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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2008, p. 732-734, Vol. 52, No. 2
0066-4804/08/$08.00+0 doi:10.1128/AAC.01012-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Unitat de Microbiologia, Facultat de Medicina i Ciéncies de la Salut, Universitat Rovira i Virgili, Reus, Spain
Received 1 August 2007/ Returned for modification 10 October 2007/ Accepted 10 November 2007
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Some in vitro studies have demonstrated variable results among the strains tested, and some authors have concluded that antifungal susceptibility is strain dependent (7, 14, 21). This could be explained by the fact that S. schenckii does not represent a single species; instead it is a complex of cryptic species. Recently, using a multilocus sequence analysis, we have demonstrated that at least six phylogenetic species are included in the complex (13), several of these species being phenotypically characterized (12). Since the antifungal susceptibility of these species is unknown, we have evaluated the in vitro activity of 12 drugs against the mycelial phase of 92 strains representing five species of the complex (Table 1), using a reference microdilution method (15). The isolates were selected to represent the widest variety of geographical regions possible.
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The antifungal agents were obtained as pure powders. AMB (USP; Rockville, MD), ITC and ketoconazole (KTC) (Janssen Pharmaceutica, Beerse, Belgium), albaconazole (ABC) (J. Uriach & Cía, Barcelona, Spain), voriconazole (VRC) (Pfizer, Inc., NY), posaconazole (PSC) (Schering-Plough, Kenilworth, NJ), ravuconazole (RVC) (Bristol-Myers Squibb Company, New Brunswick, NJ), eberconazole (EBC) (Laboratorios Salvat, S.A., Barcelona, Spain), and terbinafine (TRB) (Novartis, Basel, Switzerland) were diluted in dimethyl sulfoxide (Panreac Química S.A., Barcelona, Spain), and micafungin (MFG) (Astellas Pharma, Inc., Tokyo, Japan), flucytosine (5FC) (Sigma-Aldrich Corp., St. Louis, MO), and fluconazole (FLC) (Pfizer, Inc., Madrid, Spain) in sterile distilled water. Microplates were prepared as described in NCCLS standard M38-A (15). Final drug concentrations ranged from 128 to 0.25 µg/ml for MFG, from 64 to 0.12 µg/ml for FLC and 5FC, and from 16 to 0.03 µg/ml for the other drugs. The inoculum was prepared as recommended by the CLSI (formerly NCCLS) (15), by flooding the surface of the agar plate with sterile saline, scraping the sporulating mycelium with a culture loop, and drawing up the resultant suspension with a sterile Pasteur pipette. The suspensions were then filtered once through sterile gauze to remove hyphae. The numbers of conidia in the suspensions were adjusted to optical densities that ranged from 0.09 to 0.11, which corresponded to final concentrations of 1 x 104 to 5 x 104 CFU/ml. The viabilities of these inocula were verified by plating dilutions of the suspensions on PDA plates. The microplates were incubated at 30°C and read at 72 h. The MIC endpoint for the triazoles, AMB, MFG, and TRB was defined as the lowest concentration that produced complete inhibition of growth and for FLC, KTC, and 5FC as the lowest concentration that produced 50% growth inhibition. Approximately 80% of the tests were repeated and showed the same tendency (data not shown). However, in the few cases that did not coincide, the test was repeated and a modal MIC of the three values was considered.
The results are shown in Table 1. The MICs for the control strains agreed with the CLSI guidelines (15). TRB was the most active drug, showing a geometric mean (GM) MIC of 0.23 µg/ml for all the strains tested, followed by KTC with a GM MIC of 0.84 µg/ml. However, this latter drug was less active against Sporothrix mexicana (GM MIC of 4 µg/ml) and Sporothrix albicans (GM MIC of 3.2 µg/ml) than it was against the other species of the complex. The activity of KTC was more variable than that of TRB and depended on the species tested. PSC was the third-most-active antifungal drug tested, with a total GM MIC of 1.59 µg/ml, and the most active of the drugs for systemic use.
Although we could test only two isolates of S. mexicana, this was the species that responded least well to antifungals and only TRB showed a relatively low MIC (0.5 µg/ml) against this species.
FLC and MFG were not active against any of the isolates tested, as had already been demonstrated by other authors (11, 21). VRC showed poor activity, in agreement with the results of McGinnis et al. (14), who also obtained a high GM MIC (6.50 µg/ml) against strains of S. schenckii.
RVC and ITC only showed good activity against Sporothrix brasiliensis, whereas, for the other species tested, both drugs showed high MICs. Other authors (14) had also demonstrated poor in vitro activity of ITC. Despite these in vitro results, ITC has generally shown efficacy in the clinical setting. Conti Díaz et al. (1) successfully treated 18 cutaneous sporotrichosis patients with this drug. Sharkey-Mathis et al. (20) reported that 11 out of 15 (83%) patients with osteoarticular sporotrichosis who received ITC responded to the therapy.
In the present work, TRB has shown high activity against all the species tested. However, the therapeutic potential of TRB has been confirmed only for cutaneous and lymphocutaneous sporotrichosis (5, 6, 17, 19). By contrast, this drug has not demonstrated efficacy in the treatment of systemic sporotrichosis in a murine model (8). In severe or systemic infections, PSC constitutes a promising therapeutic agent since, in vitro, it has worked better than AMB and ITC, at least against strains of S. brasiliensis, S. albicans, and S. schenckii. Further in vivo studies are needed to confirm this activity.
Although in vitro results do not always correlate with in vivo outcome, the drugs tested showed in general very poor activity against S. albicans, Sporothrix globosa, and S. mexicana. It would be interesting to know if any drug combinations exert any activity against such species. However, no data are so far available on the activity of combined drugs against S. schenckii sensu lato.
In recent years, application of the phylogenetic species concept in different biological species of pathogenic molds has revealed different lineages that reflected species divergence (3, 10, 16). The delineation of these phylogenetic groups and the development of easy methods for their identification are crucial, since they can show different pathological behaviors and different antifungal responses (4). This study has demonstrated that S. schenckii constitutes a clear example of the latter.
Since clinical information on these new species does not yet exist, the significance of our findings is unknown. However, it seems that proper identification of the species of the S. schenckii complex involved in a given infection could be important for the appropriate treatment. For instance, in the case of a systemic infection, if the species causing the infection was S. mexicana, it is likely that the response to treatment with ITC or PSC would be poorer than if the species was S. brasiliensis.
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ACKNOWLEDGMENTS |
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This study was supported by the Spanish Ministerio de Ciencia y Tecnología, grants CGL 2004-00425/BOS and CGL 2005-07394.
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FOOTNOTES |
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Published ahead of print on 26 November 2007.
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