光動力療法（photodynamic therapy, PDT）乃是結合感光劑及適當波長之光源，經由電子或能量轉移產生能進行無特異性目標攻擊之活性氧類（reactive oxygen species），以破壞腫瘤及病原體。臨床上已使用五胺基酮戊酸（5-aminolevulinic acid, ALA）作為感光劑前驅物，針對表面傷口感染進行治療。多數病人之傷口皆能有效治癒復原，但亦有少數病人無法有效抑制細菌感染。本研究即針對四種由兩位分別接受一次和五次ALA-PDT之病人所分離而得之細菌－oxacillin-resistant Staphylococcus aureus（ORSA）, Enterococcus faecalis, Pseudomonas aeruginosa, Shewanella putrefaciens－於體外使用ALA進行光動力抑菌試驗（ALA-PDI）。我們以隨機擴增片段多型性（RAPD）方法將屬同一菌種之菌株加以分型，發現各菌種之菌株含有至少兩種以上的RAPD型別。我們接著測定各菌種之菌株對ALA-PDI之感受性，並以fluorometry測定其代謝ALA所產生具感光性之紫質衍生物的量，發現了不同菌種對ALA-PDI即有不同之反應。具不同型別的P. aeruginosa對ALA-PDI及其菌體內代謝ALA所產生的紫質衍生物之量均有劑量依存關係。不同型別的ORSA菌株則在ALA濃度約0.1 mM時表現最高的感受性，同時其菌體內所產生之porphyrin derivatives的量亦達到最高。E. faecalis菌株則對ALA-PDI無明顯反應，且其菌體內亦偵測不到紫質衍生物的產生，推測可能該菌無法有效攝取或利用ALA合成紫質衍生物而使ALA-PDI無效。較令人困惑的是，ALA-PDI對S. putrefaciens雖無殺菌效果，但我們仍能於此菌之菌體內偵測到大量紫質衍生物的產生。本研究之結果顯示不同細菌對ALA-PDI之感受度即有差異，因此，臨床上採用ALA-PDT治療細菌感染前，須先試驗各個菌株對ALA-PDI之感受性，找出最適當之感光劑濃度及光劑量，方能將光動力療法之殺菌能力發揮至最大。

　　Photodynamic therapy （PDT） combines the photosensitizers （PSs） and visible light to produce a phototoxic response that results in oxidative damages to a variety of targets including nucleic acids, proteins, and lipids. It has been noticed that bacterial strains of the same species were repeatedly isolated from the wounds of two patients who received one and five, respectively, courses of PDT with 5-aminolevulinic acid （ALA）, a precursor of photoactive porphyrin derivatives. From one of them the oxacillin-resistant Staphylococcus aureus （ORSA）, Enterococcus faecalis and Shewanella putrefaciens were isolated, while from the other patient only Pseudomonas aeruginosa was isolated. The strains of each species exhibited at least two different randomly amplified polymorphic DNA （RAPD） types. We then examined the strains of different RAPD types for their susceptibility to photodynamic inactivation（PDI） with ALA and the amount of porphyrins produced in their cells. We found that strains of ORSA and P. aeruginosa could be killed by ALA-PDI but those of E. faecalis and S. putrefaciens could not. Both the susceptibility to ALA-PDI and amounts of porphyrins synthesized from ALA showed a dose-dependent response for the P. aeruginosa strains. Strains of ORSA showed an optimal response to 0.1 mM of ALA for both the sensitivity to ALA-PDI and porphyrins production from ALA. No fluorescence representing the porphyrins produced from ALA was detected in E. faecalis, suggesting that this species may be unable to uptake or metabolize ALA to porphyrins, which leads to irresponsiveness of this bacterial species to ALA-PDI. Intriguingly, although strong fluorescence signals were detected in the cells of S. putrefaciens, strains of this species were resistant to ALA-PDI. These results suggest that the bacterial pathogens may not be equally sensitive to PDT with ALA, and it may be necessary to test each isolate to find the optimal ALA-PDT conditions.

1. Castano, A.P., Demidova, T.N. & Hamblin, M.R. Mechanisms in photodynamic therapy: part one--photosensitizers, photochemistry and cellular localization. Photodiagnosis and photodynamic therapy 1, 279-293 (2004).
