近幾年來白色念珠菌感染的發生率已有逐年增加趨勢，主要是由於頻繁的使 用抗真菌藥物治療感染，引發許多致病性真菌產生抗藥性問題，使受到真菌感染 的患者日益據增，因此發展出新型的治療方法來對抗真菌的感染，像是能減少或 是取代抗真菌藥物使用的治療方法是相當重要的議題。
透過本研究發現，光動力殺菌對白色念珠菌懸浮菌體具有良好殺菌效果。當 不論是在標準菌株，甚至是臨床抗藥性菌株上，Fluconazole 能增強光動力殺菌 對白色念珠菌的殺菌效果。另外，在另一種 azole 類藥物—Posaconazole 也發現 能夠增強光動力殺菌對白色念珠菌懸浮菌體的殺菌效果。藉由光動力殺菌結合 Fluconazole 或 Posaconazole 皆能夠對菌體造成完全殺菌效果，一方面可以減少藥 物使用劑量，降低病原菌對藥物產生抗藥性的機會。以及減低藥物副作用產生的 可能性。而在光動力殺菌結合 Fluconazole 對生物膜殺菌的應用仍有待解決問題，
像是胞外聚合物的影響或藥物作用的性質等。
未來可以繼續探討光動力殺菌結合 Fluconazole 在其他真菌、抗藥性菌株是 否也能增強殺菌效果，以及光動力殺菌結合其他種類的抗真菌藥物是否也能達到 增強殺菌效果並了解其作用機制，在生物膜方面可以嘗試將光動力殺菌結合其他 類型藥物或是透過掃描式雷射共軛焦顯微鏡觀察胞外聚合物破壞程度。
希望日後能藉由光動力殺菌和抗真菌藥物兩者能搭配出更好的治療效益，降 低藥物的使用劑量與副作用，以及減少病原菌產生抗藥性機會，有助於臨床上治 療真菌造成的感染。
結果圖表
TBO concentration (mM)
control 0.05 0.1 0.2 0.3 0.4
Log (surviving CFU/ml)
0 1 2 3 4 5 6 7 8 9
圖一、不同濃度 TBO 對白色念珠菌的光動力殺菌效果
將 TBO 0.05、0.1、0.2、0.3 和 0.4 mM 分別與 1x107 CFU/ml 白色念珠菌懸浮菌 體在避光及室溫下以平面震盪器搖晃 100 rpm 培養 30 分鐘後，離心 12000 rpm，
1 鐘重複一次，再進行照光 50 J/cm2 (630±5 nm，30 mW)，縱軸為存活菌體濃度 取 Log 值，橫軸為 TBO 濃度。(* p<0.05 ***p<0.001 as compared to control
# p<0.05 )
*
***
#
37
Incubation time (hr)
0 2 4 6 8 10 12
Log (surviving CFU/ml)
0 1 2 3 4 5 6 7 8 9
non-PDI
PDI (TBO 0.05 mM) PDI (TBO 0.075 mM) PDI (TBO 0.1 mM)
圖二、光動力作用對白色念珠菌生長的影響
將 TBO 0.05、0.075、0.1mM 分別與 1x107 CFU/ml 白色念珠菌懸浮菌體在避光 及室溫下以平面震盪器搖晃 100 rpm 培養 30 分鐘後，離心 12000 rpm，1 分鐘 重複一次，再進行照光 50 J/cm2 (630±5 nm，30 mW)，於照光後取 200 μL 菌液加
到 20 mL YPD，於 37oC 培養箱以 150 rpm 振盪培養，並在不同的培養時間點(0、
2、4、6、8、10、12 小時)取出菌液計數菌落數。縱軸為存活菌體濃度取 Log 值，
橫軸為 TBO 濃度。
Fluconazole concentration (g/ml)
control 0.125 0.25 0.5 0.75 1
Log (surviving CFU/ml)
0
Fluconazole concentration (g/ml)
control 0.125 0.25 0.5 0.75 1
Log (surviving CFU/ml)
0
圖三、不同濃度 Fluconazole 增強光動力殺菌對白色念珠菌的殺菌效果
(A)將 TBO 0.05 mM 與 1x107 CFU/ml 白色念珠菌懸浮菌體在避光及室溫下以平 面震盪器搖晃 100 rpm 培養 30 分鐘後，離心 12000 rpm，1 分鐘重複一次，進 行照光 50 J/cm2 (630±5 nm，30 mW)，再加入 0.125、0.25、0.5、0.75、1 g/ml
Fluconazole 於 37oC 培養 24 小時。(B)TBO 0.1 mM。縱軸為存活菌體濃度取 Log 值，橫軸為不同濃度 Fluconazole。(* p<0.05 ***p<0.001 as compared to control # p<0.05 ### p<0.001as compared to PDI only)
