Evaluation of Disk Diffusion Method for Determining Eberconazole Susceptibility of Dermatophytes and Influence of Culture Medium

Abstract

We have evaluated a disk diffusion method to determine the activity of eberconazole against 50 strains of dermatophytes by testing three culture media (RPMI, antibiotic medium 3, and high resolution). No differences were found among the results obtained with the three media. A significant correlation between disk diffusion and microdilution methods was observed with AM3.

The increase in dermatophytoses and the fact that some patients do not respond well to treatment make it necessary to test new therapeutic strategies (15, 17). Eberconazole (EBC) is a topical imidazole that has shown good in vitro and in vivo antimicrobial activity (18). Using a microdilution method that we had previously standardized (4), we have recently proven the excellent in vitro activity of this drug against a high number of strains of dermatophytes (3). However, agar-based methods such as Etest and disk diffusion can be good alternatives because they are simpler and faster than broth-based methods (5, 10). We have determined the usefulness of the disk diffusion method for testing the susceptibility to EBC of dermatophytes and to evaluate the influence of culture media.

A total of 50 strains belonging to five species of dermatophytes were tested (Table 1). Prior to testing, each isolate was subcultured onto potato dextrose agar (PDA) at 28°C. Quality control strains Aspergillus fumigatus NCPF 7099 and Candida parapsilosis ATCC 22019 were used, and all results were within published limits (2, 12).

TABLE 1.

In vitro activity of eberconazole against 50 strains of dermatophytes determined by the disk diffusion method testing three media and the microdilution method

Species (no. of strains)	Medium	Disk diffusion (mm)		Microdilution (μg/ml)
		Range	AMa	Range	GMb
M. canis (10)	RPMI	26-35	30.9	0.03-2.0	0.09
	AM3	25-35	29.8
	HR	31-36	33.5
M. gypseum (10)	RPMI	29-50	42.9	0.03-0.25	0.10
	AM3	40-42	41.4
	HR	34-40	35.7
T. interdigitale (10)	RPMI	32-36	33.8	0.125-0.5	0.35
	AM3	29-35	31.8
	HR	32-36	33.8
T. mentagrophytes (10)	RPMI	22-35	26.9	0.06-0.5	0.17
	AM3	22-35	26.9
	HR	23-36	31.5
T. rubrum (10)	RPMI	30-46	38.1	0.03-0.125	0.07
	AM3	35-50	40.1
	HR	23-38	33.0
Total (50)	RPMI	22-50	34.5	0.03-2.0	0.12
	AM3	22-50	34.6
	HR	23-40	33.5

^aAM, arithmetic mean of the inhibition zone diameters.

^bGM, geometric mean of the MICs.

The microdilution tests were performed as we described in a previous study (4). EBC was obtained as standard powder from Laboratorios Salvat, S. A. (Barcelona, Spain) and dissolved in 100% dimethyl sulfoxide. Drug dilutions were performed as described by the NCCLS (12). The final concentrations were from 16 to 0.01 μg/ml. Microdilution plates were incubated at 28°C and read at 7 days. The MIC was defined as the lowest concentration showing 100% growth inhibition.

The procedure for the disk diffusion method was mainly that of the document M44-P (13). The inocula were prepared as in the microdilution method and adjusted to a final concentration of 10⁴ CFU/ml. EBC disks were prepared in our laboratory. First we performed a preliminary study to find the optimal concentration (those which produced inhibition zones neither too wide nor too narrow to be read) of the drug in the disks. EBC was dissolved in 100% dimethyl sulfoxide (DMSO) to obtain stock solutions of 50, 100, 200, 400, and 800 μg/ml. Blank paper disks (Schleicher & Schuell, Spain) with a diameter of 6.0 mm were impregnated with 20 μl of each concentration of the stock solutions mentioned above. The disks were allowed to dry in the dark at room temperature. We considered 100 μg/ml as the optimal concentration, which resulted in a final concentration per disk of 2 μg. The following culture media were tested: RPMI-1640 medium buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS) and supplemented with 1.5% Bacto agar; antibiotic medium 3 (AM3) buffered to pH 7.0 with MOPS (0.165 M); and high-resolution medium (HR) buffered to pH 7.0 with phosphate buffer (0.2 M). The dried surfaces of the petri dishes with the agar media (90 mm diameter) were inoculated by streaking several times a sterile swab that was dipped into each adjusted inoculum. After 5 min, antifungal disks were dispensed onto the surface of the inoculated agar plates. The plates were inverted and incubated at 28°C, and the inhibition zone diameters (IZDs) were measured in millimeters (mm) after 5 days.

To determine the correlation between the disk diffusion method and the microdilution method, the IZDs were plotted against their respective MICs. Pearson's correlation coefficient was used to calculate a regression line for each comparison. For all tests, P values of <0.05 were considered statistically significant.

The IZDs were read after 5 days of incubation, except for Microsporum gypseum and Trichophyton interdigitale, which were read at 3 days. Table 1 shows the MICs and IZDs of EBC for the three media. When all strains were considered together, the IZDs obtained with the three media were not significantly different. Similarly, no differences were found between the IZDs obtained with RPMI and those obtained with AM3 when each species was studied separately. However, with the exception of T. interdigitale, the IZDs obtained with HR were significantly different from those obtained with AM3 and RPMI.

The IZDs obtained by disk diffusion showed an inverse correlation with those obtained with MICs by the microdilution method (r = −0.41) only with AM3 (Fig. 1). With the other two media the correlation was not significant (r = −0.25 for RPMI and r = −0.16 for HR).

FIG. 1.

Zones of inhibition around the 2-μg EBC disks on AM3 agar plotted against the MICs determined by microdilution method. The numbers inside the graph indicate the numbers of isolates.

In general, our results are difficult to compare with those of other authors who also tested dermatophytes (8, 20), because they have used either other drugs or other testing parameters. Although the NCCLS recommends using RPMI medium for the broth test and using Mueller-Hinton medium for the agar test, other less expensive media have also been used with some success (6, 9, 16). Pfaller et al. (16) proposed the use of Casitone agar for testing fluconazole against yeasts with the Etest method. They found a good correlation with the reference microdilution method. Other authors proposed AM3 for detecting resistance of Cryptococcus neoformans to amphotericin B using a microdilution method (7) and AM3 and RPMI, both supplemented with glucose, for testing micafungin against Candida albicans and Candida dubliniensis (11). Several studies on the influence of the culture media have also been performed with filamentous fungi (1, 6, 9, 19). The results from these studies generally depended on the drug-fungi-culture medium combination tested. In our case, the effect of the culture medium on IZDs also depended on the species tested. Several authors have investigated the correlation between the disk diffusion and microdilution methods for testing novel drugs. Odabasi et al. (14) found a poor correlation between the methods when testing the echinocandin anidulafungin against Candida parapsilosis. In this study, we have also obtained a good correlation for EBC between the two methods, although only with AM3. We think that the results obtained with EBC, which has proven to be one of the most active topical drugs against dermatophytes (3), is a very positive result for the development of an easy and reliable testing method. However, further studies are needed to find the conditions most appropriate for other drugs. Depending on the species/culture medium combination tested, the optimization of these tests may require adjustments.