光動力殺菌對於抗Methicillin金黃葡萄球菌與表皮葡萄球菌之懸浮菌體與生物膜之研究

Photodynamic Inactivation on Methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis Planktonic and Biofilm Cells

莊其蓁(Chi-Chen Chuang)

指導教授 ： 鄧麗珍

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00997

摘要

隨著抗藥菌株的增加以及多重抗藥菌株的出現，抗生素治療方式已經遇上瓶頸，而且生物膜對於抗生素的抗性比懸浮菌體約高出50~500倍之多，我們無法完全仰賴抗生素來對付細菌引起的感染，於是許多學者進一步發展其他治療方式，光動力殺菌(photodynamic inactivation)即為其中之一。光動力殺菌的原理是透過特定波長之可見光將光感物質(photosensitizer)激發，經過系統間的能量轉移，將氧分子激發成單態氧(singlet oxygen)、或是將能量傳給組織內的生物分子形成自由基，透過單態氧或自由基而對細菌造成傷害。由於光動力殺菌為非專一性的毒殺作用，故細菌難以產生抗性，如果成功將光動力殺菌的方式應用在抗藥菌株造成的感染，將可彌補抗生素治療的不足。

本實驗以Staphylococcus aureus與Staphylococcus epidermidis為對象，除了ATCC標準菌株外，還挑選了三株臨床抗藥或多重抗藥菌株，包括methicillin-resistant Staphylococcus aureus (MRSA)、vancomycin-intermediate Staphylococcus aureus (VISA)、methicillin-resistant Staphylococcus epidermidis (MRSE)等，並探討三種光感物質Rose Bengal (RB)、Toluidine Blue O (TBO)、Hematoporphyrin (HP)經LED可見光激發後，對於懸浮菌體與生物膜兩種狀態下的光動力殺菌成效。

我們發現對於懸浮菌體來說，所有菌株皆對光動力殺菌有很好的感受性，而RB較TBO效果佳，可能是RB的單態氧量子產率較TBO高所造成。RB與TBO除了有效殺菌，也可抑制MRSA之Lipase產量，RB的光動力作用還會對細菌膜蛋白造成損壞。雖然HP效果最差，但以微脂體包埋HP或延長照光前細菌與HP培養的時間，則會有所改善，表示減少HP聚集或增加光感物質吸附量可能有利於光動力殺菌。

而光動力作用對於生物膜的殺菌效果比懸浮菌體差，可能是包覆生物膜的多醣基質提供了屏障，故單態氧與自由基無法對菌體直接作用，且RB對生物膜的殺菌效果與TBO相近，可能是由於RB分子量較大，不易滲透到生物膜中所導致。

關鍵字

光動力 ； 金黃葡萄球菌 ； 表皮葡萄球菌 ； 生物膜 ； 光感物質

並列摘要

Antibiotic resistances are getting increasingly severe, compared to the past, and the followings are two obvious examples. First of all, antibiotic therapy may fail to inhibit multidrug resistant bacteria. Second, biofilm cells are more resistant to antibiotics than planktonic cells are. To solve these problems, the photodynamic inactivation was evaluated in this study. Photodynamic inactivation employs the visible light of certain wavelength to excite the photosensitizers. After excitation, the photosensitizers can undergo two types of reactions. Type I reaction involves electron-transfer from excited photosensitizers to biological molecules nearby and forms free radicals afterwards. Type II reaction produces excited-state singlet oxygen right after the collisions between exited photosensitizers and oxygen molecules. Free radicals and singlet oxygen will oxidize bacterial components, such as proteins, lipids and nucleotides, and finally lead to cell death. Bacterial cells rarely develop resistances to photodynamic inactivation because of the non-specific attack mediated by reactive oxidants.This will be a great benefit on clinical treatment.
In this study, we investigated the photodynamic inactivations of different photosensitizers, including rose bengal (RB), toluidine blue O (TBO) and hematoporphyrin (HP), on ATCC or clinical strains of Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA) and Methicillin-resistant Staphylococcus epidermidis (MRSE). We found that all strains exhibited good susceptibilities to RB and TBO during the planktonic stage. RB showed better efficacy of photodynamic inactivation than TBO. This result might be due to higher quantum yields of singlet oxygen by RB. Photodynamic effects of RB and TBO could also inhibit the lipase production of MRSA and induce damages to bacterial membrane proteins. Among the photpsensitizers, HP showed the least photodynamic inactivation efficacy, but its efficacy could be improved by encapsulating HP into liposomes or prolonging the incubation time with bacterial cells before irradiation.
Photodynamic inactivation was not well effective to biofilms, possibly due to the exopolysaccharides, in which the bacterial cells were embedded, which provide the barrier between active oxidants and bacterial cells. Unlike the planktonic stage, RB showed similar efficacy to TBO in the biofilm stage and the results probably due to the lower penetration efficiency into biofilms, which is related to higher molecular weight of RB.

並列關鍵字

photodynamic ； Staphylococcus aureus ； Staphylococcus epidermidis ； biofilm ； photosensitizer

參考文獻

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