光動力抗微生物療法治療抗藥性金黃色葡萄球菌角膜炎

【研究背景及目的】感染性角膜炎為眼科常見急症，常導致患者永久性視力損失，甚至須接受角膜移植或眼球剜除以控制感染。其中，抗藥性金黃色葡萄球菌是最棘手的致病菌株之一，對抗生素反應差、好發於術後患者及慢性病患、且社區感染率正逐年攀升。光動力抗微生物療法，利用特定波長光源激發光感物質，產生大量過氧化物破壞病源菌結構，已初步在體外試驗中被證實能有效抑制抗藥性菌株生長，我們的研究希望探討此類療法是否可用於治療抗藥性金黃色葡萄球菌感染性角膜炎。【研究方法與設計】我們的實驗分為三部分，第一部分嘗試提高光動力殺菌效率，找出理想的光感物質與激發光組合。以抗藥性金黃色葡萄球菌標準菌株ATCC 33592的細菌懸浮液進行光毒殺體外實驗，使用雙氫葉酚類（chlorin e6）為主光感物質成分，測試微胞包埋（micelle encapsulation）的雙氫葉酚類是否能較自由懸浮的雙氫葉酚類有更好的光毒殺效力？並測試雙氫葉酚類以405奈米紫光和663奈米紅光雷射激發，相同能量是否有不同的細菌毒殺效果？第二部分則進行光動力殺菌療法在抗藥性金黃色葡萄球菌細菌生物膜（biofilm）、新鮮牛眼角膜炎模組及兔眼角膜炎動物實驗是否也有良好殺菌效力。第三部分則探討若以光動力治療結合傳統抗生素療法，光毒殺細菌後再投予萬古黴素（vancomycin），其治療效果較傳統抗生素療法有何差異？【結果】在第一部分的體外實驗中，我們證實，（1）光原本深，不論是紫色或紅色可見光，在每平方公分20焦耳（J/cm2）以內的照射能量無抑制抗藥性金黃色葡萄球菌生長的功效；（2）微包埋的雙氫葉酚類或游離態的雙氫葉酚類，藥物本身在100微摩爾以內的濃度對抗藥性金黃色葡萄球菌無毒殺效果；（3）以微胞包埋雙氫葉酚類，可有效提高其對抗藥性金黃色葡萄球菌的光毒殺效力；（4）微胞包埋的雙氫葉酚類可有效吸收405奈米的紫色可見光及663奈米的紅色可見光，其中紫色光的光毒殺效力較佳，但兩者無顯著差異。（5）光源本身及光感物質對金黃色葡萄球菌無毒殺力，但雙氫葉酚類一旦接受紫光或紅光激發後，其在細菌懸浮液實驗中，殺菌效果顯著，若不洗去光感物質，以每平方公分10焦耳能量的紫光照射2微摩爾（μM）的微胞包埋雙氫葉酚類即可降低十的五次方抗藥性金黃色葡萄球菌菌量。在第二部分實驗中，我們發現，（1）抗藥性金黃色葡萄球菌隔夜培養形成生物膜後，光動力的殺菌效果即明顯降低；須提高光感物質濃度和照射能量，以每平方公分20焦耳紫光照射100微摩爾的微胞體包埋雙氫葉酚類可減少10的二次方至三次方菌量；（2）而在新鮮牛眼的感染性角膜炎體外實驗和兔眼角膜炎動物實驗，於牛眼或兔眼角膜基質內注射100單位抗藥性金黃色葡萄球菌培養12小時後造成發病，再刮除牛眼及兔眼角膜上皮後給予光動力治療，殺菌效率不佳，實驗組與對照組無論是菌量或臨床疾病的嚴重程度，均沒有顯著差異。在實驗的第三部分，我們試圖結合光動力及抗生素的殺菌效果。並發現，（1）在細菌懸浮液試驗中，以每平方公分10焦耳紫光照射2微摩爾微胞包埋雙氫葉酚類，再投以萬古黴素合併治療後，其細菌毒殺性與單純使用等濃度萬古黴素相當；光動力治療與抗生素治療在細菌懸浮液試驗中，無明顯拮抗或加成作用；（2）在細菌生物膜試驗中，以每平方公分20焦耳紫光照射100微摩爾微胞包埋雙氫葉酚類，再投以萬古黴素合併治療，其細菌毒殺性較單純使用等濃度萬古黴素為高，可增加一至二次方的殺菌量，光動力治療可加成抗生素毒殺細菌生物膜的效果。（3）在細菌生物膜試驗中，以每平方公分20焦耳紫光照射100微摩爾微胞包埋雙氫葉酚類，光動力抗微生物療法的細菌毒殺性在24小時後消失。（4）在牛眼試驗中，以每平方公分40焦耳紫光照射100微摩爾微胞包埋雙氫葉酚類，再投以萬古黴素合併治療，其細菌毒殺性較單純使用等濃度萬古黴素為高，可增加十倍殺菌量，光動力治療可略為加成抗生素治療的殺菌效果。【結論】光動力抗微生物療法，以405奈米紫色可見光激發微胞包埋的雙氫葉酚類，在細菌懸浮液試驗中，可有效毒殺抗藥性金黃色葡萄球菌；一旦抗藥性金黃色葡萄球菌形成細菌生物膜，光動力殺菌效力驟降，需要大幅度提高照光能量及光感物質濃度才能達成部分殺菌作用；在牛眼及兔眼感染性角膜炎試驗中治療結果更不顯著。而以光動力合併萬古黴素治療，在抗藥性金黃色葡萄球菌細菌生物膜及牛眼試驗中，可觀察到部分加成效果。考量光動力抑菌效果在24小時後會消失，此治療目前並不適用於單一治療或單次治療抗藥性金黃色葡萄球菌，未來還需要更多的實驗測試最佳使用時機。