2. Hamblin, M.R. & Hasan, T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochemical and photobiological sciences 3, 436-450 (2004).
3. Pervaiz, S. & Olivo, M. Art and science of photodynamic therapy. Clinical and experimental pharmacology and physiology 33, 551-556 (2006).
4. Foote, C.S. Definition of type I and type II photosensitized oxidation. Photochemistry and photobiology 54, 659 (1991).
5. Maisch, T. et al. The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria. Proceedings of the national academy of sciences of the united states of America 104, 7223-7228 (2007).
6. Wainwright, M. Photodynamic antimicrobial chemotherapy (PACT). The Journal of antimicrobial chemotherapy 42, 13-28 (1998).
7. van den Bergh, H. On the evolution of some endoscopic light delivery systems for photodynamic therapy. Endoscopy 30, 392-407 (1998).
8. MacCormack, M.A. Photodynamic therapy. Advances in dermatology 22, 219-258 (2006).
9. Triesscheijn, M., Baas, P., Schellens, J.H. & Stewart, F.A. Photodynamic therapy in oncology. Oncologist 11, 1034-1044 (2006).
10. Henderson, B.W. et al. Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors. Cancer research 64, 2120-2126 (2004).
11. Alster, T.S. & Surin-Lord, S.S. Photodynamic therapy: practical cosmetic applications. Journal of drugs in dermatology 5, 764-768 (2006).
12. Tschen, E.H. et al. Photodynamic therapy using aminolaevulinic acid for patients with nonhyperkeratotic actinic keratoses of the face and scalp: phase IV multicentre clinical trial with 12-month follow up. The British journal of dermatology 155, 1262-1269 (2006).
13. Tang, H.M., Hamblin, M.R. & Yow, C.M. A comparative in vitro photoinactivation study of clinical isolates of multidrug-resistant pathogens. Journal of infection and chemotherapy 13, 87-91 (2007).
14. Cunha, B.A. Antibiotic resistance. Control strategies. Critical care clinics 14, 309-327 (1998).
15. Falagas, M.E. & Bliziotis, I.A. Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era? International journal of antimicrobial agents 29, 630-636 (2007).
16. Maisch, T., Bosl, C., Szeimies, R.M., Love, B. & Abels, C. Determination of the antibacterial efficacy of a new porphyrin-based photosensitizer against MRSA ex vivo. Photochemical and photobiological sciences 6, 545-551 (2007).
17. Tegos, G.P. et al. Protease-stable polycationic photosensitizer conjugates between polyethyleneimine and chlorin(e6) for broad-spectrum antimicrobial photoinactivation. Antimicrobial agents and chemotherapy 50, 1402-1410 (2006).
18. Nitzan, Y. & Ashkenazi, H. Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a cationic hydrophilic porphyrin at various light wavelengths. Current microbiology 42, 408-414 (2001).
19. Sharma, M. et al. Toluidine blue-mediated photodynamic effects on staphylococcal biofilms. Antimicrobial agents and chemotherapy 52, 299-305 (2008).
20. Wood, S., Metcalf, D., Devine, D. & Robinson, C. Erythrosine is a potential photosensitizer for the photodynamic therapy of oral plaque biofilms. The Journal of antimicrobial chemotherapy 57, 680-684 (2006).
21. Komerik, N., Wilson, M. & Poole, S. The effect of photodynamic action on two virulence factors of gram-negative bacteria. Photochemistry and photobiology 72, 676-680 (2000).
22. Wainwright, M. Local treatment of viral disease using photodynamic therapy. International journal of antimicrobial agents 21, 510-520 (2003).
23. Mohr, H., Lambrecht, B. & Selz, A. Photodynamic virus inactivation of blood components. Immunological investigations 24, 73-85 (1995).
24. Donnelly, R.F., McCarron, P.A. & Tunney, M.M. Antifungal photodynamic therapy. Microbiological research 163, 1-12 (2008).
25. Lustigman, S. & Ben-Hur, E. Photosensitized inactivation of Plasmodium falciparum in human red cells by phthalocyanines. Transfusion 36, 543-546 (1996).
