Combination of Miconazole and Domiphen Bromide Is Fungicidal against Biofilms of Resistant Candida spp

Abstract

The occurrence and recurrence of mucosal biofilm-related Candida infections, such as oral and vulvovaginal candidiasis, are serious clinical issues. Vaginal infections caused by Candida spp., for example, affect 70 to 75% of women at least once during their lives. Miconazole (MCZ) is the preferred topical treatment against these fungal infections, yet it has only moderate antibiofilm activity. Through screening of a drug-repurposing library, we identified the quaternary ammonium compound domiphen bromide (DB) as an MCZ potentiator against Candida biofilms. DB displayed synergistic anti-Candida albicans biofilm activity with MCZ, reducing the number of viable biofilm cells 1,000-fold. In addition, the MCZ-DB combination also resulted in significant killing of biofilm cells of azole-resistant C. albicans, C. glabrata, and C. auris isolates. In vivo, the MCZ-DB combination had significantly improved activity in a vulvovaginal candidiasis rat model compared to that of single-compound treatments. Data from an artificial evolution experiment indicated that the development of resistance against the combination did not occur, highlighting the potential of MCZ-DB combination therapy to treat Candida biofilm-related infections.

Keywords: Candida; biofilms; combination treatment; domiphen bromide; drug repurposing; fungicidal; miconazole; resistance development; vulvovaginal candidiasis.

FIG 1

Combining imidazole antifungals and specific quaternary ammonium compounds results in fungicidal activity against C. albicans SC5314 biofilms. Biofilms of C. albicans SC5314 were treated with combinations of imidazoles (either 150 μM miconazole [MCZ], 300 μM ketoconazole [KTC], or 150 μM clotrimazole [CLT]) and a quaternary ammonium compound (QUAT). The maximal concentrations of the quaternary ammonium compound and the imidazole antifungal that did not result in significant killing of biofilm cells upon single-compound treatment were selected and used in the combination. The following quaternary ammonium compounds were used: 50 μM tetraethylammonium bromide (TB), 50 μM benzethonium chloride (BTC), 50 μM benzalkonium chloride (BKC), 12.5 μM dequalinium chloride (DQ), or 37.5 μM domiphen bromide (DB). The DMSO background concentration was 1%. After 24 h of treatment, the number of CFU was determined. Mean log CFU values are shown for at least 5 biological replicates. Statistical analysis was performed to assign significant differences between the combination treatment and the single-compound and DMSO-only treatment (the control treatments). A 2-way ANOVA and either Sidak’s multiple-comparison test (for MCZ) or Tukey’s multiple-comparison test (for ketoconazole and clotrimazole) were applied, and significant differences (P < 0.05) relative to the results of the control treatments are shown in orange.

FIG 2

Combining MCZ and DB results in fungicidal activity against biofilms of fluconazole-resistant clinical isolates C. albicans Tansir_082 and C. albicans Tansir_121B and of intrinsically azole-resistant isolates C. glabrata BG2 and C. auris. For each pathogen, the maximal concentrations of DB and MCZ that did not result in significant killing of biofilm cells upon single-compound treatment were selected and used in the combination. Biofilms of fluconazole-resistant C. albicans isolates or C. auris were treated with 150 μM miconazole (MCZ) combined with either 12.5 μM domiphen bromide (DB) or 37.5 μM DB, respectively. Biofilms of C. glabrata BG2 were treated with 500 μM MCZ combined with 25 μM DB. The DMSO background concentration was 1%. After 24 h (or 48 h for C. glabrata BG2) of treatment, the number of CFU was determined. Mean log CFU values are shown for at least 4 biological replicates. Statistical analysis was performed to assign significant differences between the combination treatment and the single-compound or DMSO-only treatment (the control treatments). A 2-way ANOVA and Tukey’s multiple-comparison test were applied, and significant differences (P < 0.05) relative to the results of the control treatments are shown in orange.

FIG 3

Combining MCZ and DB results in fungicidal action against planktonic stationary-phase cultures of C. albicans SC5314 and C. glabrata BG2. For each pathogen, the maximal concentrations of DB and MCZ that did not result in significant killing of planktonic cells upon single-compound treatment were selected and used in the combination. Planktonic stationary-phase cultures of C. albicans SC5314 or C. glabrata BG2 were treated with 25 μM domiphen bromide (DB) combined with 62.5 μM or 250 μM miconazole (MCZ), respectively. The DMSO background concentration was 1%. After 24 h of treatment, the number of CFU was determined. Mean log CFU values are shown for 4 biological replicates. Statistical analysis was performed to assign significant differences between the combination treatment and the single-compound or DMSO-only treatment (the control treatments). A 2-way ANOVA and Tukey’s multiple-comparison test were applied, and significant differences (P < 0.01) relative to the results of the control treatments are shown in orange.

FIG 4

Resistance against the MCZ-DB combination does not occur in MCZ-DB-evolved C. glabrata BG2 cultures. The sensitivity of three independent C. glabrata BG2 stationary-phase cultures (lineages), either evolved for 61 evolution cycles or not (cycle 0, starter culture), to 25 μM domiphen bromide (DB), 250 μM miconazole (MCZ), or the combination was assessed by determination of the number of CFU after 24 h of incubation. The DMSO background concentration was 1%. Mean log CFU values are shown for at least 4 biological replicates. Within each evolution cycle, a 2-way ANOVA and Tukey’s multiple-comparison test were performed to assign significant differences between the combination treatment and single-compound or DMSO-only treatment. Significant differences (P < 0.001) are shown in orange. Between evolution cycles, significant differences between the corresponding treatments were assessed by a 2-way ANOVA and Sidak’s multiple-comparison test. ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 5

Efficacy of treatment with 1% MCZ nitrate or the 1% MCZ nitrate and 0.144% DB combination in a C. albicans B2630 vaginitis rat model. First, 2 independent repeats were used to evaluate the effect of DB alone (8 rats in total per group). Subsequently, to evaluate the effect of treatment with MCZ or the combination, 3 independent repeats were used with a total of 24 rats per group for the treatment groups and 12 rats for the control group. The AUC, representing the cumulative infection burden over 14 days, is shown. Differences in the AUC between the 1% MCZ and 0.144% DB treatment and all other treatment groups were tested using a linear mixed model followed by a post hoc analysis with the Tukey correction for multiple testing. Significant differences compared to the results for the control are shown in orange. *, P < 0.05.

FIG 6

Histopathology of inflammation and infection determined by staining with H&E (A to C) and PAS-D (D). (A) Image of tissue with a grade I score, showing minimal inflammation with few chronic inflammatory cells; (B) image of tissue with a grade II score, showing moderate inflammation with chronic inflammatory cells; (C) image of tissue with a grade III score, showing a severe and dense organized chronic granulomatous reaction; (D) candidal infiltration was observed (magenta, black arrows) after PAS-D staining. The depth of inflammatory infiltration in the lamina propria is indicated with double-headed black arrows (A to C).