Mucormycosis is an emerging disease with high mortality rates. Few antifungal drugs are active against Mucorales.
KEYWORDS: Mucorales, zinc chelators, antifungal susceptibility testing, posaconazole
ABSTRACT
Mucormycosis is an emerging disease with high mortality rates. Few antifungal drugs are active against Mucorales. Considering the low efficacy of monotherapy, combination-therapy strategies have been described. It is known that fungi are susceptible to zinc deprivation, so we tested the in vitro effect of the zinc chelators clioquinol, phenanthroline, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine combined with amphotericin B or posaconazole against 25 strains of Mucorales. Clioquinol-posaconazole was the most active combination, although results were strain dependent.
INTRODUCTION
Mucormycosis is a rapidly progressing fungal disease associated with high mortality (1). Few antifungal drugs are active against Mucorales, and fewer are being used as monotherapy to treat these mycoses. Mucormycosis treatment is currently limited to amphotericin B (AMB) (especially in its lipid formulations) and isavuconazole, whereas posaconazole (POS) is being used as salvage and deescalation therapy (1–3). Because the efficacy of monotherapy is suboptimal, combination therapy strategies have been described, not only between antifungal drugs but also in combination with other nonantifungal agents (2, 4, 5).
It is long known that zinc (Zn) starvation inhibits microbial growth in tissues (6). Zn deficiency induces stress in fungal cells and hampers fungal development by restricting the activity of Zn-binding proteins, which are mainly transcription factors involved in many biological processes (6, 7). Published data about Zn homeostasis in fungi have inferred that compounds that interfere with this metabolic process would inhibit fungal growth. Thus, the antifungal activity of some Zn chelators has been tested against Aspergillus fumigatus strains to evaluate their clinical application, with promising results (8, 9).
The aim of this study was to evaluate the in vitro effect of the Zn chelators 5-chloro-7-iodo-quinolin-8-ol (clioquinol [CLIO]), 1,10-phenanthroline (PHEN), and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) in combination with AMB and POS against clinical Mucorales strains.
Twenty-five isolates were tested, including 18 Rhizopus microsporus, 2 Rhizopus oryzae, 2 Syncephalastrum racemosum, 1 Mucor circinelloides, 1 Lichtheimia corymbifera, and 1 Cunninghamella bertholletiae. Candida parapsilosis ATCC 22019 was used as the antifungal susceptibility testing quality control strain. Mucorales isolates were identified by internal transcribed spacer sequencing (10, 11). To choose the range of drug concentrations to be tested in the combination studies, MICs of the individual Zn chelators were first determined according to CLSI M38 guidlines (12). Interactions between antifungals and Zn chelators were studied by calculating the fractional inhibitory concentration index (FICI) (13) using the same CLSI guidelines modified for a broth microdilution checkerboard procedure. The FICI data were interpreted as synergism (FICI, ≤0.5), antagonism (FICI, >4), or no interaction (FICI, >0.5 to 4) (13). All drugs were purchased from Sigma as standard powders, dissolved in dimethyl sulfoxide, and stored at −70°C.
Each isolate was tested at least three times on different days. Sporangiospore suspensions were counted in a hemocytometer chamber and then diluted into RPMI to reach a concentration of 2 × 104 sporangiospores/ml (equivalent to 0.16 OD530 [optical density at 530 nm]). The concentration ranges tested in checkerboard plates were 0.06 to 4 μg/ml for AMB, 0.12 to 8 μg/ml for POS, and 0.03 to 16 μg/ml for the Zn chelators. Microplates were incubated at 35°C, and MICs were determined visually as the lowest drug concentration (tested alone or in combination) that had no visible growth (100% inhibition). The incubation time was extended to 48 h in the combination experiment to confirm the 100% inhibition. MIC and FICI values are expressed as the geometric means (GMs) and arithmetic means, respectively, of the results obtained for the triplicates. Off-scale MIC values were converted to the next concentration (e.g., 32 for >16 μg/ml) to obtain the GMs.