26. Akilov, O.E., Kosaka, S., O'Riordan, K. & Hasan, T. Photodynamic therapy for cutaneous leishmaniasis: the effectiveness of topical phenothiaziniums in parasite eradication and Th1 immune response stimulation. Photochemical and photobiological sciences 6, 1067-1075 (2007).
27. Carvalho, C.M. et al. Photoinactivation of bacteria in wastewater by porphyrins: bacterial beta-galactosidase activity and leucine-uptake as methods to monitor the process. Journal of photochemistry and photobiology 88, 112-118 (2007).
28. Jori, G. & Brown, S.B. Photosensitized inactivation of microorganisms. Photochemical and photobiological sciences 3, 403-405 (2004).
29. Morton, C.A. et al. Guidelines for topical photodynamic therapy: report of a workshop of the British Photodermatology Group. The British journal of dermatology 146, 552-567 (2002).
30. Juzeniene, A., Peng, Q. & Moan, J. Milestones in the development of photodynamic therapy and fluorescence diagnosis. Photochemical and photobiological sciences 6, 1234-1245 (2007).
31. Lee, D.H., Jun, W.J., Shin, D.H., Cho, H.Y. & Hong, B.S. Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli. Bioscience, biotechnology, and biochemistry 69, 470-476 (2005).
32. Gadmar, O.B., Moan, J., Scheie, E., Ma, L.W. & Peng, Q. The stability of 5-aminolevulinic acid in solution. Journal of photochemistry and photobiology 67, 187-193 (2002).
33. Kaliszewski, M. et al. Biological activity of 5-aminolevulinic acid and its methyl ester after storage under different conditions. Journal of photochemistry and photobiology 87, 67-72 (2007).
34. Hunter, G.A., Rivera, E. & Ferreira, G.C. Supraphysiological concentrations of 5-aminolevulinic acid dimerize in solution to produce superoxide radical anions via a protonated dihydropyrazine intermediate. Archives of biochemistry and biophysics 437, 128-137 (2005).
35. Claydon, P.E. & Ackroyd, R. 5-Aminolaevulinic acid-induced photodynamic therapy and photodetection in Barrett's esophagus. Diseases of the esophagus 17, 205-212 (2004).
36. Dietel, W., Pottier, R., Pfister, W., Schleier, P. & Zinner, K. 5-Aminolaevulinic acid (ALA) induced formation of different fluorescent porphyrins: a study of the biosynthesis of porphyrins by bacteria of the human digestive tract. Journal of photochemistry and photobiology. B, Biology 86, 77-86 (2007).
37. Johnsson, A., Kjeldstad, B. & Melo, T.B. Fluorescence from pilosebaceous follicles. Archives of dermatological research 279, 190-193 (1987).
38. Ramstad, S., Le Anh-Vu, N. & Johnsson, A. The temperature dependence of porphyrin production in Propionibacterium acnes after incubation with 5-aminolevulinic acid (ALA) and its methyl ester (m-ALA). Photochemical and photobiological sciences 5, 66-72 (2006).
39. Berg, K. et al. Porphyrin-related photosensitizers for cancer imaging and therapeutic applications. Journal of microscopy 218, 133-147 (2005).
40. Trilla, A. & Miro, J.M. Identifying high risk patients for Staphylococcus aureus infections: skin and soft tissue infections. Journal of chemotherapy 7 Suppl 3, 37-43 (1995).
41. Barber, M. Methicillin-resistant staphylococci. Journal of clinical pathology 14, 385-393 (1961).
42. Ayliffe, G.A. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clinical infectious diseases 24 Suppl 1, S74-79 (1997).
43. Johnson, A.P., Pearson, A. & Duckworth, G. Surveillance and epidemiology of MRSA bacteraemia in the UK. The Journal of antimicrobial chemotherapy 56, 455-462 (2005).
44. Janapatla, R.P. et al. Inducible clindamycin resistance in Staphylococcus aureus isolates causing bacteremia at a university hospital in southern Taiwan. Diagnostic microbiology and infectious disease 58, 203-209 (2007).
45. Feng, Y. et al. Evolution and pathogenesis of Staphylococcus aureus: lessons learned from genotyping and comparative genomics. FEMS microbiology reviews 32, 23-37 (2008).
46. Stuart, C.H., Schwartz, S.A., Beeson, T.J. & Owatz, C.B. Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. Journal of endodontics 32, 93-98 (2006).
