Abstract
The in vitro activities of eight antifungal drugs against 106 clinical and environmental isolates of waterborne and cutaneous Exophiala species were tested. The MICs and minimum effective concentrations for 90% of the strains tested (n = 106) were, in increasing order, as follows: posaconazole, 0.063 μg/ml; itraconazole, 0.25 μg/ml; micafungin, 1 μg/ml; voriconazole, 2 μg/ml; isavuconazole, 4 μg/ml; caspofungin, 8 μg/ml; amphotericin B, 16 μg/ml; fluconazole, 64 μg/ml.
TEXT
Exophiala is a genus of black yeasts containing numerous agents of human infection, most of which occur in otherwise healthy individuals. Infections range from asymptomatic cutaneous colonization to fatal disseminated and cerebral disease. Among the more serious opportunistic fungi are the neurotrope Exophiala dermatitidis (1), the osteotrope E. spinifera (2), and E. asiatica (3), all of which have the potential to cause fatal infections in individuals without apparent immune or metabolic disorders. The presence of melanin and the ability to assimilate alkylbenzenes have been suggested to play roles in pathogenicity and evasion of the host defense (4).
Species of Exophiala traditionally were difficult to identify, but in recent years the diagnosis has been facilitated by the use of molecular tools, particularly sequence data of rDNA internal transcribed spacer regions (5, 6). Numerous species can be recognized. Judging from phylogenetic data, waterborne Exophiala species, growing at relatively low temperatures, form a compact lineage referred to as the “salmonis clade” (4). In their natural habitat, these mesophilic species are frequently involved in disseminated infections of cold-blooded animals such as fish, frogs, crabs, and turtles (4, 7, 8), occasionally reaching epidemic proportions (9). Human infections by the same fungi involve skin and nail disorders, particularly in diabetic patients (G. Haase and P. Mayser, personal communication) or subcutaneous lesions in the elderly (10), possibly explained by poor blood circulation and concomitantly low body temperatures. The cross-kingdom infections of cold- versus warm-blooded hosts suggest a certain degree of intrinsic virulence shared between meso- and thermophilic black yeasts. Veronaea botryosa, an agent of potentially fatal disseminated infections in immunocompetent humans (11, 12), was found to be the nearest neighbor of the waterborne Exophiala species (4). As a route of transmission of numerous black fungi, carriage by indoor water systems such as bathing facilities (13) or dental unit water lines (14) has been suggested. Case reports of Exophiala species in the salmonis clade are scant in the medical literature, and no information on antifungal susceptibility is available. This study aimed to determine the in vitro susceptibilities of a large collection of clinical and environmental isolates of waterborne Exophiala species to eight antifungal drugs, including the new triazole isavuconazole, which recently received an orphan drug designation by the U.S. FDA for the treatment of invasive aspergillosis.
A total of 106 strains were obtained from the reference collection of the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center, which included Exophiala equina (n = 35), E. mesophila (n = 12), E. angulospora (n = 11), E. castellanii (n = 10), E. aquamarina (n = 9), E. opportunistica (n = 6), E. pisciphila (n = 5), E. alcalophila (n = 5), E. cancerae (n = 5), E. halophila (n = 3), E. salmonis (n = 3), and E. lacus (n = 2). The strains originated from diseased cold-blooded animals, from human clinical samples, and from the environment, particularly from drinking water (see Table S1 in the supplemental material). The set included all of the available type strains of described species and was supplemented with fresh isolates. Species identities had been confirmed by multilocus studies using Genealogical Concordance Phylogenetic Species Recognition (15).
