Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans. Unlike C. albicans, which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis, an FLC-susceptible strain (ATCC 750) (MIC ‫؍‬ 1.0 g/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 g/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 g/ml for strains grown in medium with 8.0, 32, and 128 g of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 g/ml) and terbinafine (MIC > 512 g/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 g/ml reverted to low MICs (1.0 g/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 g/ml) (128 R) reverted to 16 g/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/ day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis.

The in vitro susceptibilities of 90 clinical isolates of gram-positive and gram-negative aerobic bacteria to six cationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated by two broth microdilution methods. The first method was performed according to the procedures outlined by the National Committee for Clinical Laboratory Standards for bacteria that grow aerobically, while the second was performed according to the procedures recently proposed by the R. E. W. Hancock laboratory for testing antimicrobial peptides. Overall, the first method produced MICs two-and fourfold higher than the second method.Cationic peptides have been isolated from various biological sources (2-6, 10, 11-13, 19). In mammals, including humans, they are found in the neutrophil and on the surface of the tongue, trachea, lungs, and upper intestine. In fact, cationic peptides are thought to be major factors in antibacterial defense on mucosal surfaces (10, 11), and because of their antimicrobial potency they may have therapeutic potential in the treatment of infections (1, 6-10, 12-14, 16-18). Many of these compounds carry net positive charges, and it has been suggested that their mode of action as antimicrobial agents may be similar and may involve the formation of ion channel pores spanning the membranes without requiring a specific target receptor (10,11,16). Nevertheless, since several peptides have a tendency to precipitate and bind avidly to the surface of target cells or plastic materials, such as polystyrene, methods for evaluating the in vitro antimicrobial activities of these compounds are debated (10, 11). The main aim of this study was to compare two different broth microdilution methods to evaluate the antimicrobial activity of the cationic peptides: the first was performed according to the procedures outlined by the National Committee for Clinical Laboratory Standards (NCCLS) for bacteria that grow aerobically (15), while the second was based on the procedures recently proposed by R. E. W. Hancock (University of British Columbia, Vancouver, British Columbia, Canada) for testing antimicrobial peptides (http: //www.interchg.ubc.ca/bobh/MIC.htm). Secondarily, time-kill kinetics were determined to point out the influence of polypropylene and polystyrene in bactericidal activity.A total of 90 nonduplicate, clinical isolates were tested and were found to consist of methicillin-susceptible Staphylococcus aureus (30 strains), Pseudomonas aeruginosa (30 strains), and Escherichia coli (30 strains).Buforin II, cecropin P1, magainin II, indolicidin, nisin, and ranalexin were obtained from Sigma-Aldrich S.r.l. (Milan, Italy). The aminoglycoside amikacin (Sigma-Aldrich) was used as a control cationic antimicrobial agent. The drugs were dissolved in distilled water. Solutions were made fresh on the day of assay or stored at Ϫ80°C in the dark for short periods. The MIC of each peptide was determined by two broth microdilution methods with cation-adjusted Mueller-Hinton (MH) broth (Becton Dickinso...

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A broth microdilution method performed in accordance with the National Committee for Clinical Laboratory Standards guidelines was used to compare the in vitro activity of the new antifungal triazole SCH 56592 (SCH) to that of fluconazole (FLC), itraconazole (ITC), and ketoconazole (KETO) against 257 clinical yeast isolates. They included 220 isolates belonging to 12 different species of Candida, 15 isolates each of Cryptococcus neoformans and Saccharomyces cerevisiae, and seven isolates of Rhodotorula rubra. The MICs of SCH at which 50% (MIC 50 ) and 90% (MIC 90 ) of the isolates were inhibited were 0.06 and 2.0 g/ml, respectively. In general, SCH was considerably more active than FLC (MIC 50 and MIC 90 of 1.0 and 64 g/ml, respectively) and slightly more active than either ITC (MIC 50 and MIC 90 of 0.25 and 2.0 g/ml, respectively) and KETO (MIC 50 and MIC 90 of 0.125 and 4.0 g/ml, respectively). Our in vitro data suggest that SCH has significant potential for clinical development.

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