|Year : 2016 | Volume
| Issue : 4 | Page : 506-508
Synergism between fluconazole and methylene blue-photodynamic therapy against fluconazole-resistant Candida strains
JP Lyon1, CR Carvalho1, RR Rezende1, CJ Lima2, FV Santos1, LM Moreira2
1 Department of Natural Sciences, Campus Centro-Oeste D. Lindu, Universidade Federal de São João Del Rei, São João Del Rei, São Paulo, Brazil
2 Department of Animal Science, Instituto de Engenharia Biomédica, Universidade Camilo Castelo Branco, São Paulo, Brazil
|Date of Submission||22-Apr-2015|
|Date of Acceptance||04-Nov-2016|
|Date of Web Publication||8-Dec-2016|
J P Lyon
Department of Natural Sciences, Campus Centro-Oeste D. Lindu, Universidade Federal de São João Del Rei, São João Del Rei, São Paulo
Source of Support: None, Conflict of Interest: None
Photodynamic therapy (PDT) has been proved to be effective against fungi and it may be employed as a coadjutant to conventional antifungal agents, leading to a more effective microbial control minimising side effects. This work evaluates the combined effect of PDT and fluconazole against resistant Candida albicans, Candida glabrata and Candida krusei. The yeasts were submitted to methylene blue-PDT (MB-PDT) in sub-inhibitory concentrations. In the present work, MB-PDT combined with fluconazole was more efficient in the inhibition of the C. albicans and C. glabrata than each treatment alone, being possible to infer that the treatments are synergic.
Keywords: Antifungal photodynamic therapy, antimicrobial resistance, fluconazole, photodynamic therapy, synergism
|How to cite this article:|
Lyon J P, Carvalho C R, Rezende R R, Lima C J, Santos F V, Moreira L M. Synergism between fluconazole and methylene blue-photodynamic therapy against fluconazole-resistant Candida strains. Indian J Med Microbiol 2016;34:506-8
|How to cite this URL:|
Lyon J P, Carvalho C R, Rezende R R, Lima C J, Santos F V, Moreira L M. Synergism between fluconazole and methylene blue-photodynamic therapy against fluconazole-resistant Candida strains. Indian J Med Microbiol [serial online] 2016 [cited 2017 Aug 17];34:506-8. Available from: http://www.ijmm.org/text.asp?2016/34/4/506/195351
| ~ Introduction|| |
Several works have pointed photodynamic therapy (PDT) as an alternative treatment for fungal infections.,, Methylene blue-PDT (MB-PDT) has been associated with increased membrane permeability in Candida yeasts.,, Among the advantages of antifungal PDT, we can mention the relative selectivity to fungi  and the lack of mutagenicity avoiding the selection of resistant strains.,
Considering the increasing number of Candida isolates resistant to fluconazole and the relative toxicity of this drug as well as analysing the promising results associated to PDT, the present work evaluates the synergic effect of sub-inhibitory concentrations of MB-PDT and fluconazole on Candida albicans, Candida glabrata and Candida krusei.
| ~ Materials and Methods|| |
One fluconazole resistant strain of C. albicans, one strain of C. krusei and one strain of C. glabrata were employed in the present study. The strains were previously considered resistant to fluconazole, according to CLSI criteria. The minimum inhibitory concentration (MIC) for each strain was the following: C. albicans: 64 µg/mL; C. glabrata: 32 µg/mL. C. krusei is intrinsically resistant to fluconazole and MIC is > 64 µg/mL.
Methylene blue-photodynamic therapy
Yeast cells' suspensions of 106 cells/mL were incubated in the dark for 1 h at 37°C with MB in the concentrations of 64, 32, 16, 8 and 4 µg/mL totalising a volume of 2.0 mL. The plaques were then irradiated with LED (InGaAlP, 100 mW, 200 mW/cm 2) for 15 min. A control group consisting only of 1.0 mL yeast cells (106 cells/mL) and 1.0 mL sterile saline and a control group consisting of non-irradiated MB were added to this experiment. Each group tested was plated in Sabouraud's dextrose agar (SDA) for colony forming counting. The test showed that all the three strains were inhibited by MB-PDT with MB concentrations of 32 µg/mL or higher. It means that MB-PDT was sub-inhibitory with MB concentrations below 16 µg/mL.
