In vitro combination therapy using low dose clotrimazole and photodynamic therapy leads to enhanced killing of the dermatophyte Trichophyton rubrum

BMC Microbiology

Colin Stack¹  Mousawi Chau¹  C Oliver Morton¹ 

[1] School of Science and Health, University of Western Sydney, Campbelltown Campus, Narellan Road, Campbelltown NSW 2560, Australia

关键词: Antifungal therapy;    Trichophyton rubrum;    Photodynamic therapy;   

Others  :  1137749
DOI  :  10.1186/s12866-014-0261-z

 received in 2014-06-12, accepted in 2014-10-01,  发布年份 2014

【 摘 要 】

Background

Superficial infections of the skin and mucous membranes caused by dermatophyte fungi are amongst the most common and challenging infections to treat. Previously we demonstrated the phototoxic effects of photodynamic therapy (PDT) towards Trichophyton rubrum, using a green laser to photoactivate Rose Bengal (RB). The aim of this study was to evaluate whether we could; (1) achieve a similar effect using an inexpensive light-emitting diode (LED) to photoactivate RB and (2) to evaluate whether our PDT regime could be combined with standard antifungal drug therapy and increase its effectiveness.

Methods

We designed and built our own inexpensive green (530 nm) LED source and tested its efficacy as part our RB-PDT regime in vitro against T. rubrum. We also examined the potential benefits of incorporating PDT as part of combination therapy and whether the order in which this was done had an impact. First we subjected spore suspensions to sub-inhibitory concentrations of a number of antifungal agents (CLT, MCZ and TRB) for 72 hours followed by RB-PDT. Secondly we subjected spore suspensions to sub-inhibitory PDT followed by drug treatment and evaluated if there were any changes to the minimum inhibitory concentrations (MICs) of the drugs tested.

Results

The optimal conditions for photoinactivation of T. rubrum using RB-PDT alone were 140 μM of RB and 24 J/cm² of LED (equating to a 30-minute exposure). These parameters also caused a 100% reduction in the viability of the pathogenic yeast Candida albicans and the model fungus Saccharomyces cerevisiae. By combining our RB-PDT regime as an adjunct to antifungal drugs we were able to dramatically reduce the exposure times. Treatment of spore suspensions using a sub-inhibitory dose of clotrimazole (CLT) followed by RB-PDT, this order was critical, significantly reduced the exposure times required to achieve 100% inhibition of T. rubrum to 15 minutes as compared to RB-PDT alone.

Conclusions

The combination of antifungal drug and RB-PDT represents an attractive alternative to the current antifungal therapies used to treat superficial fungal diseases. Our approach has the potential to reduce treatment times and drug dosages which can also reduce drug toxicity and improve patient compliance.