關鍵字

光動力；光動力抗微生物療法；抗藥性金黃色葡萄球菌；微胞；雙氫葉酚類；萬古黴素；生物膜

並列摘要

【Background and Study Purpose】 Infectious keratitis is a common ocular emergency and can lead to permanent visual loss. Occasionally, corneal transplant or evisceration is necessary for infection control in severe cases. Among the common pathogens, methicillin-resistant Staphylococcus aureus （MRSA） is one of the most notorious. MRSA has poor response to traditional antibiotics and is usually found in post-operative infections and in patients of chronic diseases. The community acquired MRSA infection rate is increasing. Photodynamic antimicrobial therapy （PACT）uses light of certain wavelength to activate the photosensitizer and produces abundant oxygenated products killing the bacteria. PACT has been proved to be effective in photoinactivation of both gram positive and gram negative bacteria in vitro. Our study aims to prove the possible application of PACT in treating MRSA keratitis. 【Material and Method】 Our studies can be divided in to three parts. First, we have tried to increase the efficiency of PACT in order to find the ideal combination of light source and photosensitizer. We apply PACT on MRSA ATCC 33592 bacterial suspension. We test the different efficacy between free form chlorin e6（Ce6）and micelle encapsulated Ce6. We also test the different photoinactivation ability between 405nm purple laser 663nm red laser. Second, we construct MRSA biofilms, ex vivo bovine eye keratitis model, and in vivo rabbit keratitis models. We titrate the PACT protocol and test the photoinactivation results in different models. Third, we combined PACT with vancomycin and test that whether PACT pre-treatment can have synergestic effect with vancomycin therapy. 【Results】 In the first part of our study, we have proved （1）the light itselt, neither purple laser nor red laser, has photoinactivation effect of MRSA 33592 within 20J/cm2 irradiation.（2） micelle Ce6 and free form Ce6 have no toxicity to MRSA 33592 below 100μM.（3）micelle encapsulation can increase the photoinactivation efficiency of chlorin e6. （4） micelle Ce6 can absorb both 405nm purple laser and 663nm red laser efficiently, but 405nm purple laser may be a better light source due to its stability and slightly higher efficiency. （5） with laser irradiation（purple or red laser）, the photoinactivation effect of micelle Ce6 is significant. We can decrease 5 log MRSA 33592 in suspension with 10J/cm2 irradiation of 2μM micelle Ce6. In the second part of our study, we have found （1）the photoinactivation ability of our PACT decreases dramatically in MRSA ATCC 33592 biofilm model. With 20J/cm2 irradiation on 100μM micelle Ce6 can only decrease 2 to 3 log bacteria load. （2）using this protocol to treat ex vivo keratitis model of bovine eyes and in vivo rabbit keratitis model, the effecs are both poor without significant difference than the placebo group. In the third part of our study, we have tried to combine PACT with vancomycin treatment. We have found （1）PACT (10 J/cm2 irradiation on 2μM micelle Ce6）pretreating can not increase the vancomycin therapeutic effect in MRSA ATCC 33592 suspension. （2）PACT （20 J/cm2 irradiation on 100μM micelle Ce6）pretreating can increase the vancomycin therapeutic effect in MRSA ATCC 33592 biofilm. （3）PACT （20 J/cm2 irradiation on 100μM micelle Ce6）effects on MRSA biofilm is temporary and disappears after 24 hours incubation. （4）PACT （40 J/cm2 irradiation on 100μM micelle Ce6） pretreating can mildly incrase the vancomycin therapeutic effect in MRSA keratits in ex vivo bovine eye model. 【Conclusion】 Our PACT uses 405nm purple laser combined with micelle encapsulated chlorine e6. This regimen has significant photoinactivation effect in MRSA bacterial suspension. However, the therapeutic effect decreases in biofilm model and is not significant in either ex vivo bovine keratitis model or in vivo rabbit keratitis model. Combination of PACT and vancomycin treatment can have partial syngestic effect in treating MRSA biofilm and bovine keratitis model. Our study has proved that PACT may be effective in MRSA infection control, but the photoinhibition effect only lasts for less than 24 hours. Therefore, PACT may not be suitable for single treatment or once treatment for MRSA keratitis. Furhter studies are necessary to find the clinical useful regimen of PACT as a new modality for treating MRSA keratitis.

並列關鍵字

photodynamic therapy ； photodynamic antimicrobial therapy ； methicillin-resistant Staphylococcus aureus ； micelle ； chlorin e6 ； biofilm

參考文獻

1. Bennett HGB, Hay J, Devonshire P, et al. Antimicrobial management of presumed microbial keratitis: guidelines for treatment of central and peripheral ulcers. Br J Ophthalmol. 1998;82:137-145.

2. Bertoloni G, Lauro FM, Cortella G, et al. Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells. Biochim Biophys Act. 2000;1475:169-174.

3. Blomquist PH. Methicillin-resistant Staphylococcus aureus infections of the eye and orbit. Trans Am Ophthalmol Soc 2006;104:322-345.

4. Bodor N, Buchwald P. Ophthalmic drug design based on the metabolic activity of the eye: soft drugs and chemical delivery systems. AAPS J. 2005;7:820-833.

5. Bisland SK, Chien C, Wilson BC, et al. Pre-clinical in vitro and in vivo studies to examine the potential use of photodynamic therapy I the treatment of osteomyelitis. Photochem photobiol Sci. 2006;5:31-38.

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光動力抗微生物療法治療抗藥性金黃色葡萄球菌角膜炎

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