原子力显微镜观测血卟啉单甲醚对细菌光动力杀伤作用

Observation of bacteria photodynamic inactivation induced by hematoporphvrin monomethyl ether by atomic force microscope

【目的】探讨血卟啉单甲醚(Hematoporyrin monomethyl Ether,HMME)对革兰氏阳性(G+)、阴性(G-)菌的光动力杀伤作用。【方法】通过平板菌落计数法和原子力显微镜(AFM),观察细菌与HMME作用前后形貌的变化。【结论】当HMME浓度为50μg/mL,可见光(光功密度为200mW/cm2)光照30min时90%以上的金黄色葡萄球菌(Staphylococcus aureus)能被杀死,无光照时对S.aureus杀灭效果显著。同等条件下,无论光照还是无光照,HMME对大肠杆菌(E.coli)无明显的杀伤作用。AFM图像显示,S.aureus细菌表面破坏严重,完全碎裂成鱼鳞状的片状堆积。对HMME作用后的E.coli扫描可见,菌体原来光滑的表面变成网格状的裂纹排列。【讨论】HMME对G+有明显的光失活效应,而对G-效果不明显。AFM的超微图像显示HMME对细菌细胞的攻击位点主要在细胞膜上。AFM为我们研究光敏剂对细菌的光动力损伤作用机制的可视化提供了依据。

[Objective] To study the photodynamic inactivation of Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli by hematoporyrin monomethyl ether. [Methods] Bacteria incubated with different concentrations of hematoporyrin monomethyl ether （HMME） and then irradiated with visual light for 30minutes, bacteria inactivation efficiency was detected with the reduction of colony unit, and morphological changes were observed with atomic force microscope （AFM）. [Results] Results indicated that 90% of Staphylococcus aureus was photoinactivated by illumination with visible light for 30 min （power density 200 mW/cm^2） in the presence of 50 μg/mL HMME. The bacteria killing efficiency to Staphylococcus aureus with light irradiation was much obvious than that in dark at the same concentration of HMME, although without noticeable damage to E. coli with illumination or in dark. AFM ultrastructure images showed that the cells surface of photodynamic inactivated bacteria was all damaged seriously without the leakage of cell contents. [Conclusion] We concluded that the attacked sites to bacteria cells by hematoporyrin monomethyl ether were bacteria membrane structure. Atomic force microscopy provides us a visual technique to study the mechanism of bacteria reacted with photosensitizers.