In vitro antifungal activity of Candida culture extracts against Trichophyton rubrum and Trichophyton mentagrophytes

Onychomycoses are nail infections caused primarily by dermatophytes fungi, yeasts, and other filamentous fungi, characterized by persistent infections, prolonged therapy, and high recurrence rates. In clinical practice, some of these occurrences present two or more microorganisms, and the interactions among them can change the chemical environment mediated by small diffusible molecules, producing a competitive niche. The aim of this study was to evaluate the antifungal activity of individual extracts of pure cultures of Candida albicans and C. parapsilosis against dermatophytes. To obtain the fungal extracts, cultures were filtered through a 0.2 μm membrane and submitted to liquid-liquid extraction using ethyl acetate. The Minimal Inhibitory Concentration (MIC) of each extract was evaluated by broth microdilution method and checkerboard assay with fluconazole against clinical isolates of Trichophyton rubrum and T. mentagrophytes. The invertebrate model of Galleria mellonella was used to evaluate the toxicity of the extracts. As results, the extracts of C. albicans and C. parapsilosis showed antifungal activity with MICs between 31,2 – 2000 μg/mL. In association with fluconazole, synergistic effect was detected for all combinations. The extracts presented low toxicity in G. mellonella. In the future, isolation and identification of the extract compounds may allow new therapeutic approaches in the control of fungal infections.


Introduction
Onychomycoses are nail infections caused by dermatophytes, yeasts, and other non-dermatophytes moulds. They are responsible for 50% of all nail diseases and 30% of cutaneous fungal infections [1]. It is a public health problem, which generates high cost to the health services and psychosocial impact, decreasing work productivity and quality of life to the patients [2, 3]. These nail infections are characterized by high-rate recurrence with a prolonged therapy, with irreversible damage to the nail plate. This event contributes to the emergence of microorganisms resistant to conventional antifungals, which may cause refractory fungal diseases [4,5]. In addition, the limited antifungal drugs arsenal and the potential side effects, including hepatotoxic damage, contribute to high morbidity rates [6][7][8].
Dermatophytes are the main etiologic agents of onychomycosis, especially Trichophyton mentagrophytes and T. rubrum species. Moreover, Candida, mostly C. albicans and C. parapsilosis, can also be associated with nail infections, especially fingernails [9]. Interactions between yeasts and dermatophytes in nails diseases may occur in cases of mixed infections or colonization, in which competition or cooperation can alter disease progression. Small diffusible molecules are produced and can mediate these interactions inside nail matrix and affect the survival of these microorganisms in this environment [10]. The mechanisms involved should be explored, once these antifungal molecules can constitute therapeutic alternatives against fungal infections [11][12][13]. The aim of this work was to evaluate the in vitro antifungal activity of Candida extracts, and their synergistic interaction with fluconazole, against Trichophyton mentagrophytes and Trichophyton rubrum, and to assess their toxicity in vivo model, Galleria mellonella.

Preparation of the inoculum and extracts
Each yeast was cultured in Sabouraud Dextrose Agar (SDA, DIFCO®) and incubated at 35 °C for 24 hours. Then, inoculums were prepared in 500 mL Sabouraud Dextrose Broth (DIFCO®, SDB), incubated at 35 °C for 48 hours, and filtered through a 0.2 μm Millipore® membrane and submitted to liquid-liquid extraction using ethyl acetate (Synth®) as a counter-phase. The procedure was repeated three times, allowing the total extraction of the fungal metabolites from the culture media. The ethyl acetate phase was completely dried using a rotary evaporator and subsequently solubilized in sterile distilled water with 10% dimethyl sulfoxide (Synth®, DMSO).

In vitro antifungal determination of Candida extracts against dermatophytes isolates
The antifungal activity of Candida extracts against dermatophytes was evaluated by the broth microdilution technique according to the M38-A2 document protocol, with modifications [14].
The inoculums of dermatophytes were performed after seven days of incubation on potato dextrose agar (Sigma-Aldrich®, PDA) at 28ºC. The fungal colonies were covered with 5 mL of distilled water, and the suspensions were made by scraping the surface with the tip of a sterile loop. The mixture of conidia and hyphal fragments was transferred to sterile tubes and the optical density of the suspensions, at 530 nm, adjusted to 70% of transmittance. Each suspension was diluted (1:50) with RPMI to obtain the final inoculum concentration of 0.4 to 5 x 104 cells/mL. Aliquots of 100 µL were inoculated into 96 well plates containing 100 µL of specific extract concentration. Controls were included for the fungal growth and sterility control of culture medium, Candida extracts, and solvent. The microplates were incubated at 37 ºC for 120h. After incubation, the absorbance of the samples was measured by spectrophotometer (Biospectro, SP22, Curitiba, Brazil). The following formula was applied to calculate the percentage inhibition: I=1-(AbsT-AbsC/AbscGC)x 100 where: I = percentage inhibition; AbsT = absorbance of the inoculum with extract; AbsSC = absorbance of sterility control; AbsGC = absorbance of growth control. The MIC of extracts was defined as the lowest concentration inhibiting at least 80% of the control growth. An aliquot from each well that showed antifungal activity was plated in Petri dishes containing SDA, to determine the minimum fungicidal concentration (MFC). The MFC was defined as the lowest concentration of the extracts that allowed no visible growth on the solid medium [12]. All tests were carried out in triplicate.

