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

Abstract

BackgroundSuperficial 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.MethodsWe 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0261-z) contains supplementary material, which is available to authorized users.

Rose Bengal– and Riboflavin-Mediated Photodynamic Therapy to Inhibit Methicillin-Resistant Staphylococcus aureus Keratitis Isolates

“…12,13 The efficacy of rose bengal, a commonly used ocular stain, as an alternate photosensitizer has been investigated for inhibiting the growth of microbial isolates. [14][15][16] One group found the minimum concentration of rose bengal needed to inhibit fungal isolates to be 280 µM (0.03%), which was used as one of the concentrations of rose bengal in our study. 16 It was hypothesized that PDT is effective in eradicating multidrugresistant MRSA strains in vitro.…”

“…[14][15][16] One group found the minimum concentration of rose bengal needed to inhibit fungal isolates to be 280 µM (0.03%), which was used as one of the concentrations of rose bengal in our study. 16 It was hypothesized that PDT is effective in eradicating multidrugresistant MRSA strains in vitro. To test this, two clinical MRSA strains were exposed to 0.03% and 0.1% rose bengal-mediated PDT and 0.1% riboflavin-mediated PDT and observed for growth inhibition.…”

Biomedical Applications of Photo- and Sono-Activated Rose Bengal: A Review

“…The effectiveness of photo-activated RB against Fusarium keratoplasticum fungi in keratitis both in vitro and in vivo was also demonstrated by dropping RB solution into the patient's eye and irradiating it in the operating room, 41 and against Trichophyton rubrum. 2,42 As discussed by Spagnul et al, 3 RB for antimicrobial PDT can be incorporated into natural polymers, such as in chitosan nanoparticles or on to cellulose acetate surfaces. Manoil et al 43 discussed the use of RB-Acetate against Enterococcus faecalis, resulting in reduced bacterial viability, whereas Decraene et al 44 demonstrated reduced environmental contamination on clinical surfaces by using cellulose RB-Acetate coatings.…”

Searching new antifungals: The use of in vitro and in vivo methods for evaluation of natural compounds

“…In vitro methods using PDT have been widely performed against several species of microorganisms, including bacteria, viruses, protozoa, and fungi (Arboleda et al, 2014;Brooks et al, 1994;Choi et al, 2014;Lim et al, 2012;Lyon et al, 2011;Makarov et al, 2014;Morton et al, 2014;Rossoni et al, 2014;Sabino et al, 2015;Soares et al, 2011;Song et al, 2011;Takahashi et al, 2014). Silva et al (2015) report that after cancer, infections represent the next most frequent application of PDT.…”