A wide range of MIC values were obtained when drugs were tested alone (AMB, 0.25 to 4 μg/ml; POS, 0.50 to 4.00 μg/ml; TPEN, 0.25 to >16 μg/ml; PHEN, 2 to 8 μg/ml; and CLIO, 0.5 to >16 μg/ml) (Table 1). For AMB and POS, similar GMs were obtained (1.12 and 1.21 μg/ml, respectively). TPEN was the most active Zn chelator (MIC GM, 0.47 μg/ml), followed by PHEN (MIC GM, 3.68 μg/ml) and CLIO (MIC GM, 8 μg/ml). The studied polyene showed lower MIC values than for POS for all the species except R. microsporus (AMB versus POS GMs, 1.36 versus 1.08 μg/ml) and C. bertholletiae (4.00 μg/ml for both agents). Regarding the activity of Zn chelators, TPEN showed low MIC values for most of the strains. In contrast, MIC values for PHEN were elevated for all tested strains, whereas CLIO acted in a strain-dependent manner, with low MICs only for S. racemosum and L. corymbifera isolates. C. bertholletiae was the only isolate totally resistant to all of the Zn chelators.
TABLE 1.
Antifungal susceptibility testing results for strains used in study
| Strain | Species | MIC (μg/ml)a |
||||
|---|---|---|---|---|---|---|
| AMB | POS | CLIO | PHEN | TPEN | ||
| LMDM-156 | R. microsporus | 2.00 | 1.00 | 4.00 | 2.00 | 0.25 |
| LMDM-157 | R. microsporus | 1.00 | 0.50 | 4.00 | 4.00 | 0.50 |
| LMDM-158 | R. microsporus | 2.00 | 0.50 | 8.00 | 4.00 | 0.50 |
| LMDM-159 | R. microsporus | 2.00 | 1.00 | 4.00 | 2.00 | 0.25 |
| LMDM-164 | R. microsporus | 2.00 | 1.00 | 8.00 | 4.00 | 0.25 |
| LMDM-165 | R. microsporus | 2.00 | 1.00 | 8.00 | 2.00 | 0.50 |
| LMDM-166 | R. microsporus | 1.00 | 2.00 | 4.00 | 4.00 | 0.25 |
| LMDM-167 | R. microsporus | 2.00 | 1.00 | 2.00 | 4.00 | 0.25 |
| LMDM-168 | R. microsporus | 2.00 | 1.00 | 8.00 | 4.00 | 0.25 |
| LMDM-175 | R. microsporus | 1.00 | 2.00 | >16 | 4.00 | 0.25 |
| LMDM-176 | R. microsporus | 2.00 | 1.00 | 16.00 | 4.00 | 0.50 |
| LMDM-184 | R. microsporus | 2.00 | 1.00 | 8.00 | 4.00 | 0.25 |
| LMDM-185 | R. microsporus | 1.00 | 2.00 | >16 | 4.00 | 0.25 |
| LMDM-379 | R. microsporus | 4.00 | 1.00 | >16 | 4.00 | 0.50 |
| LMDM-596 | R. microsporus | 1.00 | 1.00 | 8.00 | 4.00 | 0.50 |
| LMDM-1073 | R. microsporus | 0.50 | 1.00 | 8.00 | 4.00 | 0.50 |
| LMDM-1074 | R. microsporus | 0.25 | 1.00 | >16 | 4.00 | 1.00 |
| LMDM-1127 | R. microsporus | 1.00 | 2.00 | >16 | 4.00 | 0.50 |
| LMDM-597 | R. oryzae | 2.00 | 2.00 | >16 | 4.00 | 0.50 |
| LMDM-1075 | R. oryzae | 0.25 | 1.00 | >16 | 4.00 | 0.25 |
| LMDM-1123 | S. racemosum | 0.25 | 1.00 | 1.00 | 2.00 | 0.25 |
| LMDM-1124 | S. racemosum | 0.50 | 2.00 | 1.00 | 4.00 | 1.00 |
| LMDM-1019 | M. circinelloides | 1.00 | 2.00 | 4.00 | 4.00 | 0.50 |
| LMDM-1121 | L. corymbifera | 0.25 | 1.00 | 0.50 | 4.00 | 1.00 |
| LMDM-1291 | C. bertholletiae | 4.00 | 4.00 | >16.00 | 8.00 | >16 |
MIC values were obtained on 3 different days by using the protocol published by CLSI (document M38 [12]) and are presented as geometric means. AMB, amphotericin B; POS, posaconazole; CLIO, clioquinol (5-chloro-7-iodo-quinolin-8-ol); PHEN, 1,10-phenanthroline; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine.
