engleski

Optimization of inoculum size of Malassezia pachydermatis and its use in disk diffusion method for antifungal susceptibility testing

During the last 15 years, antifungal susceptibility testing of Malassezia pachydermatis has been explored using various modified protocols. However, there is no standardised method that would ensure inter-laboratory consistency and results. In susceptibility testing, the turbidity method is the standard for the preparation of the inoculum suspension. A butyrous consistency of M. pachydermatis represents a well-known obstacle in the preparation of a homogenous suspension. In this study, we wanted to compare turbidity measurements with the viable plate count, when the inoculum suspension was prepared with lipids. In addition, the reproducibility of the results and the use in the disk diffusion method were evaluated. A total of thirty isolates of M. pachydermatis were subcultured for 72 h at 37 °C on Sabouraud dextrose agar. An inoculum with sterile saline containing 0.04% Tween 80 aiming concentration of 1 to 5x107 CFU/mL was prepared. Different turbidity values and their ten-fold serial dilutions were measured and expressed in McFarland units (McF). For the disk diffusion method, miconazole, clotrimazole and itraconazole tablets (10 μg) and Mueller-Hinton agar with the addition of 2% glucose and 0.5 μg/L methylene blue were used. The plates were incubated at 37 °C and inhibition zones were read after 24, 48, 72, 96 and 120 h. The inoculum suspensions adjusted to 3.5 McF corresponded to the average of 1.27x107 CFU/mL, providing reproducible results. During the first 48 hr, most of the strains demonstrated a weak growth that became confluent after 72 h, allowing the measuring of diameters of clearly visible inhibition zones. There was a statistically significant difference in the readings of the inhibition zones between 48 and 72 h, while the readings after 96 and 120 h remained consistent. The use of 3.5 McF corresponded to the recommended range of yeast cell density for antifungal testing, and the optimal reading time at 72 h showed a consistent reproducibility, which represents the first step in the standardisation of the method.

Malassezia pachydermatis, inoculum, antifungal susceptibility testing

nije evidentirano