47. Tendolkar, P.M., Baghdayan, A.S. & Shankar, N. Pathogenic enterococci: new developments in the 21st century. Cellular and molecular life sciences 60, 2622-2636 (2003).
48. Amyes, S.G. Enterococci and streptococci. International journal of antimicrobial agents 29 Suppl 3, S43-52 (2007).
49. Jean, S.S. et al. Invasive infections due to vancomycin-resistant enterococci in adult patients. Journal of microbiology, immunology, and infection 34, 281-286 (2001).
50. Chou, Y.Y. et al. Vancomycin-resistant enterococcal bacteremia: comparison of clinical features and outcome between Enterococcus faecium and Enterococcus faecalis. Journal of microbiology, immunology, and infection 41, 124-129 (2008).
51. Cunha, B.A., Mickail, N. & Eisenstein, L. E. faecalis vancomycin-sensitive enterococcal bacteremia unresponsive to a vancomycin tolerant strain successfully treated with high-dose daptomycin. Heart and lung 36, 456-461 (2007).
52. Uchiyama, J. et al. Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C as a therapeutic candidate. FEMS microbiology letters 278, 200-206 (2008).
53. Davies, J.C. & Rubin, B.K. Emerging and unusual gram-negative infections in cystic fibrosis. Seminars in respiratory and critical care medicine 28, 312-321 (2007).
54. Saiman, L. Clinical utility of synergy testing for multidrug-resistant Pseudomonas aeruginosa isolated from patients with cystic fibrosis: 'the motion for'. Paediatric respiratory reviews 8, 249-255 (2007).
55. Gomez, M.I. & Prince, A. Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis. Current opinion in pharmacology 7, 244-251 (2007).
56. Ryder, C., Byrd, M. & Wozniak, D.J. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Current opinion in microbiology 10, 644-648 (2007).
57. Navon-Venezia, S., Ben-Ami, R. & Carmeli, Y. Update on Pseudomonas aeruginosa and Acinetobacter baumannii infections in the healthcare setting. Current opinion in infectious diseases 18, 306-313 (2005).
58. MacDonell, M. & Colwell, R. Phylogeny of the Vibrionaceae, and recommendation for two new genera, Listonella and Shewanella. Systematic and applied microbiology 6, 171-182 (1985).
59. Chen, Y.S. et al. Skin and soft-tissue manifestations of Shewanella putrefaciens infection. Clinical infectious diseases 25, 225-229 (1997).
60. Tsai, T.H. & You, H.Y. Necrotizing fasciitis caused by Shewanella putrefaciens in a uremic patient. Journal of microbiology, immunology, and infection 39, 516-518 (2006).
61. Martin-Gil, J., Ramos-Sanchez, M.C. & Martin-Gil, F.J. Shewanella putrefaciens in a fuel-in-water emulsion from the Prestige oil spill. Antonie Van Leeuwenhoek 86, 283-285 (2004).
62. Pagani, L. et al. Soft tissue infection and bacteremia caused by Shewanella putrefaciens. Journal of clinical microbiology 41, 2240-2241 (2003).
63. Wang, I.K., Lee, M.H., Chen, Y.M. & Huang, C.C. Polymicrobial bacteremia caused by Escherichia coli, Edwardsiella tarda, and Shewanella putrefaciens. Chang Gung medical journal 27, 701-705 (2004).
64. Holt, H.M., Gahrn-Hansen, B. & Bruun, B. Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics. Clinical microbiology and infection 11, 347-352 (2005).
65. Kohen, R. & Nyska, A. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicologic pathology 30, 620-650 (2002).
66. Lee, J.H. Methicillin (Oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans. Applied and environmental microbiology 69, 6489-6494 (2003).
67. Ben Omar, N. et al. Functional and safety aspects of Enterococci isolated from different Spanish foods. Systematic and applied microbiology 27, 118-130 (2004).
68. Ruiz, L., Dominguez, M.A., Ruiz, N. & Vinas, M. Relationship between clinical and environmental isolates of Pseudomonas aeruginosa in a hospital setting. Archives of medical research 35, 251-257 (2004).
69. Vogel, B.F. et al. Homogeneity of Danish environmental and clinical isolates of Shewanella algae. Applied and environmental microbiology 66, 443-448 (2000).