Antifungal susceptibility testing was performed as described in CLSI document M38-A2, with some modifications (16, 17–20). Briefly, isolates were cultured on potato dextrose agar in the dark (25°C) for up to 7 days to induce sporulation. Inocula were prepared by scraping the surface of the fungal colonies with a cotton swab moistened with sterile physiological saline containing 0.05% Tween 40. Large particles were allowed to settle for 5 min, and the spore suspension was adjusted to 68 to 71% transmission at 530 nm with a spectrophotometer (Spectronic 20D; Milton Roy, Rochester, NY) and diluted 10-fold to yield a final inoculum of 1.5 × 104 to 5 ×104 CFU/ml, as verified by colony counting. Stock solutions of antifungals were prepared to a final concentration of 3,200 mg/liter with a dimethyl sulfoxide solution, while fluconazole was dissolved in sterile distilled water. The antifungal agents were diluted in filter-sterilized (0.22-μm filter) RPMI 1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma) with l-glutamine without bicarbonate to yield two times their concentrations and dispensed into 96-well flat-bottom microdilution trays at final concentrations of 0.016 to 16 μg/ml for amphotericin B (Bristol-Myers Squibb, Woerden, The Netherlands), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer Central Research, Sandwich, United Kingdom), isavuconazole (Basilea, Basel, Switzerland), and posaconazole (MSD, Haarlem, The Netherlands); 0.063 to 64 μg/ml for fluconazole (Pfizer); and 0.08 to 8 μg/ml for micafungin (Astellas Pharma, Ibaraki, Japan) and caspofungin (MSD). Paecilomyces variotii (ATCC 22319), Candida parapsilosis ATCC 22019, and C. krusei ATCC 6258 served as quality control strains. About half of the isolates could be read after incubation for 72 h, and the other half needed incubation for 144 h in the dark at 25°C. Agitation of plates was not used. MICs of amphotericin B, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole were determined visually with an inverted mirror by comparison of growth in the wells containing the drug with that of the drug-free control. Minimum effective concentrations (MECs) of caspofungin and micafungin were read with a plate microscope (Olympus SZX9; Olympus Nederland, Zoeterwoude, The Netherlands) at ×25 to ×50 magnification and defined as the lowest concentrations of drug that lead to the growth of small, rounded, compact hyphal forms compared with the long, unbranched hyphal clusters that were seen in the growth controls.
The species-specific MICs and MECs determined for all waterborne Exophiala species are listed in Table 1. Four species with sufficient numbers of isolates (n = ≥10) were included to calculate the MICs for 50 and 90% of the isolates tested (MIC50s and MIC90s, respectively), viz., E. equina, E. mesophila, E. angulospora, and E. castellanii (Table 1). All waterborne Exophiala isolates had low MIC/MEC90s (≤1 μg/ml) of itraconazole, posaconazole, and micafungin. Less active drugs (MIC/MEC90s, ≥2 μg/ml) were amphotericin B, fluconazole, voriconazole, isavuconazole, and caspofungin. The highest geometric mean values were 35.45 μg/ml for fluconazole and 2.2 μg/ml for amphotericin B. Much lower geometric mean MIC/MECs were obtained for the following expanded-spectrum triazoles and echinocandins: isavuconazole, 1.783 μg/ml; caspofungin, 1.827 μg/ml; voriconazole, 0.624 μg/ml; itraconazole, 0.078 μg/ml; micafungin, 0.093 μg/ml; posaconazole, 0.035 μg/ml. The MIC/MEC90 differences between any groups of isolates did not exceed 1 dilution step. No significant differences in antifungal susceptibility between the different haplotypes of E. equina were observed.