Arithmetic means of FICI results and their interpretation are described in Table 2. AMB exhibited no interaction with the Zn chelators against most of the isolates (22 out of 25 strains). Synergism was only observed when the polyene was combined with CLIO against M. circinelloides and 2 R. microsporus isolates. However, it was remarkable that borderline FICI values were obtained against the R. oryzae strains with AMB+CLIO (FICI, 0.51 and 0.56), even when their AMB MICs were not similar (2 and 0.25 μg/ml) and both strains had CLIO MIC values of >16 μg/ml. The same was observed for 2 R. microsporus strains (LMDM-164 and LMDM-1127), with borderline FICI values and high CLIO MICs.
TABLE 2.
Fractional inhibitory concentration index results for all isolates and drugs in study
| Isolate | FICI for a: |
|||||
|---|---|---|---|---|---|---|
| AMB+ |
POS+ |
|||||
| CLIO | PHEN | TPEN | CLIO | PHEN | TPEN | |
| LMDM-156 | 0.88 (Ni) | 0.75 (Ni) | 1.06 (Ni) | 0.37 (S) | 4.01 (A) | 1.06 (Ni) |
| LMDM-157 | 0.75 (Ni) | 0.76 (Ni) | 1.06 (Ni) | 0.25 (S) | 0.32 (S) | 0.52 (Ni) |
| LMDM-158 | 0.64 (Ni) | 0.76 (Ni) | 1.02 (Ni) | 0.29 (S) | 1.38 (Ni) | 1.27 (Ni) |
| LMDM-159 | 1.00 (Ni) | 0.51 (Ni) | 1.06 (Ni) | 1.00 (Ni) | 1.00 (Ni) | 1.03 (Ni) |
| LMDM-164 | 0.54 (Ni) | 0.75 (Ni) | 1.09 (Ni) | 0.42 (S) | 0.26 (S) | 1.06 (Ni) |
| LMDM-165 | 0.66 (Ni) | 0.75 (Ni) | 1.00 (Ni) | 0.54 (Ni) | 2.00 (Ni) | 1.28 (Ni) |
| LMDM-166 | 1.13 (Ni) | 1.00 (Ni) | 1.06 (Ni) | 1.00 (Ni) | 0.50 (S) | 1.03 (Ni) |
| LMDM-167 | 1.00 (Ni) | 1.01 (Ni) | 1.06 (Ni) | 4.13 (A) | 0.56 (Ni) | 1.03 (Ni) |
| LMDM-168 | 1.28 (Ni) | 0.63 (Ni) | 0.94 (Ni) | 1.01 (Ni) | 2.50 (Ni) | 1.00 (Ni) |
| LMDM-175 | 0.63 (Ni) | 1.00 (Ni) | 1.06 (Ni) | 4.13 (A) | 1.00 (Ni) | 1.06 (Ni) |
| LMDM-176 | 1.38 (Ni) | 0.76 (Ni) | 0.70 (Ni) | 1.36 (Ni) | 0.67 (Ni) | 1.05 (Ni) |
| LMDM-184 | 1.53 (Ni) | 0.63 (Ni) | 0.84 (Ni) | 0.45 (S) | 1.00 (Ni) | 1.06 (Ni) |
| LMDM-185 | 0.25 (S) | 2.13 (Ni) | 0.59 (Ni) | 4.13 (A) | 1.00 (Ni) | 0.63 (Ni) |
| LMDM-379 | 2.05 (Ni) | 0.75 (Ni) | 1.06 (Ni) | 0.39 (S) | 2.00 (Ni) | 1.06 (Ni) |
| LMDM-596 | 0.75 (Ni) | 2.06 (Ni) | 0.56 (Ni) | 0.63 (Ni) | 1.50 (Ni) | 0.63 (Ni) |
| LMDM-1073 | 0.63 (Ni) | 1.00 (Ni) | 0.63 (Ni) | 0.69 (Ni) | 2.00 (Ni) | 0.53 (Ni) |
| LMDM-1074 | 0.34 (S) | 0.75 (Ni) | 0.83 (Ni) | 1.18 (Ni) | 1.54 (Ni) | 0.52 (Ni) |
| LMDM-1127 | 0.55 (Ni) | 0.81 (Ni) | 0.94 (Ni) | 0.75 (Ni) | 1.31 (Ni) | 0.63 (Ni) |
| LMDM-597 | 0.51 (Ni) | 0.77 (Ni) | 1.03 (Ni) | 1.58 (Ni) | 2.00 (Ni) | 1.38 (Ni) |
| LMDM-1075 | 0.56 (Ni) | 1.01 (Ni) | 1.03 (Ni) | 0.69 (Ni) | 1.01 (Ni) | 0.43 (S) |
| LMDM-1123 | 1.03 (Ni) | 1.01 (Ni) | 0.75 (Ni) | 0.77 (Ni) | 0.52 (Ni) | 1.51 (Ni) |