(B)
Log (CFU/ml)
Log (surviving CFU/ml)
0 1 2 3 4 5 6 7 8 9
0 g/ml 0.125 g/ml 0.25 g/ml 0.5 g/ml 0.75 g/ml 1 g/ml
圖四、不同濃度 Fluconazole 對不同起始菌數白色念珠菌的影響
將 0.125、0.25、0.5、0.75、1 g/ml Fluconazole 分別與 107、106、105、104CFU/ml
白色念珠菌懸浮菌體於 37oC 培養 24 小時。縱軸為存活菌體濃度取 Log 值，橫 軸為不同濃度 Fluconazole。
7 6 5 4
TBO concentration (mM)
control 0.05 0.075 0.1
Log (surviving CFU/ml)
0 1 2 3 4 5 6 7 8 9
PDI only
PDI+Fluconazole 0.25 g/ml
圖五、不同濃度 TBO 結合 Fluconazole 對白色念珠菌的殺菌效果
將 TBO 0.05、0.075、0.1 mM 分別與 1x107 CFU/ml 白色念珠菌懸浮菌體於避光 及室溫培養 30 分鐘，離心 12000 rpm，1 分鐘重複一次，進行照光 50 J/cm2 (630±5 nm，30 mW)，再加入 0.25 g/ml Fluconazole 於 37oC 培養 24 小時。縱軸為存活 菌體濃度取 Log 值，橫軸為不同濃度 TBO。(*p<0.05 ***p<0.001 as compared to control # p<0.05 ### p<0.001as compared to TBO 0.075 mM)
*** *** ***
*** ***
###
Log (surviving CFU/ml)
圖六、不同濃度 Fluconazole 結合光動力殺菌對白色念珠菌殺菌的效果
將 0.25、0.5、0.75、1 g/ml Fluconazole 分別與 1x107 CFU/ml 白色念珠菌懸浮菌
體於 37oC 培養 24 小時，離心 12000 rpm，1 分鐘重複一次，再與 TBO 0.05 mM 於避光及室溫下以平面震盪器搖晃 100 rpm 培養 30 分鐘後，離心 12000 rpm，1 分鐘重複一次，進行照光 50 J/cm2 (630±5 nm，30 mW)。縱軸為存活菌體濃度取 Log 值，橫軸為不同濃度 Fluconazole。(*p<0.05 ***p<0.001 as compared to control
# p<0.05 ##p<0.01 ### p<0.001as compared to PDI only )
－ － 0.25 0.5 0.75 1
Fluconazole(g/ml)
－ ＋ ＋ ＋ ＋ ＋
PDI(TBO 0.05mM)
*** *** ***
The timing added Fluconazole after PDI (hr)
control 0 2 4 6 8 10 12
Log surviving (CFU/ml)
0 1 2 3 4 5 6 7 8 9
non-PDI
PDI (TBO 0.1 mM)
圖七、光動力作用後，不同的時間點加入 Fluconazole 對白色念珠菌的影響
將 TBO 0.1 mM 與 1x107 CFU/ml 白色念珠菌懸浮菌體在避光及室溫下以平面震 盪器搖晃 100 rpm 培養 30 分鐘後，離心 12000 rpm，1 分鐘重複一次，進行照 光 50 J/cm2 (630±5 nm，30 mW)後於 37oC 培養，並在不同時間點(0、2、4、6、8、
10、12 小時)加入 70 l Fluconazole(最終濃度 0.25 g/ml)培養 24 小時，縱軸為存 活菌體濃度取 Log 值，橫軸為不同培養時間點。(* p<0.05 ***p<0.001 as compared to PDI only)
*** ***
***
Posaconazole concentration (g/ml)
control 0.125 0.25 0.5 0.75 1
Log (surviving CFU/ml)
0
PDI (TBO 0.05mM)
Posaconazole concentration (g/ml) control 0.125 0.25 0.5 0.75 1
Log (survivng CFU/ml)