Table 1.
MICs/MECs of eight antimycotic agents for waterborne E. species
| Organism (no. of strains) and drug | MIC/MEC (μg/ml) |
|||
|---|---|---|---|---|
| Range | For 50% of strains | For 90% of strains | Geometric mean | |
| All (106) | ||||
| Amphotericin B | 0.125–16 | 2 | 16 | 2.2 |
| Fluconazole | 8–64 | 32 | 64 | 35.45 |
| Itraconazole | ≤0.016–8 | 0.063 | 0.25 | 0.08 |
| Voriconazole | 0.125–4 | 0.5 | 2 | 0.62 |
| Posaconazole | ≤0.016–0.5 | 0.031 | 0.063 | 0.03 |
| Isavuconazole | 0.25–16 | 2 | 4 | 1.78 |
| Caspofungin | ≤0.016–8 | 2 | 8 | 1.82 |
| Micafungin | ≤0.008–0.016 | 0.125 | 1 | 0.09 |
| E. equina (35) | ||||
| Amphotericin B | 0.25–8 | 2 | 4 | 1.6 |
| Fluconazole | 16–64 | 32 | 64 | 35.33 |
| Itraconazole | ≤0.016–0.5 | 0.063 | 0.25 | 0.07 |
| Voriconazole | 0.125–4 | 1 | 2 | 0.87 |
| Posaconazole | ≤0.016–0.125 | 0.016 | 0.063 | 0.03 |
| Isavuconazole | 0.5–8 | 2 | 4 | 1.55 |
| Caspofungin | 0.25–8 | 4 | 8 | 2.97 |
| Micafungin | ≤0.008–2 | 0.125 | 0.5 | 0.12 |
| E. mesophila (12) | ||||
| Amphotericin B | 1–8 | 4 | 8 | 3 |
| Fluconazole | 16–64 | 32 | 64 | 28.5 |
| Itraconazole | 0.031–0.5 | 0.063 | 0.125 | 0.08 |
| Voriconazole | 0.125–1 | 0.25 | 1 | 0.35 |
| Posaconazole | ≤0.016–0.5 | 0.031 | 0.063 | 0.04 |
| Isavuconazole | 0.5–4 | 2 | 4 | 1.88 |
| Caspofungin | 0.125–8 | 2 | 4 | 1.88 |
| Micafungin | 0.016–2 | 0.125 | 1 | 0.21 |
| E. angulospora (11) | ||||
| Amphotericin B | 0.25–16 | 4 | 16 | 3.52 |
| Fluconazole | 16–64 | 64 | 64 | 49.74 |
| Itraconazole | 0.031–0.5 | 0.063 | 0.5 | 0.08 |
| Voriconazole | 0.125–1 | 0.5 | 1 | 0.53 |
| Posaconazole | ≤0.016–0.25 | 0.063 | 0.063 | 0.048 |
| Isavuconazole | 0.25–2 | 1 | 2 | 1.13 |
| Caspofungin | 0.25–4 | 2 | 4 | 1.76 |
| Micafungin | ≤0.008–2 | 0.125 | 2 | 0.15 |
| E. castellanii (10) | ||||
| Amphotericin B | 0.25–8 | 1 | 4 | 1.32 |
| Fluconazole | 8–64 | 16 | 64 | 22.63 |
| Itraconazole | ≤0.016–0.25 | 0.063 | 0.125 | 0.055 |
| Voriconazole | 0.125–0.5 | 0.25 | 0.5 | 0.26 |
| Posaconazole | ≤0.016–0.125 | 0.031 | 0.063 | 0.029 |
| Isavuconazole | 0.5–4 | 1 | 4 | 1.23 |
| Caspofungin | 0.5–2 | 0.5 | 2 | 1.07 |
| Micafungin | ≤0.008–0.5 | 0.063 | 0.063 | 0.039 |
| E. aquamarina (9) | ||||
| Amphotericin B | 2–16 | NCa | NC | 5.88 |
| Fluconazole | 32–64 | NC | NC | 40.32 |
| Itraconazole | 0.031–0.25 | NC | NC | 0.12 |
| Voriconazole | 0.5–1 | NC | NC | 0.86 |
| Posaconazole | 0.063–0.125 | NC | NC | 0.07 |
| Isavuconazole | 1–8 | NC | NC | 4.32 |
| Caspofungin | 0.5–8 | NC | NC | 2.94 |
| Micafungin | ≤0.008–1 | NC | NC | 0.1 |
| E. opportunistica (6) | ||||
| Amphotericin B | 0.125–2 | NC | NC | 0.89 |
| Fluconazole | 16–64 | NC | NC | 35.92 |
| Itraconazole | ≤0.016–0.25 | NC | NC | 0.05 |
| Voriconazole | 0.125–2 | NC | NC | 0.5 |
| Posaconazole | ≤0.016–0.063 | NC | NC | 0.03 |
| Isavuconazole | 0.25–4 | NC | NC | 1.12 |
| Caspofungin | 0.25–8 | NC | NC | 1.78 |
| Micafungin | 0.016–2 | NC | NC | 0.11 |
| E. pisciphila (5) | ||||
| Amphotericin B | 1–16 | NC | NC | 2.64 |
| Fluconazole | 16–64 | NC | NC | 27.86 |
| Itraconazole | ≤0.016–1 | NC | NC | 0.08 |
| Voriconazole | 0.25–0.5 | NC | NC | 0.43 |
| Posaconazole | 0.25–0.5 | NC | NC | 0.04 |
| Isavuconazole | 1–4 | NC | NC | 1.51 |
| Caspofungin | 0.125–8 | NC | NC | 1 |
| Micafungin | ≤0.008–1 | NC | NC | 0.08 |
| E. alcalophila (5) | ||||
| Amphotericin B | 0.25–1 | NC | NC | 0.5 |
| Fluconazole | 32–64 | NC | NC | 36.76 |