70. Nitzan, Y., Salmon-Divon, M., Shporen, E. & Malik, Z. ALA induced photodynamic effects on gram positive and negative bacteria. Photochemical and photobiological sciences 3, 430-435 (2004).
71. Szocs, K., Gabor, F., Csik, G. & Fidy, J. delta-Aminolaevulinic acid-induced porphyrin synthesis and photodynamic inactivation of Escherichia coli B. Journal of photochemistry and photobiology 50, 8-17 (1999).
72. Chen, C.Y., Nace, G.W. & Irwin, P.L. A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli. Journal of microbiological methods 55, 475-479 (2003).
73. Power, E.G. RAPD typing in microbiology--a technical review. The Journal of hospital infection 34, 247-265 (1996).
74. Yoshikawa, T.T. Antimicrobial resistance and aging: beginning of the end of the antibiotic era? Journal of the american geriatrics society 50, S226-229 (2002).
75. Wilson, B.C. & Patterson, M.S. The physics, biophysics and technology of photodynamic therapy. Physics in medicine and biology 53, R61-109 (2008).
76. Jori, G. et al. Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers in surgery and medicine 38, 468-481 (2006).
77. Fotinos, N., Campo, M.A., Popowycz, F., Gurny, R. & Lange, N. 5-Aminolevulinic acid derivatives in photomedicine: Characteristics, application and perspectives. Photochemistry and photobiology 82, 994-1015 (2006).
78. Krammer, B. & Plaetzer, K. ALA and its clinical impact, from bench to bedside. Photochemical and photobiological sciences 7, 283-289 (2008).
79. Kelty, C.J., Brown, N.J., Reed, M.W. & Ackroyd, R. The use of 5-aminolaevulinic acid as a photosensitiser in photodynamic therapy and photodiagnosis. Photochemical and photobiological sciences 1, 158-168 (2002).
80. Atienzar, F.A. & Jha, A.N. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutation research 613, 76-102 (2006).
81. Lin, H.Y., Chen, C.T. & Huang, C.T. Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells. Applied and environmental microbiology 70, 6453-6458 (2004).
82. Embleton, M.L., Nair, S.P., Cookson, B.D. & Wilson, M. Selective lethal photosensitization of methicillin-resistant Staphylococcus aureus using an IgG-tin (IV) chlorin e6 conjugate. The Journal of antimicrobial chemotherapy 50, 857-864 (2002).
83. Usacheva, M.N., Teichert, M.C. & Biel, M.A. Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram-positive and gram-negative microorganisms. Lasers in surgery and medicine 29, 165-173 (2001).
84. Fotinos, N., Convert, M., Piffaretti, J.C., Gurny, R. & Lange, N. Effects on gram-negative and gram-positive bacteria mediated by 5-aminolevulinic Acid and 5-aminolevulinic acid derivatives. Antimicrobial agents and chemotherapy 52, 1366-1373 (2008).
85. Hancock, L.E. & Gilmore, M.S. The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall. Proceedings of the national academy of sciences of the united states of america 99, 1574-1579 (2002).
86. Juzenas, P., Iani, V., Bagdonas, S., Rotomskis, R. & Moan, J. Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light. Journal of photochemistry and photobiology. B, Biology 61, 78-86 (2001).
87. Qin, Y. et al. Toluidine blue-mediated photoinactivation of periodontal pathogens from supragingival plaques. Lasers in medical science 23, 49-54 (2008).
88. King, N.D. & O'Brian, M.R. Identification of the lrp gene in Bradyrhizobium japonicum and its role in regulation of delta-aminolevulinic acid uptake. Journal of bacteriology 179, 1828-1831 (1997).
89. Carter, R.A. et al. dpp genes of Rhizobium leguminosarum specify uptake of delta-aminolevulinic acid. Molecular plant-microbe interactions 15, 69-74 (2002).
90. Paulsen, I.T. et al. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299, 2071-2074 (2003).
91. Lyon, D.Y., Brunet, L., Hinkal, G.W., Wiesner, M.R. & Alvarez, P.J. Antibacterial activity of fullerene water suspensions (nC60) is not due to ROS-mediated damage. Nano letters 8, 1539-1543 (2008).