0 PDI (TBO 0.1mM)
圖八、不同濃度 Posaconazole 增強光動力殺菌對白色念珠菌的殺菌效果
(A)將 TBO 0.05 mM 與 1x107 CFU/ml 白色念珠菌懸浮菌體在避光及室溫下以平 面震盪器搖晃 100 rpm 培養 30 分鐘後，離心 12000 rpm，1 分鐘重複一次，進 行照光 50 J/cm2 (630±5 nm，30 mW)，再加入 0.125、0.25、0.5、0.75、1 g/ml
Posaconazole 於 37oC 培養 24 小時。(B)TBO 0.1 mM。縱軸為存活菌體濃度取 Log 值，橫軸為不同濃度 Posaconazole。(* p<0.05 ***p<0.001 as compared to control #
(A)
control F PDI PDI+F
Log (surviving CFU/ml)
0
control P PDI PDI+P
Log (surviving CFU/ml)
0
g/ml Posaconazole 於 37oC 培養 24 小時。(A)0.25 g/ml Fluconazole (B)0.5 g/ml Posaconazole。縱軸為存活菌體濃度取 Log 值。(* p<0.05 **p<0.01 ***p<0.001 as compared to SC5314 # p<0.05 ##p<0.01 ### p<0.001 as compared to PDI only)
(A)
(B)
** ##
*** ###
TBO concentration (mM)
control 0.1 0.2 0.4
Log (surviving CFU/ml)
0 1 2 3 4 5 6 7 8 9
PDI only
PDI+Fluconazole 0.25 g/ml
圖十、不同濃度 TBO 結合 Fluconazole 對白色念珠菌抗藥性菌株的殺菌效果 將 TBO 0.1、0.2、0.4 mM 分別與 1x107 CFU/ml 白色念珠菌抗藥性菌株於避光及 室溫培養 30 分鐘，離心 12000 rpm，1 分鐘重複一次，進行照光 50 J/cm2 (630±5 nm，30 mW)，再加入 0.25 g/ml Fluconazole 於 37oC 培養 24 小時。縱軸為存活 菌體濃度取 Log 值，橫軸為不同濃度 TBO。(*p<0.05 **p<0.01 ***p<0.001 as compared to control # p<0.05 ### p<0.001as compared to TBO 0.2 mM)
** ***
***
*** ***
###
TBO concentration (mM)
control 0.25 0.5 1 2.5 5 10
Log (surviving CFU/cm2 )
0 1 2 3 4 5 6 7 8 9
圖十一、光動力殺菌對白色念珠菌生物膜的殺菌效果
將不同濃度 TBO 與白色念珠菌生物膜在避光及室溫下培養 30 分鐘後，進行照 光 50 J/cm2 (630±5 nm，30 mW)。縱軸為存活菌體取 Log 值，橫軸為 TBO 濃度。
(* p<0.05 ***p<0.001 as compared to control)
*** *** ***
*** *** ***
Fluconazole concentration (g/ml) Log surviving(CFU/cm2 )
0 1 2 3 4 5 6 7 8 9
non-PDI
PDI (TBO 2.5 mM)
圖十二、光動力殺菌結合 Fluconazole 對白色念珠菌生物膜的殺菌效果
將 TBO 2.5mM 與白色念珠菌生物膜在避光及室溫下培養 30 分鐘後，進行照光
50 J/cm2 (630±5 nm，30 mW)，再加入不同濃度 Fluconazole 培養 24 小時。縱軸 為存活菌體取 Log 值，橫軸為 Fluconazole 濃度。
(* p<0.05 ***p<0.001 as compared to control)
0 10 25 50 75 100 150 200
***
*** *** *** *** *** *** ***
附錄
附錄一、白色念珠菌生物膜形成
Adapted from
Nobile CJ, Johnson AD., Candida albicans biofilms and human disease. Annu Rev Microbiol, 2015. 69: p. 71-92.