| LMDM-1124 | 1.03 (Ni) | 1.00 (Ni) | 0.63 (Ni) | 2.00 (Ni) | 2.00 (Ni) | 0.60 (Ni) |
| LMDM-1019 | 0.50 (S) | 1.01 (Ni) | 0.56 (Ni) | 0.63 (Ni) | 2.00 (Ni) | 1.01 (Ni) |
| LMDM-1121 | 1.03 (Ni) | 1.00 (Ni) | 1.03 (Ni) | 0.76 (Ni) | 1.00 (Ni) | 1.57 (Ni) |
| LMDM-1291 | 1.00 (Ni) | 1.00 (Ni) | 0.88 (Ni) | 1.38 (Ni) | 3.06 (Ni) | 0.16 (S) |
| Average FICI | 0.87 | 0.94 | 0.90 | 1.22 | 1.45 | 0.92 |
| Synergy (n [%])b | 3 (12%) | 0 (0%) | 0 (0%) | 6 (24%) | 3 (12%) | 2 (8%) |
AMB, amphotericin B; POS, posaconazole; CLIO, clioquinol (5-chloro-7-iodo-quinolin-8-ol); PHEN, 1,10-phenanthroline; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine. FICI values are presented as arithmetic means of at least 3 repetitions performed on different days. FICI value interpretations are as follows (shown in parenthesis): S, synergy; A, antagonism; and Ni, no interaction.
Number of isolates for which combination showed synergism.
When POS was combined with CLIO, PHEN, and TPEN, both synergism and antagonism were seen: FICI values of ≤0.5 were observed for 6 (all R. microsporus), 3 (all R. microsporus), and 2 (1 R. oryzae and 1 C. bertholletiae) strains, respectively. On the other hand, antagonism was observed for R. microsporus isolates when tested against POS+CLIO (3 strains) and POS+PHEN (1 strain). Again, borderline FICI values resulted for POS+Zn chelator combinations against 5 R. microsporus strains and 1 S. racemosum strain.
Overall, the results obtained with AMB and Zn chelator combinations were discouraging, because no combination showed clear synergistic behavior. On the other hand, POS+CLIO showed promising results, especially against R. microsporus. However, the POS+Zn chelator combinations acted in a strain-dependent manner, as described earlier for AMB and POS (14). However, a Zn-chelator susceptibility pattern could potentially be established if a larger collection of strains was studied.
It is known that metal-chelating agents are able to inhibit biological processes that are essential in every cellular system. Zn-chelator concentration-related toxicity has been described and should be taken into consideration (15). It is thus clear that a Zn-depletion-based strategy for mucormycosis therapy would be plausible only if undesired effects of ion sequestration could be avoided with the development of fungal-specific ion chelators. This concept should be added to the drug development pipeline.
ACKNOWLEDGMENTS
This study was supported by Science, Technology and Productive Innovation Ministry (MinCyT-Argentina) grant PICT2013/1571 to S.G. and G.G.-E.
F.L. and D.M. have fellowships from CONICET (Argentina). C.D. and M.S.C. have postdoctoral fellowships from CONICET.
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