臺灣博碩士論文加值系統

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。

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.

英文摘要………………………………………………………………………… 1
中文摘要………………………………………………………………………… 3
緒論……………………………………………………………………………… 5
實驗設計與目的………………………………………………………………… 11
材料與方法……………………………………………………………………… 12
[第一部份]…………………………………………………………………… 12
[第二部份]…………………………………………………………………… 17
[第三部份]…………………………………………………………………… 19
[第四部份]…………………………………………………………………… 24
結果……………………………………………………………………………… 27
[第一部份]…………………………………………………………………… 27
[第二部份]…………………………………………………………………… 28
[第三部份]…………………………………………………………………… 30
[第四部份]…………………………………………………………………… 32
討論……………………………………………………………………………… 34
實驗結果之附圖表…………………………………………………………………… 42
參考文獻………………………………………………………………………… 70
附錄……………………………………………………………………………… 77

Adam, B., Baillie, G.S., and Douglas, L.J. (2002) Mixed species biofilms of Candida albicans and Staphylococcus epidermidis. J Med Microbiol 51: 344-349.
Allen, T.M. (1994) Long-circulating (sterically stabilized) liposomes for targeted drug delivery. Trends Pharmacol Sci 15: 215-220.
Bachor, R., Scholz, M., Shea, C.R., and Hasan, T. (1991) Mechanism of photosensitization by microsphere-bound chlorin e6 in human bladder carcinoma cells. Cancer Res 51: 4410-4414.
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.
Bonnett, R., Djelal, B.D., Hamilton, P.A., Martinez, G., and Wierrani, F. (1999) Photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP) and the corresponding chlorin (m-THPC) and bacteriochlorin (m-THPBC). A comparative study. J Photochem Photobiol B 53: 136-143.
Brown, M.R., Allison, D.G., and Gilbert, P. (1988) Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother 22: 777-780.
Carpentier, B., and Cerf, O. (1993) Biofilms and their consequences, with particular reference to hygiene in the food industry. J Appl Bacteriol 75: 499-511.
Chakraborty, N., and Tripathy, B.C. (1992) Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts. Plant Physiol 98: 7-11.
Chambers, H.F. (1997) Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev 10: 781-791.
Christensson, B., Fehrenbach, F.J., and Hedstrom, S.A. (1985) A new serological assay for Staphylococcus aureus infections: detection of IgG antibodies to S. aureus lipase with an enzyme-linked immunosorbent assay. J Infect Dis 152: 286-292.
Costerton, J.W., Stewart, P.S., and Greenberg, E.P. (1999) Bacterial biofilms: a common cause of persistent infections. Science 284: 1318-1322.
Dahl, T.A., Valdes-Aguilera, O., Midden, W.R., and Neckers, D.C. (1989) Partition of rose bengal anion from aqueous medium into a lipophilic environment in the cell envelope of Salmonella typhimurium: implications for cell-type targeting in photodynamic therapy. J Photochem Photobiol B 4: 171-184.
Davies, M.J. (2003) Singlet oxygen-mediated damage to proteins and its consequences. Biochem Biophys Res Commun 305: 761-770.
Demidova, T.N., and Hamblin, M.R. (2004) Photodynamic therapy targeted to pathogens. Int J Immunopathol Pharmacol 17: 245-254.
Demidova, T.N., and Hamblin, M.R. (2005) Effect of cell-photosensitizer binding and cell density on microbial photoinactivation. Antimicrob Agents Chemother 49: 2329-2335.
Diekema, D.J., BootsMiller, B.J., Vaughn, T.E., Woolson, R.F., Yankey, J.W., Ernst, E.J., Flach, S.D., Ward, M.M., Franciscus, C.L., Pfaller, M.A., and Doebbeling, B.N. (2004) Antimicrobial resistance trends and outbreak frequency in United States hospitals. Clin Infect Dis 38: 78-85.
Drago, L., De Vecchi, E., Lombardi, A., Nicola, L., Valli, M., and Gismondo, M.R. (2002) Bactericidal activity of levofloxacin, gatifloxacin, penicillin, meropenem and rokitamycin against Bacillus anthracis clinical isolates. J Antimicrob Chemother 50: 1059-1063.
Dunne, W.M., Jr. (2002) Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev 15: 155-166.
Farrell, A.M., Foster, T.J., and Holland, K.T. (1993) Molecular analysis and expression of the lipase of Staphylococcus epidermidis. J Gen Microbiol 139: 267-277.
Ferro, S., Ricchelli, F., Mancini, G., Tognon, G., and Jori, G. (2006) Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by liposome-delivered photosensitising agents. J Photochem Photobiol B 83: 98-104.
