甲殼素輔助光動力抑菌與細菌細胞壁之關聯性探討

The effect of combining chitosan with photodynamic inactivation on bacterial cell wall

林明璇(Ming-Hsuan Lin)

指導教授 ： 陳進庭

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10700

摘要

由於抗生素的廣泛使用，再加上使用方式的不當，使得一些抗藥性病原菌的出現有越來越多的趨勢。這些抗藥性菌株的出現，讓許多研究學者開始關注，並尋求其他的治療方式，如利用噬菌體、抗菌胜肽以及光動力治療。而將光動力作用在微生物上，以達到抑制或殺菌之效果即稱之為PDI (photodynamic inactivation)。光動力殺菌有別於傳統抗菌藥物治療微生物感染的方式，在光動力殺菌過程中，光感物質可迅速地聚集在菌體表面或裡面，而後在經由特定波長的光激發後，產生單態氧及自由基而對微生物細胞形成不可逆的傷害。本實驗室在先前研究發現，甲殼素應用在光動力作用上，能提升光動力作用抑制細菌生長的效果。
在本研究中，首先針對革蘭氏陽性菌之金黃色葡萄球菌和革蘭氏陰性菌之綠膿桿菌菌株，以及其臨床抗藥性菌株進行光動力作用，發現都具有殺菌效果。其次，在探討甲殼素增強光動力殺菌之作用機制，發現增加甲殼素濃度及培養時間，對S. aureus有增強光動力殺菌之效果。另外，本研究進一步發現菌體在進行光動力殺菌後，如果不於適當時間內加入甲殼素作用，菌體會有重新生長且甲殼素將無法發揮協同殺菌的效果。以穿透式電子顯微鏡觀察S. aureus菌體在經過光動力作用後加入甲殼素的型態變化，發現菌體表面都有受到明顯的破壞且呈現不平整的現象，顯示光動力作用會對菌體表面造成破壞，使甲殼素能進一步達到協同殺菌的效果。最後，在光動力作用與細胞壁相關性的研究上，發現光動力殺菌效果與細胞壁厚度沒有明顯的關係，而是和光感物質與菌體的結合量多寡有相關。

關鍵字

光動力殺菌 ； 甲殼素 ； 抗藥性菌株 ； 微生物細胞壁

並列摘要

Drug-resistant bacteria have become an important issue in health. Antimicrobial photodynamic inactivation (PDI) has emerged as a promising treatment modality for microbial infections. Previously, we have found that chitosan can augment Toluidine blue O (TBO) mediated PDI against Staphylococcus aureus and Pseudomonas aeruginosa within 30-min incubation after PDI.
In this study, we further investigated the augmented antimicrobial effect of chitosan after PDI by changing the concentration, incubation time, and its molecular weight against Gram positive S. aureus and Gram negative P. aeruginosa. We found that the increase of concentration or incubation time could enhance the antimicrobial activity of chitosan in both bacteria strains.
Furthermore, we examined the growth curve of PDI-treated S. aureus. The growth rate of PDI-treated bacteria was lower than untreated ones. However, when bacteria grew back to the log-phase after PDI treatment, the augmented antimicrobial activity of chitosan would decrease. These results indicate that surviving bacteria could recover or repaired from the PDI-induced damages when additional incubation proceeded. According to transmission electron microscopy (TEM), bacterial cell wall was further disrupted after PDI in the presence of chitosan and finally caused bacteria death. Beside, we also found that the more TBO binding to bacteria, the better PDI effect could be found. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.

並列關鍵字

Photodynamic inactivation ； Chitosan ； Antibiotics resistant bacteria ； Microbial cell wall

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