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Antimicrobial Agents and Chemotherapy, June 2006, p. 2222-2224, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01443-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effect of Culture Medium on the Disk Diffusion Method for Determining Antifungal Susceptibilities of Dermatophytes

Belkys Fernández-Torres,1 Alfonso Carrillo-Muñoz,2 Isabel Inza,1 and Josep Guarro1*

Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, 43201 Reus, Spain,1 Department of Microbiology, Asesoría Científica y de Investigación Aplicada, 08080 Barcelona, Spain2

Received 9 November 2005/ Returned for modification 21 December 2005/ Accepted 21 March 2006


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ABSTRACT
 
We have evaluated a disk diffusion method to determine the activities of five drugs against 50 strains of dermatophytes and to assess the influence of the culture medium (antibiotic medium 3, high-resolution medium, and RPMI) on the inhibition zone diameters (IZD). There were no differences among the medium/drug combinations, except for itraconazole-RPMI, which showed the narrowest IZD.


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TEXT
 
The increase in recalcitrant dermatophytoses and the fact that some patients do not respond well to therapy make it necessary to test new therapeutic strategies (16, 18). In vitro testing can help to determine the activities of new drugs and to find the right therapy. However, at present there are no in vitro reference methods for dermatophytes. Agar-based methods are quick and easy and could be good options (8, 11). A disk diffusion method to test yeasts has recently been standardized (15). However, data on the use of the disk diffusion method for testing dermatophytes are scarce (3, 6). The aim of this study is to evaluate the usefulness of this method for testing dermatophytes and to investigate the influence of culture media on the inhibition zone diameters (IZD).

Fifty strains belonging to five species of dermatophytes were tested (Table 1). Species identification was performed by standard methods (7). Tests were performed using a disk diffusion method mainly following the CLSI (formerly NCCLS) document M44-A (15) and using the optimal conditions reported in a previous multicenter study (5). Inoculum suspensions were prepared as described in a previous study (5), adjusted to a concentration of 106 CFU/ml, and diluted 1:100 in RPMI to achieve a final concentration of 104 CFU/ml. Itraconazole, micafungin, ravuconazole, terbinafine, and voriconazole were obtained from manufacturers as standard powder. All drugs were dissolved in dimethyl sulfoxide to obtain stock solutions of 50, 100, 200, 400, and 800 µg/ml, except for terbinafine stock solutions, which were 6.25, 12.5, 25, 50, and 100 µg/ml. Disks for all antifungal agents were prepared in our laboratory. First, we performed a preliminary study to find the optimal concentrations (those which produced inhibition zones neither too wide nor too narrow to be read) of the drugs in the disks. Blank paper disks (Schleicher & Schuell, Spain) were impregnated with 20 µl of each of the concentrations of the stock solutions. We considered as optimal concentrations 0.25 µg/ml for terbinafine and 100 µg/ml for the other drugs. This resulted in a final concentration per disk of 0.005 µg or 2 µg, respectively. Three culture media were tested: (i) RPMI 1640 medium with L-glutamine and without bicarbonate (Gibco BRL, Barcelona, Spain), buffered to pH 7.0 with 0.165 MOPS (morpholinepropanesulfonic acid) and supplemented with 1.5% Bacto agar; (ii) antibiotic medium 3 (AM3) (Difco, Spain) buffered to pH 7.0 with MOPS (0.165 M); and (iii) high-resolution medium (HR) (Sigma, Spain) buffered to pH 7.0 with phosphate buffer (0.2 M). The surfaces of the agar media were inoculated by streaking several times a sterile swab that was dipped into each adjusted inoculum. After 5 to 10 min, disks were dispensed onto the surfaces of the inoculated agar plates. The plates were incubated at 28°C, and the IZD were measured in millimeters.


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  		TABLE 1. Inhibition zone diameters obtained for each culture medium/drug/species combination

The ranges and arithmetic means of the IZD were calculated for each combination of species, culture medium, and drug. The significance of differences in mean values was determined by Student's t test. P values of <0.05 were considered statistically significant.

The IZD were read at 5 days of incubation, except for Microsporum gypseum and Trichophyton interdigitale, whose IZD were read at 3 days. All strains showed measurable inhibition zones, without microcolonies inside them. With AM3, the sharp zone edges were well defined and the fungal growth was faster and more confluent than with the other media.

The results are summarized in Table 1. In general, when considering all strains together, we observed no significant differences among the different medium/drug combinations. The only exception was for itraconazole, since RPMI showed a diameter (23.3 mm) narrower than that of either AM3 (24.9 mm) or HR (24.6 mm) (P < 0.05). Voriconazole showed the widest IZD, while micafungin did not show any inhibition zone (data not shown).