附錄二、光化學反應
Adapted from
Ochsner, M., Photophysical and photobiological processes in the photodynamic therapy of tumours. J Photochem Photobiol B, 1997. 39(1): p. 1-18.
附錄三、TBO (Toluidine Blue O)的分子結構
Adapted from
Sigma-Aldrich (St. Louis MO, USA)
Fluconazole
Posaconazole
附錄四、Fluconazole 與 Posaconazole 的分子結構
Adapted from
Nagappan V, Deresinski S., Reviews of anti-infective agents: posaconazole: a broad-spectrum triazole antifungal agent. Clin Infect Dis, 2007. 45(12):p.1610-1617.
附錄五、光動力殺菌與 Fluconazole 對白色念珠菌之間交互作用
TBO
參考文獻
1. Sørum,H.and M. Sunde, Resistance to antibiotics in the normal flora of animals.
Vet. Res, 2001. 32 (3-4): p. 227–241.
2. Patil S, et al., Clinical Appearance of Oral Candida Infectionand Therapeutic Strategies. Front Microbiol, 2015. 6(1391): p.1-10.
3. Akpan A. and Morgan R., Oral candidiasis. Postgrad Med J, 2002.78(922): p.
455–459.
4. 藍志堅, 院內感染管制：原理與實用.合記, 2000.
5. Richards MJ, et al., Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System. Crit Care Med, 1999. 27(5): p. 887-892.
6. 衛生署疾病管制局傳染病統計暨監視年報. 2014.
7. LoH.J.,et al., Nonfilamentous C. albicans Mutants Are Avirulent. Cell, 1997.
90(5): p939–949.
8. Ruhnke M., Epidemiology of Candida albicans infections and role of non-Candida-albicans yeasts. Curr Drug Targets, 2006. 7(4): p.495-504.
9. Wey, S.B., et al., Hospital-acquired candidemia. The attributable mortality and excess length of stay. Arch Intern Med, 1988. 148(12): p. 2642-2645.
10. Chen, Y.C., et al., Stable susceptibility of Candida blood isolates to fluconazole despite increasing use during the past 10 years. J Antimicrob Chemother, 2003.
52(1): p. 71-77.
11. 吳綺容, 李欣純, 柯文謙, 念珠菌菌血症臨床處置的新進展. 內科學誌, 2003.
14(5): p. 224-231.
12. O'Toole G, Kaplan HB, Kolter R., Biofilm formation as microbial development.
Annu Rev Microbiol, 2000. 54: p. 49-79.
13. Kojic, E.M. and R.O. Darouiche, Candida infections of medical devices. Clin Microbiol Rev, 2004. 17(2): p. 255-267.
14. Seneviratne, C.J., L. Jin, and L.P. Samaranayake, Biofilm lifestyle of Candida: a mini review. Oral Dis, 2008. 14(7): p. 582-590.
15. Baillie, G.S. and L.J. Douglas, Role of dimorphism in the development of Candida albicans biofilms. J Med Microbiol, 1999. 48(7): p. 671-679.
16. Mukherjee P.K., Chandra J., Candida biofilm resistance. Drug Resist Updat, 2004.
7(4-5):p. 301-309.
17. Gristina, A.G., et al., The glycocalyx, biofilm, microbes, and resistant infection.
Semin Arthroplasty, 1994. 5(4): p. 160-170.