【 授权许可】

   
2014 Morton et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Burmester A, Shelest E, Glockner G, Heddergott C, Schindler S, Staib P, Heidel A, Felder M, Petzold A, Szafranski K, Feuermann M, Pedruzzi I, Priebe S, Groth M, Winkler R, Li W, Kniemeyer O, Schroeckh V, Hertweck C, Hube B, White TC, Platzer M, Guthke R, Heitman J, Wöstemeyer J, Zipfel PF, Monod M, Brakhage AA: Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi. Genome Biol 2011, 12(1):R7. BioMed Central Full Text
• [2]Peres NT, Maranhao FC, Rossi A, Martinez-Rossi NM: Dermatophytes: host-pathogen interaction and antifungal resistance. An Bras Dermatol 2010, 85(5):657-667.
• [3]Nenoff P, Kruger C, Ginter-Hanselmayer G, Tietz HJ: Mycology - an update. Part 1: Dermatomycoses: causative agents, epidemiology and pathogenesis. J Deutschen Dermatol Gesellschaft = J German Soc Dermatol: JDDG 2014, 12(3):188-209. quiz 210, 188–211; quiz 212
• [4]Crawford F, Hollis S: Topical treatments for fungal infections of the skin and nails of the foot. Cochrane Database Syst Rev 2007., 3
• [5]Meis JF, Verweij PE: Current management of fungal infections. Drugs 2001, 61(Suppl 1):13-25.
• [6]El-Gohary M, van Zuuren EJ, Fedorowicz Z, Burgess H, Doney L, Stuart B, Moore M, Little P: Topical antifungal treatments for tinea cruris and tinea corporis. Cochrane Database Syst Rev 2014., 8
• [7]Crowley PD, Gallagher HC: Clotrimazole as a pharmaceutical: past, present and future. J Appl Microbiol 2014, 117(3):611-617.
• [8]Prabagar B, Yoo BK, Woo JS, Kim JA, Rhee JD, Piao MG, Choi HG, Yong CS: Enhanced bioavailability of poorly water-soluble clotrimazole by inclusion with beta-cyclodextrin. Arch Pharm Res 2007, 30(2):249-254.
• [9]Bell-Syer SE, Khan SM, Torgerson DJ: Oral treatments for fungal infections of the skin of the foot. Cochrane Database Syst Rev 2012., 10
• [10]Odds FC, Brown AJ, Gow NA: Antifungal agents: mechanisms of action. Trends Microbiol 2003, 11(6):272-279.
• [11]Piscitelli SC, Rodvold KA, Pai MP: Drug Interactions in Infectious Diseases. Humana Press, NJ, USA; 2011.
• [12]Ting PC, Walker SS: New agents to treat life-threatening fungal infections. Curr Top Med Chem 2008, 8(7):592-602.
• [13]Niewerth M, Korting HC: Management of onychomycoses. Drugs 1999, 58(2):283-296.
• [14]Panzarini E, Inguscio V, Dini L: Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy. Cell Death Dis 2011, 2:e169.
• [15]Dougherty TJ: An update on photodynamic therapy applications. J Clin Laser Med Surg 2002, 20(1):3-7.
• [16]Calzavara-Pinton PG, Venturini M, Sala R: A comprehensive overview of photodynamic therapy in the treatment of superficial fungal infections of the skin. J Photochem Photobiol B 2005, 78(1):1-6.
• [17]Lyon JP, Moreira LM, de Moraes PC, dos Santos FV, de Resende MA: Photodynamic therapy for pathogenic fungi. Mycoses 2011, 54(5):e265-e271.
• [18]Calzavara-Pinton P, Rossi MT, Sala R, Venturini M: Photodynamic antifungal chemotherapy. Photochem Photobiol 2012, 88(3):512-522.
• [19]Dai T, Fuchs BB, Coleman JJ, Prates RA, Astrakas C, St Denis TG, Ribeiro MS, Mylonakis E, Hamblin MR, Tegos GP: Concepts and principles of photodynamic therapy as an alternative antifungal discovery platform. Front Microbiol 2012, 3:120.
• [20]Cronin L, Moffitt M, Mawad D, Morton OC, Lauto A, Stack C: An in vitro study of the photodynamic effect of rose bengal on trichophyton rubrum. J Biophotonics 2014, 7(6):410-417.
• [21]Cronin LJ, Mildren RP, Moffitt M, Lauto A, Morton CO, Stack CM: An investigation into the inhibitory effect of ultraviolet radiation on Trichophyton rubrum. Lasers Med Sci 2014, 29(1):157-163.
• [22]De Flora S: Genotoxicity and carcinogenicity of the light emitted by artificial illumination systems. Arch Toxicol 2013, 87(3):403-405.
• [23]Chen BK, Friedlander SF: Tinea capitis update: a continuing conflict with an old adversary. Curr Opin Pediatr 2001, 13(4):331-335.
• [24]Bottiroli G, Croce AC, Balzarini P, Locatelli D, Baglioni P, Lo Nostro P, Monici M, Pratesi R: Enzyme-assisted cell photosensitization: a proposal for an efficient approach to tumor therapy and diagnosis. The rose bengal fluorogenic substrate. Photochem Photobiol 1997, 66(3):374-383.
