In Vitro Activity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions and In Vivo Efficacy in a Murine Model of Oral Candidiasis

Abstract

The generation of a new antifungal against Candida albicans biofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potent in vitro activity against multiple fluconazole-susceptible and -resistant C. albicans clinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibits C. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library of C. albicans transcription factor mutants provided additional insight into the activity of miltefosine against C. albicans growing under planktonic and biofilm conditions. Finally, we demonstrate the in vivo efficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis.

FIG 1

Induction of in vitro development of resistance to drugs by continuous exposure. Both strains of Candida albicans, SC5314 and 6482, were challenged daily with fresh culture medium (control; blue line), increasing subinhibitory concentrations of miltefosine (MLT; red line), and increasing subinhibitory concentrations of fluconazole (FLC; green line), for 30 days. (a and b) Susceptibility of the three groups to miltefosine over time; (c and d) susceptibility of the three groups to fluconazole over time.

FIG 2

In vivo evaluation of the protective effect of miltefosine against oral candidiasis. Treated groups received 50 mg/kg of miltefosine, twice a day, by topical administration. Miltefosine treatment was initiated the day before infection (day −1). The control group received saline twice a day, also by topical administration. (a) Clinical score of candidiasis progression, showing that miltefosine has a protective role, preventing the development of disease. **, P < 0.01; statistical analysis by nonparametric Mann-Whitney test. (b) Extent of C. albicans colonization on tongues from control mice and mice treated with miltefosine. Only one animal from each group was sacrificed at the end of day 3. Bars indicate the standard deviations. The graph shows the reduction in C. albicans colonizing the tongues of animals treated with miltefosine in comparison to the untreated control group. Statistical analysis by nonparametric Mann-Whitney test (4th day), P = 0.0952, and Mann-Whitney U test, 4.000. (c) Visual analysis of the extension of tongue colonization by C. albicans in mice from the control group (left) and the group treated with miltefosine (right) at the end of day 4. Control tongues showed a dense biofilm covering the entire tongue surface on day 4 (left), and miltefosine treatment showed a protective role by reducing the tongue colonization and reducing the biofilm formation (right). (d to g) Histological sections of tongues from control mice (d and e) and mice treated with miltefosine (f and g) sacrificed on the 4th day and stained with Grocott-Gomori stain and silver methanamine. The administration of miltefosine reduced tissue colonization, inhibited hypha formation, and reduced invasive behavior at the infection site (f and g).