18. Kumamoto C.A., Candida biofilms. Curr Opin Microbiol, 2002. 5(6): p. 608-611.
19. Ramage, G., et al., Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother, 2002. 49(6): p. 973-980.
20. LaFleur M.D., Kumamoto C.A., et al., Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob Agents Chemother, 2006. 50(11): p.
3839-3846.
21. Allison D.G., Gilbert P., Modification by surface association of antimicrobial susceptibility of bacterial populations. J Ind Microbiol, 1995. 15(4):p. 311-317.
22. Dougherty, T.J., et al., Photodynamic therapy. J Natl Cancer Inst, 1998. 90(12): p.
889-905.
23. Raab, O., Uber die Wirkung fluoreszierender Stoffe auf Infusorien. Zeitung Biol, 1900. 39: p. 524-526.
24. Roelandts, R., A new light on Niels Finsen, a century after his nobel prize.
Photodermatol Photoimmunol Photomed, 2005. 21(3): p.115-117.
Muench Med. Wochenschr, 1903. 47: p. 2042-2044.
26. Diamond, I., et al., Photodynamic therapy of malignant tumors. The Lancet, 1972.
300(7788): p. 1175-1177.
27. Kelly, J.F. and M.E. Snell, Hematoporphyrin derivative : possible aid in diagnosis and therapy of carcinoma of bladder. J Urol, 1976. 115(2): p.150-151.
28. Ochsner, M., Photophysical and photobiological processes in the photodynamic therapy of tumours. J Photochem Photobiol B, 1997. 39(1): p. 1-18.
29. Soukos, N.S., et al., Photodynamic effects of toluidine blue on human oral keratinocytes and fibroblasts and Streptococcus sanguis evaluated in vitro. Lasers Surg Med, 1996. 18(3): p. 253-259.
30. Aoki A, Takeuchi Y, et al., Antimicrobial photodynamic therapy suppresses dental plaque formation in healthy adults: a randomized controlled clinical trial. BMC Oral Health, 2014. 14: p. 152-161.
31. Donnelly, R.F., P.A. McCarron, and M.M. Tunney, Antifungal photodynamic therapy. Microbiological Research, 2008. 163(1): p. 1-12.
32. Cohen, B.E., Amphotericin B toxicity and lethality: a tale of two channels.
International Journal of Pharmaceutics, 1998. 162(1-2): p. 95-106.
33. Mehta, R., et al., Liposomal amphotericin B is toxic to fungal cells but not to mammalian cells. Biochim Biophys Acta, 1984. 770(2): p. 230-234.
34. Vermes, A., H.J. Guchelaar, and J. Dankert, Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J Antimicrob Chemother, 2000. 46(2): p. 171-179.
35. Bennett J.T., Dismukes W.E., Duma R.T., et al, A comparison of amphotericin B alone and combined with flucytosine in the treatment of cryptococcal meningitis.
N Engl J Med, 1979. 301(3): p. 126-131.
buccal epithelial cells of oral Candida dubliniensis isolates subsequent to limited exposure to amphotericin B, ketoconazole and fluconazole. J Investig Clin Dent, 2015. 6(3): p. 186-192.
37. Orozco, A.S., et al., Mechanism of fluconazole resistance in Candida krusei.
Antimicrob Agents Chemother, 1998. 42(10): p. 2645-2649.
38. Nagappan V, Deresinski S., Reviews of anti-infective agents: posaconazole: a broad-spectrum triazole antifungal agent. Clin Infect Dis, 2007. 45(12): p.
1610-1617.
39. Moudgal V., Sobel J., Antifungals to treat Candida albicans. Expert Opin Pharmacother, 2010. 11(12): p. 2037-2048.
40. Hoang A., Caspofungin acetate: an antifungal agent. Am J Health Syst Pharm, 2001. 58(13): p. 1206-1214.
41. White, T.C., K.A. Marr, and R.A. Bowden, Clinical, cellular, and molecularfactors that contribute to antifungal drug resistance. Clin Microbiol Rev, 1998. 11(2): p.