Methylene blue-photodynamic therapy and fluconazole synergism
The MB-PDT was applied to the Candida strains following the protocol described above and employing MB concentrations of 16, 8 and 4 µg/mL. The yeast cells' suspensions were incubated in Sabouraud's dextrose broth with fluconazole in concentrations ranging from 64 to 2 µg/mL for 24 h at 37°C for 24 h. Then, each group was plated in SDA for colony forming counting. Each set of experiments were made in triplicates. For each strain tested, the following controls were included: control 1 - cells did not receive MB-PDT or fluconazole. This control group corresponds to the maximum yeast growth and is considered as 100% of growth; control 2 - cells received only MB-PDT; control 3 - cells received only fluconazole. [Figure 1] summarises the treatment and control groups.
|Figure 1: Organisation of control groups and treatment group for each sample to be tested. Each set of experiments was made in triplicate. C1 - group of cells that did not receive methylene blue or light treatment. C2 - group of cells that were submitted to methylene blue-photodynamic therapy. C3 - group of cells that were submitted only to fluconazole. Treatment cells that received methylene blue-photodynamic therapy plus fluconazole|
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The results were submitted to the analysis of variance and to Tukey test. A P < 0.001 was considered as statistically significant.
| ~ Results|| |
The analysis of variance demonstrated that the best results were achieved when MB was employed at the concentration of 16 µg/mL. Given that, this concentration of MB was employed for the statistical analysis. Considering the C. albicans strain, the growth in control 2 and control 3 groups was 78.25% and 87.11%, respectively, when compared to control 1 group. These results represent the inhibition of MB-PDT and fluconazole alone against the resistant strains tested and the difference between these two groups and the control group was not considered as statistically significant employing the Tukey's test. Regarding the effect of the synergic treatment MB-PDT (16 µg/mL) plus fluconazole, the growth was 12.93% when compared to control 1 and the difference between the treatment and all the control groups was statistically significant (P < 0.0001), evidencing the effectiveness of the synergic treatment for the C. albicans fluconazole resistant strain.
Regarding the resistant C. glabrata strain, the growth in control 2 and control 3 groups was 52. 07% and 91.85%, respectively, being that the difference between control 1 and control 3 groups was not statistically significant. The treatment group presented growth of 19.04% in comparison with control 1 group and the difference between the treatment and all the control groups was statistically significant (P < 0.0001).
For C. krusei, the growth in control 2 and control 3 groups was 55.78% and 98.02%, respectively, being that the difference between control 1 and control 3 groups was not statistically significant. The treatment group presented growth of 50.90% in comparison with control 1 (P < 0.0001). The difference between the treatment and control 2 group was not statistically significant either, evidencing that the synergism between fluconazole and PDT was not effective against C. krusei.
| ~ Discussion|| |
Fluconazole acts by selective inhibition of ergosterol synthesis, which affects membrane permeability. According to Donnelly et al., phenothiazinium photosensitisers can be localised in the membrane of fungal cells. Interestingly, Giroldo et al. demonstrated that MB-PDT increases the cell permeability in C. albicans, which can diminish the resistance of this microorganism to other drugs. In this context, the synergic effect obtained by the combination of MB-PTD and fluconazole could be explained by the action of both agents on the membrane permeability.
Recently, Snell et al. verified that the exposure to miconazole after MB-PDT, enhanced C. albicans killing. These authors tested the same protocol with fluconazole but found that this antifungal drug has neither enhanced C. albicans killing nor induced fungistatic effect. In the present study, however, the combination of MB-PDT and fluconazole showed a synergic interaction. Regarding C. krusei, it is important to notice that the employment of MB-PDT alone achieved approximately the same inhibition effect than the synergic treatment demonstrating that the killing action on C. krusei is due to the PDT action rather than to the combined effect.
| ~ Conclusion|| |
The association of PDT and conventional antifungal therapy with fluconazole against C. albicans and C. glabrata fluconazole-resistant strains demonstrated to be more effective in the inhibition of these microorganisms than both treatments when employed alone.
We would like to thank FAPEMIG and CNPq for financial support.
Financial support and sponsorship
We thank to FAPEMIG and CNPq for financial support.
Conflicts of interest
There are no conflicts of interest.
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