Antifungal susceptibility testing for fluconazole
The antifungal susceptibility test for fluconazole (Sigma-Aldrich®) was performed according to the M27-A3 document for yeasts and M38-A2 for dermatophytes, with modifications. Regarding the yeasts, the inoculums were prepared according to the turbidity of a 0.5 McFarland standard (corresponding to 1~5 × 10 6 colony forming units (CFU/mL) in Roswell Park Memorial Institute (RPMI) 1640 medium. The suspension was diluted with RPMI 1640 to prepare a final inoculum suspension (1 × 10 3 CFU/mL). The test was performed in pre-sterilized, flat-bottom 96-well polystyrene plates. The ATCC strains (C. albicans and C. parapsilosis) were used as control. The dermatophyte inoculums suspensions were prepared as described above. Fluconazole MIC was defined as the lowest concentration inhibiting at least 50% for yeast, and 80% for dermatophytes of the growth, in the relation to the control. According to the CLSI criteria, isolates with fluconazole MIC of ≥ 64 μg/mL were considered as resistant [15].

Checkerboard assay
The Checkerboard testing method was used to evaluate the combinatory effects of the Candida spp. extracts and fluconazole against T. mentagrophytes and T. rubrum by a microdilution technique and a spectrophotometric method. Candida

Toxicity test in Galleria mellonella
The in vivo toxicity of Candida extracts was tested in G. mellonella model. A total of 10 larvae (200 ± 25 mg each) in the sixth-instar of development and absence of cuticle pigmentation were artificially injected with Hamilton micro-syringe 7000.5KH of 10 µL volume. Each larva was injected with 5 µL of Candida extracts at the concentration of 8 mg/mL into de hemocoel through the last right proleg. Additionally, each larva was bathed for two seconds into the Candida extracts at the same concentration to evaluate the superficial toxicity.
The positive control of the experiment was the injection and bath with quaternary ammonium solution. The Candida extracts solvent control was 10% DMSO. After the injection and bath, the larvae (treated and control) were incubated at 28°C, deprived of feed and direct illumination. Throughout the experiment and every 24 h, the larvae were removed from the pre-pupae, in order to delay their metamorphosis. Statistical analyses and graphics were performed by the Log-rank (Mantel-Cox) method in Prism 5 software (GraphPad®) [17].

Minimum Inhibitory Concentration of Candida extracts against dermatophytes
The antifungal activity of Candida extracts is shown in Table 1  Considering the species in general, according to the geometric means (GM), the best antifungal activity was observed for the C. parapsilosis extracts (GM of 383 μg/mL). The CP4 extract presented the lowest mean against dermatophytes (GM of 134 μg/mL). On the other hand, C. albicans extracts showed less activity (GM of 732 μg/mL). The best activity of Candida albicans extracts was observed by CA5 extract (GM of 406 μg/mL) ( Table 2). The Candida extracts against dermatophytes in general showed fungicidal properties (MFC) with a value higher than the MIC (range of 500 ->2000 μg/mL, and GM of 1858 μg/mL) ( Table 1).

Antifungal susceptibility testing of clinical isolates for fluconazole
The MIC values for fluconazole are shown in Table 3. Candida albicans and C. parapsilosis isolates showed MIC values between 0.25 -2 μg/mL. Among the dermatophytes, higher values were found for T. mentagrophytes (16 -64 μg/mL), in comparison with T. rubrum (4 -8 μg/mL). According to CLSI interpretive guidelines, isolates of C. albicans, C. parapsilosis and T. rubrum were characterized as susceptible (S) to fluconazole. For T. mentagrophytes, isolates were characterized as susceptible dose-dependent or resistant (Table 3).
The results showed decreased in the MIC ranges for the most combinations tested (Table  4). Consequently, a predominantly synergistic effect (FICI ≤ 0.5) could be observed in the combinations evaluated. Considering the results against T. rubrum and T. mentagrophytes together, general GM of FICI was 0.42. The best antifungal synergistic activities were observed for CA3, CA5, and CA4 with GM of FICI of 0.26, 0.28 and 0.3, respectively (Table 5).

Toxicity test in Galleria mellonella
In the experiments with larvae bathed in the Candida extracts, there were no statistical differences among extracts, except when compared to the ammonium quaternary toxicity control (p> 0.05, with 95% confidence) ( Figure  3). Regarding the experiment with the Candida extracts injections, the CA5 extract was the only one that show some toxicity, being able to kill 40% of the larvae (p= 0.023) (Figure 4). Thus, results show nontoxicity of Candida of the great majority of the extracts at 8 mg/mL in both injection and bath procedure in G. mellonella model.      from Aspergillus nidulans [27,37].
In this study, the extracts produced in these specific conditions showed Candida species. However, the prolonged use has toxicity effects including headache, nausea, rash, abdominal pain, and elevation of transaminases, rarely associated with liver injury or failure [38].
Fluconazole was tested in this study because is an alternative to terbinafine and itraconazole, with advantages in the therapy (lower risk of treatment discontinuation and less adverse events) [39][40][41]. Thus, the combination of new antimicrobial agents and known drugs has become an alternative to increase the antifungal spectrum and efficacy of these drugs, with reduction of toxicity [42][43][44].