382-402.
42. Subden, R.E., et al., Eburicol, lichesterol, ergosterol, and obtusifoliol from polyene antibiotic-resistant mutants of Candida albicans. Can J Microbiol, 1977.
23(6): p. 751-754.
43. Lamb, D.C., et al., The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem, 1997. 272(9): p. 5682-5688.
44. Sanglard, D., et al., Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother, 1996. 40(10): p. 2300-2305.
45. Perlin, D.S., Resistance to echinocandin-class antifungal drugs. Drug Resist Updat,
46. Hamblin, M.R.and Hasan, T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem Photobiol Sci, 2004. 3(5): p. 436-450.
47. 林明璇,甲殼素輔助光動力抑菌與細菌細胞壁之關聯性探討. 2011, 國立臺灣 大學.
48. Kato IT, Prates RA, et al., Antimicrobial photodynamic inactivation inhibits Candida albicans virulence factors and reduces in vivo pathogenicity. Antimicrob
Agents Chemother, 2013. 57(1): p.445-451.
49. Sharma S.K., Dai T., et al., Drug discovery of antimicrobial photosensitizers using animal models. Curr Pharm Des, 2011. 17(13): p.1303-1319.
50. Bliss JM, Bigelow CE, Foster TH, Haidaris CG. Susceptibility of Candida species to photodynamic effects of photofrin. Antimicrob Agents Chemother, 2004. 48(6):
p.2000–2006.
51. Lambrechts SA, Aalders MC, Van Marle J., Mechanistic study of the photodynamic inactivation of Candida albicans by a cationic porphyrin.
Antimicrob Agents Chemother, 2005. 49(5): p. 2026-2034.
52. Navarathna DH, Hornby JM, et al., Enhanced pathogenicity of Candida albicans pre-treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis. J Antimicrob Chemother, 2005. 56(6): p.1156-1159.
53. Casalinuovo IA, Di Francesco P, Garaci E. Fluconazole resistance in Candida albicans: a review of mechanisms. Eur Rev Med Pharmacol Sci, 2004. 8(2):
p.69-77.
54. Soysal A. Prevention of invasive fungal infections in immunocompromised patients: the role of delayed-release posaconazole. Infect Drug Resist, 2015. 8:
p.321-331.
55. Franz, R., Ruhnke M., and Morschhäuser J. Molecular aspects of fluconazole
56. Baillie, G. S., and L. J. Douglas. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol, 1999. 310: p.644–656.
57. Ramage, G., et al., Candida biofilms: an update. Eukaryot Cell, 2005. 4(4): p.
633-638.
58. Sardi Jde C, Pitangui Nde S, et al., Highlights in pathogenic fungal biofilms. Rev Iberoam Micol, 2014. 31(1): p. 22-29.
59. Dovigo LN, Carmello JC, et al., Photodynamic inactivation of clinical isolates of Candida using Photodithazine® . Biofouling, 2013. 29(9): p. 1057-67.
60. Mukherjee, P. K., Chandra J, et al., Mechanism of fluconazole resistance in Candida albicans biofilms: phasespecific role of efflux pumps and membrane
sterols. Infect. Immun, 2003. 71: p. 4333–4340.
61. Chandra J, Kuhn DM, et al., Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J. Bacteriol, 2001.
183(18): p.5385-5394.
62. Denning DW. Echinocandin antifungal drugs. Lancet, 2003. 362(9390): p.
1142–1151.
63. Kuhn, D. M., George T., et al., Antifungal susceptibility of Candida biofilms:
unique efficacy of amphotericin B lipid formulations and echinocandins.
Antimicrob Agents Chemother, 2002. 46(6): p. 1773-1780.
64. Li DD, Xu Y, et al., Fluconazole assists berberine to kill fluconazole-resistant Candida albicans. Antimicrob Agents Chemother, 2013. 57(12): p.6016-6027.
65. Chou TC., Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res, 2010. 70(2): p.440-446.