• [25]Croce AC, Supino R, Lanza KS, Locatelli D, Baglioni P, Bottiroli G: Photosensitizer accumulation in spontaneous multidrug resistant cells: a comparative study with Rhodamine 123, Rose Bengal acetate and Photofrine. Photochem Photobiol Sci: Offic J Eur Photochem Assoc Eur Soc Photobiol 2002, 1(1):71-78.
• [26]Bottone MG, Soldani C, Fraschini A, Alpini C, Croce AC, Bottiroli G, Pellicciari C: Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells. Histochem Cell Biol 2007, 127(3):263-271.
• [27]Coelho LM, Aquino-Ferreira R, Maffei CM, Martinez-Rossi NM: In vitro antifungal drug susceptibilities of dermatophytes microconidia and arthroconidia. J Antimicrob Chemother 2008, 62(4):758-761.
• [28]Evans EG: The rationale for combination therapy. Br J Dermatol 2001, 145(Suppl 60):9-13.
• [29]Cruz FS, Lopes LA, De Souza W, Moreno SN, Mason RP, Docampo R: The photodynamic action of rose bengal on Trypanosoma cruzi. Acta Trop 1984, 41(2):99-108.
• [30]Dahl TA, Midden WR, Neckers DC: Comparison of photodynamic action by Rose Bengal in gram-positive and gram-negative bacteria. Photochem Photobiol 1988, 48(5):607-612.
• [31]Lyon JP, de Resende MA: Correlation between adhesion, enzyme production, and susceptibility to fluconazole in Candida albicans obtained from denture wearers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006, 102(5):632-638.
• [32]Cuenca-Estrella M: Antifungal drug resistance mechanisms in pathogenic fungi: from bench to bedside. Clin Microbiol Infect 2014, 20(Suppl 6):54-59.
• [33]Arboleda A, Miller D, Cabot F, Taneja M, Aguilar MC, Alawa K, Amescua G, Yoo SH, Parel JM: Assessment of rose bengal versus riboflavin photodynamic therapy for inhibition of fungal keratitis isolates. Am J Ophthalmol 2014, 158(1):64-70. e62
• [34]Dovigo LN, Pavarina AC, Mima EG, Giampaolo ET, Vergani CE, Bagnato VS: Fungicidal effect of photodynamic therapy against fluconazole-resistant Candida albicans and Candida glabrata. Mycoses 2011, 54(2):123-130.
• [35]Mima EG, Pavarina AC, Dovigo LN, Vergani CE, Costa CA, Kurachi C, Bagnato VS: Susceptibility of Candida albicans to photodynamic therapy in a murine model of oral candidosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010, 109(3):392-401.
• [36]Teichert MC, Jones JW, Usacheva MN, Biel MA: Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002, 93(2):155-160.
• [37]Snell SB, Foster TH, Haidaris CG: Miconazole induces fungistasis and increases killing of Candida albicans subjected to photodynamic therapy. Photochem Photobiol 2012, 88(3):596-603.
• [38]Coleman JJ, Okoli I, Tegos GP, Holson EB, Wagner FF, Hamblin MR, Mylonakis E: Characterization of plant-derived saponin natural products against Candida albicans. ACS Chem Biol 2010, 5(3):321-332.
• [39]Chabrier-Rosello Y, Giesselman BR, De Jesus-Andino FJ, Foster TH, Mitra S, Haidaris CG: Inhibition of electron transport chain assembly and function promotes photodynamic killing of Candida. J Photochem Photobiol B 2010, 99(3):117-125.
• [40]Giroldo LM, Felipe MP, de Oliveira MA, Munin E, Alves LP, Costa MS: Photodynamic antimicrobial chemotherapy (PACT) with methylene blue increases membrane permeability in Candida albicans. Lasers Med Sci 2009, 24(1):109-112.
• [41]Lu Z, Dai T, Huang L, Kurup DB, Tegos GP, Jahnke A, Wharton T, Hamblin MR: Photodynamic therapy with a cationic functionalized fullerene rescues mice from fatal wound infections. Nanomedicine (Lond) 2010, 5(10):1525-1533.
• [42]Toomey P, Kodumudi K, Weber A, Kuhn L, Moore E, Sarnaik AA, Pilon-Thomas S: Intralesional injection of rose bengal induces a systemic tumor-specific immune response in murine models of melanoma and breast cancer. PLoS One 2013, 8(7):e68561.
• [43]Tan CY, Neuhaus SJ: Novel use of Rose Bengal (PV-10) in two cases of refractory scalp sarcoma. ANZ J Surg 2013, 83(1–2):93.
• [44]Wachter E, Dees C, Harkins J, Scott T, Petersen M, Rush RE, Cada A: Topical rose bengal: pre-clinical evaluation of pharmacokinetics and safety. Lasers Surg Med 2003, 32(2):101-110.