微脂粒包埋血紫質對於A群鏈球菌及甲苯胺藍對多重抗藥細菌之光動力殺菌能力

Photobactericidal Activity of Liposome-hematoporphyrin on Group A Streptococcus and Efficacy of Toluidine Blue O Against Multidrug-resistant Bacteria

羅庭萱(Ting-Hsuen Lo)

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.2007.01004

摘要

近年來因為抗生素的濫用而產生了多重抗藥的細菌，學者開始研究其他對抗微生物的新方法，光動力治療就是其中之一。光動力治療(Photodynamic therapy, PDT)的基本原理是利用特定的光感物質，在適合的波長光線照射之下，將光感物質激發並與四周的氧氣產生光化學反應，進而破壞細胞。
本論文實驗首先是以Hematoporphyrin (Hp)以及微脂粒包覆的Hp (Lipo-Hp及PEG接枝的Lipo-Hp)作為光感物質，研究光動力作用對於A群鏈球菌的影響，當光感物質濃度為5μM ，在照光強度90 J/cm2下無論是使用Hp或是微脂粒包覆的Hp皆可使A群鏈球菌存活率下降7.4個log以上，而在光感物質為0.5μM，相同照光強度下，PEG-Lipo-Hp可使細菌存活率下降7.2個log，Lipo-Hp可使細菌存活率下降4.4個log，而Hp則只能使細菌存活率下降0.3個log。結果顯示使用liposome包埋的Hp作為光感物質可以提高對A群鏈球菌光動力殺菌的效果，同時也發現在光動力殺菌作用下會使A群鏈球菌產生毒性因子：蛋白酶的能力下降。
我們也針對抗萬古黴素腸球菌( vancomycin-resistant Enterococcus)、多重抗藥綠膿桿菌(Multidrug-resistant Pseudomonas aeruginosa)以及鮑氏不動桿菌(Acinetobacter baumannii)以Toluidine blue O (TBO)或Rose Bengal (RB)作為光感物質觀察光動力殺菌之效果，結果發現使用TBO加上光照可以有效的毒殺抗藥菌株，推論光動力殺菌作用並不會受到細菌本身是否具有對抗生素有抗性的所影響。最後我們利用快速篩檢的方法比較33株多重抗藥綠膿桿菌及19株未抗藥綠膿桿菌的臨床菌株，以TBO為光感物質找出光動力殺菌所需的最低光感物質濃度，結果發現綠膿桿菌對於光動力殺菌的感受性不會受到抗生素多重抗藥的影響。
由上述結果顯示使用低濃度的liposome包埋Hp可以增進對A群鏈球菌的光動力殺菌的效果，此種光動力殺菌作用並不會因為細菌對抗生素有抗藥性而失效；利用光動力殺菌的作用方式，在目前苦無新藥可對抗抗藥菌株的情形之下的確是一個具有潛力的選擇，往後會針對A群鏈球菌觀察光動力殺菌是否會對細胞膜蛋白以及核酸造成損傷，希望對於光動力殺菌作用機轉能夠有清楚的了解。

關鍵字

光動力治療 ； 光感物質 ； 微脂粒 ； A群鏈球菌 ； 抗藥菌株

並列摘要

The worldwide rise of antibiotic resistance in bacteria has led to the development of new anti-microbial strategies. Photodynamic therapy (PDT) is one of the choices. Photodynamic therapy is based on the combination of photosensitizer, oxygen and harmless visible light to produce cytotoxicity via generation of reactive oxygen species.
We studied the photodynamic inactivation (PDI) effects of hematoporphyrin (Hp) and liposome-encapsulated-Hp (Lipo-Hp and polyethylene glycol (PEG) coating Lipo-Hp) on Group A streptococcus (GAS). PDI with 5 μM of Hp or Lipo-Hp followed by light irradiation of 90 J/cm2, resulted in more than 7.4 log killing for GAS. At the same concentration of photosensitizers (0.5μM), and light dose (90 J/cm2), PEG-Lipo-Hp attained 7.2 log killing and Lipo-Hp attained 4.4 log killing for GAS, but Hp only exhibited 0.3 log killing for GAS. The results showed that using lipo-Hp or PEG coating Lipo-Hp as photosensitizers can promote a more efficient photoinactivation of GAS. At the same time, we found out Hp-PDI reduced virulence factor- protease production of GAS.
We also studied the toluidine blue O (TBO) and rose bengal (RB) mediated PDI in clinical multidrug resistant bacteria such as vancomycin-resistant Enterococcus (VRE), multidrug-resistant Pseudomonas aeruginosa (MDRPA), and Acinetobacter baumannii. In this study, we show that of TBO-PDT is effective against all strains we used. PDI efficacies were independent of the antibiotic resistance pattern of the given microbial strains. At last, we used rapidly screening method to demonstrate the TBO-PDI activity in 33 multidrug resistant Pseudomonas aeruginosa and 19 susceptible Pseudomonas aeruginosa clinical isolates. We found that minimal lethal drug concentrations were not different between clinical multidrug resistant and susceptible bacteria strains.
Our studies have demonstrated that low dose liposome-encapsulated-Hp can enhance the photobactericidal activity of Hp on GAS, and its effect against antibiotic resistant bacteria was independent of their resistance pattern. These promising results suggest that PDI has the potential to be a possible alternative to antibiotic in clinical applications. In the future, we will focus on membrane proteins and nucleic acid damage during Hp-PDT on Group A streptococcus. We hope that we can better understand the mechanisms of photoinactivation for bacteria.

並列關鍵字

Photodynamic therapy ； photosensitizer ； liposome ； Group A streptococcus ； multidrug resistant bacteria

參考文獻

Ashbaugh,C.D., Warren,H.B., Carey,V.J., and Wessels,M.R. (1998) Molecular analysis of the role of the group A streptococcal cysteine protease, hyaluronic acid capsule, and M protein in a murine model of human invasive soft-tissue infection. J Clin Invest 102: 550-560.

Balchum,O.J., Doiron,D.R., and Huth,G.C. (1984) Photoradiation therapy of endobronchial lung cancers employing the photodynamic action of hematoporphyrin derivative. Lasers Surg Med 4: 13-30.

Bangham,A.D. (1993) Liposomes: the Babraham connection. Chem Phys Lipids 64: 275-285.

Bertoloni,G., Lauro,F.M., Cortella,G., and Merchat,M. (2000) Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells. Biochim Biophys Acta 1475: 169-174.

Bertoloni,G., Rossi,F., Valduga,G., Jori,G., and van,L.J. (1990) Photosensitizing activity of water- and lipid-soluble phthalocyanines on Escherichia coli. FEMS Microbiol Lett 59: 149-155.