Potent Synergistic Effect of Doxycycline with Fluconazole against Candida albicans Is Mediated by Interference with Iron Homeostasis

Abstract

Doxycycline was found to act synergistically with the antifungal fluconazole against Candida albicans. Combination with doxycycline converts fluconazole from fungistatic to fungicidal, prevents the onset of drug resistance, and is also effective against a clinical isolate characterized by elevated resistance to fluconazole. Investigation of the interactions between the two drugs by way of checkerboard assays indicated that doxycycline had an influence on the MIC for fluconazole, as defined by CLSI standards, only at high concentrations (200 g/ml). However, lower concentrations were effective at eliminating residual cell growth at supra-MICs of fluconazole. Using MIC-0, defined as a drug combination resulting in optically clear wells, as an endpoint, doxycycline was found to be synergistic with fluconazole at a concentration as low as 25 g/ml, with a fractional inhibitory concentration index of <0.5. Doxycycline-mediated growth inhibition can be reversed by externally added iron, indicating that iron depletion may account for the synergism. Consistently, we confirmed old literature data about iron-chelating activity of doxycycline. Synergism of fluconazole with doxycycline does not appear to be mediated by calcineurin, since doxycycline further aggravates the susceptibility to fluconazole of mutants lacking the catalytic or the regulatory subunits of calcineurin. Growth in the presence of fluconazole and doxycycline is restored by an elevated dosage of ERG11 in Saccharomyces cerevisiae but not in C. albicans, despite the full competence of the pathogen's protein to act as a suppressor in baker's yeast. Infections by Candida albicans have been on the rise in the past decades, mostly due to changes in the clinical practice, such as the increased use of immunosuppressants and broad-spectrum antibacterial agents (26,29,47). Fungal infections represent a challenge for clinicians, because of the scarcity and sometimes the limited efficacy of antifungal drugs (1, 44). Fluconazole and other azole antifungals, targeting ergosterol biosynthesis (23), have been extensively used in the recent clinical practice thanks to their reduced toxicity and lower cost, compared to amphotericin B in its conventional and lipidic forms, respectively (44). The major disadvantage of azole antifungals, however, is their fungistatic nature, an aspect that favors the onset of drug resistance. The development of isogenic strains of C. albicans characterized by stepwise increased tolerance to fluconazole in patients undergoing continued treatment with this drug has been documented in several cases (34,56,57). For these reasons, the conversion of fluconazole from fungistatic to fungicidal via combinations with other drugs is highly desirable.In a different perspective, tools for the detailed study of the molecular genetics of C. albicans have been greatly improved in recent times (6,7,40). Regulatable promoters are available to the research community, to induce or repress gene expression in C. albicans. Most of them, such as the PCK1 and the MAL2...

diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays

“…Fluconazole, the primary antifungal used to treat candidiasis, is fungistatic: it inhibits fungal growth but does not kill the majority of the cells. Combination therapies, in which a second drug is used together with fluconazole, have been reported to increase the potential therapeutic efficacy of fluconazole (Liu et al, 2014, Butts & Krysan, 2012Butts et al, 2013;Fiori & Van Dijck, 2012;Liu et al, 2014;Zeidler et al, 2013, and references within). However, the degree to which these different agents influence tolerance rather than resistance is usually not determined in a manner that permits quantitative comparison between different drug combination therapy regimes.…”

In Vitro Synergism Observed with Azithromycin, Clarithromycin, Minocycline, or Tigecycline in Association with Antifungal Agents against Pythium insidiosum

“…Since then, several studies demonstrated the in vitro synergisms between amphotericin B or fluconazole with azithromycin, clarithromycin, doxycycline, minocycline, or tetracycline against Aspergillus spp., Candida spp., and Fusarium spp. (18)(19)(20)(21)(22)(23), as well as other combinations of antibacterial and antifungal agents against pathogenic fungi, as reviewed by Afeltra and Verweij (24) and Liu et al (25).…”

Quercetin Sensitizes Fluconazole-Resistant Candida albicans To Induce Apoptotic Cell Death by Modulating Quorum Sensing

“…Fluconazole (FCZ) is most widely used to treat candidiasis infections because of its high bioavailability and low toxicity (11)(12)(13). However, excessive and indiscriminate clinical use of FCZ has led to the emergence of multiple-drug-resistant (MDR) strains of C. albicans (5,14,15).…”