| Itraconazole | ≤0.016–8 | NC | NC | 0.07 |
| Voriconazole | 0.5–1 | NC | NC | 0.66 |
| Posaconazole | ≤0.016–0.063 | NC | NC | 0.03 |
| Isavuconazole | 0.25–2 | NC | NC | 0.76 |
| Caspofungin | 0.25–4 | NC | NC | 1.51 |
| Micafungin | ≤0.008–0.25 | NC | NC | 0.07 |
| E. cancerae (5) | ||||
| Amphotericin B | 1–4 | NC | NC | 2.3 |
| Fluconazole | 16–64 | NC | NC | 32 |
| Itraconazole | ≤0.016–0.125 | NC | NC | 0.05 |
| Voriconazole | 0.125–1 | NC | NC | 0.5 |
| Posaconazole | ≤0.016–0.063 | NC | NC | 0.03 |
| Isavuconazole | 0.5–4 | NC | NC | 1.51 |
| Caspofungin | 0.5–4 | NC | NC | 1.74 |
| Micafungin | ≤0.008–0.5 | NC | NC | 0.08 |
| E. halophila (3) | ||||
| Amphotericin B | 0.5–2 | NC | NC | 0.79 |
| Fluconazole | 32–64 | NC | NC | 40.32 |
| Itraconazole | 0.031 | NC | NC | 0.03 |
| Voriconazole | 0.5–1 | NC | NC | 0.63 |
| Posaconazole | ≤0.016–0.063 | NC | NC | 0.02 |
| Isavuconazole | 1–4 | NC | NC | 2 |
| Caspofungin | 2 | NC | NC | 2 |
| Micafungin | ≤0.008–0.016 | NC | NC | 0.01 |
| E. salmonis (3) | ||||
| Amphotericin B | 0.5–2 | NC | NC | 1 |
| Fluconazole | 16–32 | NC | NC | 25.4 |
| Itraconazole | 0.063 | NC | NC | 0.06 |
| Voriconazole | 0.25–1 | NC | NC | 0.5 |
| Posaconazole | 0.031 | NC | NC | 0.031 |
| Isavuconazole | 1–4 | NC | NC | 2 |
| Caspofungin | 1–2 | NC | NC | 1.26 |
| Micafungin | ≤0.008–0.125 | NC | NC | 0.02 |
| E. lacus (2) | ||||
| Amphotericin B | 2–16 | NC | NC | 5.65 |
| Fluconazole | 32–64 | NC | NC | 45.25 |
| Itraconazole | 0.031–0.25 | NC | NC | 0.09 |
| Voriconazole | 1 | NC | NC | 1 |
| Posaconazole | ≤0.016–0.063 | NC | NC | 0.032 |
| Isavuconazole | 1–4 | NC | NC | 2 |
| Caspofungin | 1–8 | NC | NC | 2.83 |
| Micafungin | ≤0.008–0.25 | NC | NC | 0.04 |
NC, not calculated (fewer than 10 isolates).
Posaconazole, itraconazole, and micafungin were the drugs with the best overall activity against waterborne Exophiala species. These data are in agreement with previous reports on black yeasts and relatives, i.e., thermophilic Exophiala species (21), Cladophialophora carrionii (17), V. botryosa (11), and species of Phialophora (18), Cyphellophora (18), Fonsecaea (20), and Rhinocladiella (22).
In the present study, no significant MIC/MEC differences between strains isolated from cold-blooded animals, clinical samples, or the environment could be detected and there was no geographic structuring. Furthermore, variation in growth (72 to 144 h) between and within species was observed. Fluconazole is known to have poor activity against melanized fungi (23), which was confirmed in the present study with a MIC50 of 32 μg/ml. E. dermatitidis is susceptible to amphotericin B, with MICs ranging between 0.016 and 0.5 μg/ml (21), but species of the salmonis clade are less sensitive, with MICs ranging between 0.125 and 16 μg/ml. In general, our results suggest that infections with the mesophilic Exophiala species of the salmonis clade can be treated with the newer triazoles and micafungin, while amphotericin B, fluconazole, and caspofungin are less active.
Supplementary Material
ACKNOWLEDGMENTS
M. J. Najafzadeh was supported by the Deputy of Research, Mashhad University of Medical Sciences, Mashhad, Iran (grant 911163).
J. F. Meis has received research grants from Astellas, Merck, Basilea, and Schering-Plough and is a consultant to Astellas, Basilea, and Merck. The rest of us have nothing to declare.
Footnotes
Published ahead of print 7 October 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01629-13.
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