In vitro activity of zinc chelators combined with antifungals against Mucormycetes

Abstract

Background: Mucormycetes are resistant to most antifungals and new treatment approaches are needed. Different combination therapies for mucormycoses were reported, with variable results. Zinc deprivation by hosts during infection represents an important antifungal mechanism. Thus, combination of chelators with antifungals may be a plausible chemotherapeutic alternative. This strategy has been tested against other fungal pathogens, with promising results. In this work we study in vitro interactions between amphotericin B, posaconazole and the zinc chelators Clioquinol (5-chloro-7-iodo-quinolin-8-ol), N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) and 1,10-Phenantroline against six Mucormycetes species. Methods & Materials: Twenty-seven Mucormycetes isolates were studied: 2 Rhizopus oryzae, 18 Rhizopus microsporus, 1 Mucor spp, 1 Lichteimia cornymbifera, 4 Syncephalastrum racemosum and 1 Cunninghamella sp. MIC values (no visible growth) for amphotericin B, posaconazole and the chelators Phenantroline, Clioquinol and TPEN were obtained using the CLSI M38-A2 broth microdilution method. All drugs were purchased at Sigma in the form of standard powders, dissolved in 100% dimethylsulphoxide to make stock solutions and stored at -70 °C until required. The dilution ranges were 0.007 to 4 mg/L for amphotericin, 0.015 to 8 mg/L for posaconazole and 0.03 to 16 mg/L for the chelators. The interactions between the antifungals and the chelators were studied by determining the fractional inhibitory concentration index (FICI) using a checkerboard method. The data was interpreted as synergy (FICI ≤ 0.5), antagonism (FICI > 4) or no interaction (FICI > 0.5–4). Results: Amphotericin and posaconazole MICs ranged between 0.25-4 and 0.5-2 mg/L, respectively. Chelators MICs ranged between 0.25 and > 16 mg/L. The FICI results were strain dependent. TPEN and posaconazole were synergistic against Cunninghamella sp. Clioquinol demonstrated synergy with both antifungals against 41% of R. microsporus isolates. Phenantroline and TPEN with either posaconazole and amphotericin demonstrated no interaction for most isolates (80%). Conclusion: Clioquinol proved to be the most effective zinc chelator against the isolates in our Mucormycetes collection. Combination of posaconazole with this drug showed promising results for further in vivo studies in a model such as Galleria mellonella.