and Biofilm Culture in vitro

作　　者：吴苏敏[1] 王颖[1] 顾瑛[1] 

机构地区：[1]解放军总医院激光医学科,北京市100853

出　　处：《中国激光医学杂志》2014年第2期59-63,112,共6页Chinese Journal of Laser Medicine & Surgery

基　　金：国家自然科学基金(81171633);北京市自然科学基金(4132079)

摘　　要：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 Examine the ability of Ru-PDT to eradicate both planktonic and biofilm cultures ofPseudomonas aeruginosa in vitro. Observe the change of biofilm structure before and after PDT. Methods Bacterial suspension of P. acruginosa PAO1 was prepared to a concentration of - 10^8 CFU/ml. Biofilm model in vitro was established. Bacterial samples were divided into 4 groups. PDT group ： different concentrations Of photosensitizer and bacterial samples were mixed and incubated in dark for 30 min. Illumination of 457 nm laser was then performed for 10 min, the power density being 40 mW/cm^2. Photosensitizer group： PS was incubated with bacterial samples for 30 min, no illumination followed. Light group： the bacterial samples were exposed to 40 mW/cm^2 457 nm laser light in the absence of PS. Blank control group： bacterial samples mixed with PBS, without PS or illumination. The survival of plaktonic bacteria were calculated with counting colony-forming units （CFUs）. Survival rates of bacteria of biofilm culture were measured with MTI＂ assay. Microscopic observation was performed before and after PDT on biofilms. Results Neither 457 nm laser nor PS had any effect on the viability of Pseudomonas aeruginosa. The killing effect on planktonic and biofilm culture increased as the concentration of PS rose. The viability of planktonic bacteria fell more than 4 Log and 6 Log at 10 μM and 25μM, which represents a value greater than 99.99% and 99. 9999% of cellular inactivation respectively. Living bacteria in bio- film culture reduced to 39. 9% and 34. 1%. The disruption of biofilm structure wasconfirmed through microscopic observation. Conclusions This study shows that PDT is effective in eradicating beth planktonic and biofilm cultures of P. aeruginosa PAO1 strain. PDT can not only make the bacteria inside the biofilm cultureinactive, but also destroy the structure of biofilm.

关 键 词：光动力疗法 铜绿假单胞菌 生物被膜 钌化合物 

分 类 号：R378[医药卫生—病原生物学]