Fluit, A.C., Wielders, C.L., Verhoef, J., and Schmitz, F.J. (2001) Epidemiology and susceptibility of 3,051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study. J Clin Microbiol 39: 3727-3732.
Girotti, A.W. (1990) Photodynamic lipid peroxidation in biological systems. Photochem Photobiol 51: 497-509.
Girotti, A.W. (2001) Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms. J Photochem Photobiol B 63: 103-113.
Girotti, A.W., and Kriska, T. (2004) Role of lipid hydroperoxides in photo-oxidative stress signaling. Antioxid Redox Signal 6: 301-310.
Gribbon, E.M., Cunliffe, W.J., and Holland, K.T. (1993) Interaction of Propionibacterium acnes with skin lipids in vitro. J Gen Microbiol 139: 1745-1751.
Hamblin, M.R., O''Donnell, D.A., Murthy, N., Contag, C.H., and Hasan, T. (2002) Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging. Photochem Photobiol 75: 51-57.
Henderson, B.W. (1992) Photodynamic Therapy: Basic Principles and Clinical Applications. Marcel Dekker.
Hiramatsu, K., Cui, L., Kuroda, M., and Ito, T. (2001) The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol 9: 486-493.
Hoebeke, M., and Damoiseau, X. (2002) Determination of the singlet oxygen quantum yield of bacteriochlorin a: a comparative study in phosphate buffer and aqueous dispersion of dimiristoyl-L-alpha-phosphatidylcholine liposomes. Photochem Photobiol Sci 1: 283-287.
Hsueh, P.R., Chen, M.L., Sun, C.C., Chen, W.H., Pan, H.J., Yang, L.S., Chang, S.C., Ho, S.W., Lee, C.Y., Hsieh, W.C., and Luh, K.T. (2002) Antimicrobial drug resistance in pathogens causing nosocomial infections at a university hospital in Taiwan, 1981-1999. Emerg Infect Dis 8: 63-68.
Hsueh, P.R., Teng, L.J., Chen, W.H., Pan, H.J., Chen, M.L., Chang, S.C., Luh, K.T., and Lin, F.Y. (2004) Increasing prevalence of methicillin-resistant Staphylococcus aureus causing nosocomial infections at a university hospital in Taiwan from 1986 to 2001. Antimicrob Agents Chemother 48: 1361-1364.
Huang, Z. (2005) A review of progress in clinical photodynamic therapy. Technol Cancer Res Treat 4: 283-293.
Inhoffen, H.H., Brockmann, H., and Bliesener, K.M. (1969) Protoporphyrine und ihre Umwandlung in Spirographis-sowie Isospirographis-porphyrin. Liebig’s Ann. Chem. 730: 173-185.
Iwamoto, Y., Itoyama, T., Yasuda, K., Morita, T., Shimizu, T., Masuzawa, T., and Yanagihara, Y. (1993) Photodynamic DNA strand breaking activities of acridine compounds. Biol Pharm Bull 16: 1244-1247.
Janzon, L., and Arvidson, S. (1990) The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. Embo J 9: 1391-1399.
Korytowski, W., Bachowski, G.J., and Girotti, A.W. (1992) Photoperoxidation of cholesterol in homogeneous solution, isolated membranes, and cells: comparison of the 5 alpha- and 6 beta-hydroperoxides as indicators of singlet oxygen intermediacy. Photochem Photobiol 56: 1-8.
Kullik, I., Giachino, P., and Fuchs, T. (1998) Deletion of the alternative sigma factor sigmaB in Staphylococcus aureus reveals its function as a global regulator of virulence genes. J Bacteriol 180: 4814-4820.
Lacey, J.A., and Phillips, D. (2002) The photobleaching of disulfonated aluminium phthalocyanine in microbial systems. Photochem Photobiol Sci 1: 120-125.
Lawrence, R.C., Fryer, T.F., and Reiter, B. (1967) Rapid method for the quantitative estimation of microbial lipases. Nature 213: 1264-1265.
Leski, T.A., and Tomasz, A. (2005) Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A. J Bacteriol 187: 1815-1824.
Lin, H.Y., Chen, C.T., and Huang, C.T. (2004) Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells. Appl Environ Microbiol 70: 6453-6458.
Lowy, F.D. (1998) Staphylococcus aureus infections. N Engl J Med 339: 520-532.
Maisch, T. (2006) Anti-microbial photodynamic therapy: useful in the future? Lasers Med Sci 22: 83-91.
Mang, T.S., Dougherty, T.J., Potter, W.R., Boyle, D.G., Somer, S., and Moan, J. (1987) Photobleaching of porphyrins used in photodynamic therapy and implications for therapy. Photochem Photobiol 45: 501-506.