In general, the IZDs obtained for each drug/medium combination were dependent on the species tested (Fig. 1). For example, in the case of itraconazole the influence of the culture medium was observed only for Microsporum canis and Trichophyton mentagrophytes (P < 0.05). For the other drugs, the culture medium significantly affected the results of all species tested, with the exception of the combinations ravuconazole/Trichophyton rubrum, voriconazole/M. gypseum, and terbinafine/M. gypseum.


Figure 1
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  		FIG. 1. Comparison of the IZD obtained with AM3, HR, and RPMI for evaluating the activities of itraconazole (ITC), ravuconazole (RVC), voriconazole (VRC), and terbinafine (TRB) against 50 strains of the following species: M. canis (MC), M. gypseum (MG), T. interdigitale (TI), T. mentagrophytes (TM), and T. rubrum (TR). The following results are based on the paired-differences Student t test: (A) the IZD for ITC were smaller in RPMI than in AM3 for MC and TM (P < 0.05); (B) the IZD for RVC were smaller in RPMI than in HR for MC, the smallest in HR for MG, the smallest in RPMI for TI, and smaller in RPMI and AM3 than in HR for TM (P < 0.05); (C) the IZD for VRC were smaller in RPMI than in HR for MC, the smallest in RPMI for TI, and the smallest in HR for TM and TR (P < 0.05); and (D) the IZD for TRB were smaller in HR and RPMI than in AM3 for MC, smaller in AM3 and HR than in RPMI for TI, smaller in AM3 and RPMI than in HR for TM, and smaller in RPMI than in HR for TR (P < 0.05).

Though in general all of the drugs tested (except micafungin) were active, we should point out the particularly good activities of voriconazole and ravuconazole. Our results are difficult to compare with those of other authors either because the other authors used different testing parameters (3, 21) or, in the case of ravuconazole or micafungin, there are no in vitro data on the activities of the drugs against dermatophytes. Venugopal and Venugopal (21) also used a disk diffusion method for evaluating the activity of terbinafine in nutrient agar and obtained wider IZD than we did.

It has been demonstrated that the composition of the culture medium exerts an important influence on the antimicrobial susceptibilities of bacteria, yeasts, and filamentous fungi (1, 12, 17). Some authors (13) have indicated that agar-based methods are not recommendable for testing azoles because these drugs can diffuse poorly in the agar. However, the disk diffusion and Etest methods have shown a good correlation with the broth-based reference methods for testing yeasts (15, 20). Although the reference methods recommend the use of RPMI medium for broth testing and Mueller-Hinton medium for agar testing, other less-expensive media have also been used with success (9, 14, 17). Pfaller et al. (17) proposed the use of Casitone agar for testing fluconazole against yeasts with the Etest method and found a good correlation with the reference microdilution method. Other authors (14) used a microdilution method and proposed AM3 and RPMI, both supplemented with glucose, for testing micafungin against Candida albicans and Candida dubliniensis. The effects of culture medium on in vitro antifungal activity have also been evaluated for filamentous fungi with contradictory results (1, 2, 12, 17). For instance, using a microdilution method to test caspofungin against isolates of Aspergillus and Fusarium species, Arikan et al. (2) obtained lower MICs with AM3 than with RPMI or RPMI supplemented with glucose. Also, using a microdilution method, Llop et al. (9) compared five media to test dematiaceous fungi. They demonstrated that the media had an influence on the MICs of amphotericin B, ketoconazole, and flucytosine. However, opposite results were reported by Tortorano et al. (19) and Manavathu et al. (10), who found no differences when comparing RPMI and AM3 for testing amphotericin B and itraconazole against Aspergillus fumigatus. The only exception was for amphotericin B in peptone-yeast extract, which gave higher MICs than those obtained with RPMI and AM3. In our study, when all strains were considered together, no differences were observed among the media, except for itraconazole, which showed wider diameters with both AM3 and HR than with RPMI. However, as we found in a previous study using RPMI and the Etest method (4), our results were dependent on the drug/species combinations tested.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI 020114).


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FOOTNOTES
 
* Corresponding author. Mailing address: Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201 Reus, Spain. Phone: 34-977-759359. Fax: 34-977-759322. E-mail: josep.guarro{at}urv.net. Back


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Antimicrobial Agents and Chemotherapy, June 2006, p. 2222-2224, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01443-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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