細菌細胞壁組成對光動力治療抑制革蘭氏陽性菌與革蘭氏陰性菌效果之影響

Abstract

不同於傳統治療方式，光動力治療 (photodynamic therapy, PDT) 是以造成細胞氧化傷害達到微生物防治的目的。然而革蘭氏陰性菌對於能抑制革蘭氏陽性菌之光感物質所引起之光毒性普遍具有抗性。故本研究利用乙二胺四乙酸 (ethylenediaminetetracetic acid, EDTA) 或溶菌酶 (lysozyme) 處理之原生質體 (spheroplasts) 或勝任細胞 (competent cells) 探討革蘭氏陽性菌與革蘭氏陰性菌於細胞壁結構與細胞膜通透性之差異，對光動力治療效果之影響，並評估此方法應用於生物膜 (biofilm) 系統之可行性。也藉由穿透式電子顯微鏡 (transmission electron microscope, TEM) 觀察微生物細胞製備成原生質體或勝任細胞後形態之改變。本研究以亞甲基藍 (methylene blue) 為光感物質，利用發光二極體產生波長為 630 ± 10 nm 紅光照射後所引起之光動力作用能抑制 108 CFU/mL 革蘭氏陽性菌 Staphylococcus aureus 與 Staphylococcus epidermidis，但對抑制相同濃度之革蘭氏陰性菌 Pseudomonas aeruginosa 與 Klebsiella pneumoniae 效果不彰，Escherichia coli 與 Enterobacter aerogenes 則具有強烈抗性無法被抑制。經由 TEM 觀察 E. coli 與 E. aerogenes 發現，正常的細胞均呈現桿狀且具備完整而連續的雙層膜，但是經 lysozyme 處理後會變得接近圓球形，且肽聚醣 (peptidoglycan) 結構被破壞，但細胞膜與外膜仍保持完整；以 EDTA 處理將導致外膜受損且結構不穩定；製備成勝任細胞後造成細胞表面不平整。革蘭氏陰性菌懸浮細胞分別製備成原生質體或勝任細胞後均可大幅提升光動力作用之殺菌效果。即使具有強烈抗性之 E. coli 與 E. aerogenes 亦能在 25 mM 亞甲基藍與 60 J/cm2 照光劑量下達到完全抑菌的效果；提高作用條件也能有效殺死 107 CFU/cm2 之 E. aerogenes 生物膜。結果顯示，細菌之細胞壁結構與細胞膜通透性確實是導致光動力殺菌效果不彰的主因。本研究所使用的方法不僅能提升光動力作用對懸浮細胞之殺菌效果，亦能用於生物膜防治，使光動力治療應用於微生物防治上更具潛力。

Photodynamic therapy (PDT) is based on the concept that a non-toxic dye known as a photosensitizer (PS) can be accumulated in target cells and activated by low intensity visible light of the appropriate wavelength in the presence of oxygen, and consequently the reaultant singlet oxygen and free radicals are toxic to target cells. In the 1990s, many studies have demonstrated that Gram-positive bacteria are particularly susceptible to photodynamic inactivation (PDI), while Gram-negative bacteria are significantly resistant under the same PDI condition. The major difference between the Gram-positive and Gram-negative species is the composition of cell wall. In this study, the roles of the outer membrane and peptidoglycan in the Gram-negative cell walls as well as the permeability of the cell membrane in methylene blue mediated PDI (MB-PDI) were investigated. Meanwhile, the cell structure of Gram-negative bacteria was observed by the transmission electron microscope (TEM). The Gram-negative bacterial cell walls were treated with lysozyme and EDTA to disrupt the peptidoglycan and the outer membrane, respectively. The cell membrane permeability was increased by making Gram-negative bacterial competent. No viable cells were detected when higher concentration of methylene blue (25 mM to 50 mM) and a light dose of 120 J/cm2 or more was used in Gram-positive bacteria, however, no significant antimicrobial efficacy was found in this condition for Gram-negative species. The cell wall disruption and morphologic change in cell membrane was observed by TEM after lysozyme, EDTA and competent cell treatment. The antimicrobial efficacy of MB-PDI against Gram-negative bacteria was enhanced both in planktonic cell and biofilm once the cell wall and membrane was disrupted. The results suggested that the outer membrane and the peptidoglycan play an important role in Gram-negative bacteria to make a diffusion barrier to methylene blue, and hence lead to the difference in MB-PDI efficacy between Gram-positive and Gram-negative bacteria.