Massey, R.C., Horsburgh, M.J., Lina, G., Hook, M., and Recker, M. (2006) The evolution and maintenance of virulence in Staphylococcus aureus: a role for host-to-host transmission? Nat Rev Microbiol 4: 953-958.
Metcalf, D., Robinson, C., Devine, D., and Wood, S. (2006) Enhancement of erythrosine-mediated photodynamic therapy of Streptococcus mutans biofilms by light fractionation. J Antimicrob Chemother 58: 190-192.
Morgan, A.R., and Tertel, N.C. (1986) Observations on the synthesis and spectroscopic characteristics of purpurins. J. Org. Chem. 51: 1347-1350.
Nickel, J.C., Ruseska, I., Wright, J.B., and Costerton, J.W. (1985) Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother 27: 619-624.
Nieuwint, A.W., Aubry, J.M., Arwert, F., Kortbeek, H., Herzberg, S., and Joenje, H. (1985) Inability of chemically generated singlet oxygen to break the DNA backbone. Free Radic Res Commun 1: 1-9.
Nostro, A., Bisignano, G., Angela Cannatelli, M., Crisafi, G., Paola Germano, M., and Alonzo, V. (2001) Effects of Helichrysum italicum extract on growth and enzymatic activity of Staphylococcus aureus. Int J Antimicrob Agents 17: 517-520.
Patel, R. (2005) Biofilms and antimicrobial resistance. Clin Orthop Relat Res 437: 41-47.
Peng, H.L., Novick, R.P., Kreiswirth, B., Kornblum, J., and Schlievert, P. (1988) Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus. J Bacteriol 170: 4365-4372.
Pitts, B., Willse, A., McFeters, G.A., Hamilton, M.A., Zelver, N., and Stewart, P.S. (2001) A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms. J Appl Microbiol 91: 110-117.
Potempa, J., Watorek, W., and Travis, J. (1986) The inactivation of human plasma alpha 1-proteinase inhibitor by proteinases from Staphylococcus aureus. J Biol Chem 261: 14330-14334.
Potempa, J., Dubin, A., Korzus, G., and Travis, J. (1988) Degradation of elastin by a cysteine proteinase from Staphylococcus aureus. J Biol Chem 263: 2664-2667.
Potempa, J., Fedak, D., Dubin, A., Mast, A., and Travis, J. (1991) Proteolytic inactivation of alpha-1-anti-chymotrypsin. Sites of cleavage and generation of chemotactic activity. J Biol Chem 266: 21482-21487.
Pottier, R., Bonneau, R., and Joussot-Dubien, J. (1975) pH dependence of singlet oxygen production in aqueous solutions using toluidine blue as a photosensitizer. Photochem Photobiol 22: 59-61.
Pourkomaillan, B. (1998) Possible link between a 35 kDa membrane protein and osmolyte transport in Staphylococcus aureus. Lett Appl Microbiol 26: 149-152.
Prokesova, L., Potuznikova, B., Potempa, J., Zikan, J., Radl, J., Hachova, L., Baran, K., Porwit-Bobr, Z., and John, C. (1992) Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus. Immunol Lett 31: 259-265.
Raab, O. (1900) Uber die wirkung fluoriziender stoffe auf infusorien. Zeit Biol 39: 524-546.
Recsei, P., Kreiswirth, B., O''Reilly, M., Schlievert, P., Gruss, A., and Novick, R.P. (1986) Regulation of exoprotein gene expression in Staphylococcus aureus by agar. Mol Gen Genet 202: 58-61.
Reszka, K.J., Denning, G.M., and Britigan, B.E. (2006) Photosensitized oxidation and inactivation of pyocyanin, a virulence factor of Pseudomonas aeruginosa. Photochem Photobiol 82: 466-473.
Rodriguez, H.B., Lagorio, M.G., and San Roman, E. (2004) Rose Bengal adsorbed on microgranular cellulose: evidence on fluorescent dimers. Photochem Photobiol Sci 3: 674-680.
Rollof, J., Braconier, J.H., Soderstrom, C., and Nilsson-Ehle, P. (1988) Interference of Staphylococcus aureus lipase with human granulocyte function. Eur J Clin Microbiol Infect Dis 7: 505-510.
Romanova, N.A., Brovko, L.Y., Moore, L., Pometun, E., Savitsky, A.P., Ugarova, N.N., and Griffiths, M.W. (2003) Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence. Appl Environ Microbiol 69: 6393-6398.
Rosenstein, R., and Gotz, F. (2000) Staphylococcal lipases: biochemical and molecular characterization. Biochimie 82: 1005-1014.
Rotomskis, R., Bagdonas, S., and Streckyte, G. (1996) Spectroscopic studies of photobleaching and photoproduct formation of porphyrins used in tumour therapy. J Photochem Photobiol B 33: 61-67.
Sayed, Z., Harris, F., and Phoenix, D.A. (2005) A study on the bacterial photo-toxicity of phenothiazinium based photosensitisers. FEMS Immunol Med Microbiol 43: 367-372.
Schafer, M., Schmitz, C., Facius, R., Horneck, G., Milow, B., Funken, K.H., and Ortner, J. (2000) Systematic study of parameters influencing the action of Rose Bengal with visible light on bacterial cells: comparison between the biological effect and singlet-oxygen production. Photochem Photobiol 71: 514-523.
Sharma, M., Bansal, H., and Gupta, P.K. (2005) Virulence of Pseudomonas aeruginosa cells surviving photodynamic treatment with toluidine blue. Curr Microbiol 50: 277-280.
Stewart, P.S. (1996) Theoretical aspects of antibiotic diffusion into microbial biofilms. Antimicrob Agents Chemother 40: 2517-2522.
Stewart, P.S., and Costerton, J.W. (2001) Antibiotic resistance of bacteria in biofilms. Lancet 358: 135-138.
Talon, R., Dublet, N., Montel, M.C., and Cantonnet, M. (1995) Purification and characterization of extracellular Staphylococcus warneri lipase. Curr Microbiol 30: 11-16.
Tang, H.M., Hamblin, M.R., and Yow, C.M. (2007) A comparative in vitro photoinactivation study of clinical isolates of multidrug-resistant pathogens. J Infect Chemother 13: 87-91.
Tanielian, C., Schweitzer, C., Mechin, R., and Wolff, C. (2001) Quantum yield of singlet oxygen production by monomeric and aggregated forms of hematoporphyrin derivative. Free Radic Biol Med 30: 208-212.
Tegos, G.P., and Hamblin, M.R. (2006) Phenothiazinium antimicrobial photosensitizers are substrates of bacterial multidrug resistance pumps. Antimicrob Agents Chemother 50: 196-203.
Tremblay, J.F., Dussault, S., Viau, G., Gad, F., Boushira, M., and Bissonnette, R. (2002) Photodynamic therapy with toluidine blue in Jurkat cells: cytotoxicity, subcellular localization and apoptosis induction. Photochem Photobiol Sci 1: 852-856.
Tuomanen, E., Cozens, R., Tosch, W., Zak, O., and Tomasz, A. (1986) The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol 132: 1297-1304.
Verhoeven, J.W. (1996) Glossary of terms used in photochemistry (IUPAC Recommendations 1996). Pure Appl. Chem. 68: 2223-2286.
von Tappeiner, H. (1904) Zur kenntis der lichtwirkenden (fluoreszierenden) stoffe 1: 579–580.
Vuong, C., and Otto, M. (2002) Staphylococcus epidermidis infections. Microbes Infect 4: 481-489.
Wachter, E., Dees, C., Harkins, J., Scott, T., Petersen, M., Rush, R.E., and Cada, A. (2003) Topical rose bengal: pre-clinical evaluation of pharmacokinetics and safety. Lasers Surg Med 32: 101-110.
Wainwright, M. (1998) Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 42: 13-28.
Walsh, C. (2000) Molecular mechanisms that confer antibacterial drug resistance. Nature 406: 775-781.
Wang, J.L., Tseng, S.P., Hsueh, P.R., and Hiramatsu, K. (2004) Vancomycin heteroresistance in methicillin-resistant Staphylococcus aureus, Taiwan. Emerg Infect Dis 10: 1702-1704.
Weigel, L.M., Clewell, D.B., Gill, S.R., Clark, N.C., McDougal, L.K., Flannagan, S.E., Kolonay, J.F., Shetty, J., Killgore, G.E., and Tenover, F.C. (2003) Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 302: 1569-1571.
Wong, T.W., Wang, Y.Y., Sheu, H.M., and Chuang, Y.C. (2005) Bactericidal effects of toluidine blue-mediated photodynamic action on Vibrio vulnificus. Antimicrob Agents Chemother 49: 895-902.
Wyatt, T.D., Ferguson, W.P., Wilson, T.S., and McCormick, E. (1977) Gentamicin resistant Staphylococcus aureus associated with the use of topical gentamicin. J Antimicrob Chemother 3: 213-217.
Yamakoshi, Y., Umezawa, N., Ryu, A., Arakane, K., Miyata, N., Goda, Y., Masumizu, T., and Nagano, T. (2003) Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2-* versus 1O2. J Am Chem Soc 125: 12803-12809.
Zanin, I.C., Goncalves, R.B., Junior, A.B., Hope, C.K., and Pratten, J. (2005) Susceptibility of Streptococcus mutans biofilms to photodynamic therapy: an in vitro study. J Antimicrob Chemother 56: 324-330.