Parameters affecting the reliable measurement of fluconazole tolerance using disk diffusion assays with Candida...

Disk diffusion assays (DDAs) are widely used to determine antimicrobial susceptibility, which is measured as the size of the zone of inhibition (ZOI) after 24 h of growth, and is inversely proportional to the minimal inhibitory concentration (MIC). DDAs can also be used to measure the level of antifungal drug tolerance, defined as the degree to which a subpopulation of cells grows slowly at drug concentrations above the MIC. Tolerance occurs via one or more mechanisms that involve stress responses and that are distinguishable from mechanisms of drug resistance. Measurements of antifungal drug tolerance are generally more sensitive to growth parameters, such as temperature and pH. Here, we investigated parameters used for executing DDAs and asked if they affect the reproducibility of fluconazole susceptibility and tolerance measurements using Candida albicans isolates. Specifically, we tested drug format, media composition, and inoculum parameters. Drug format had little effect on DDA measurements, whereas media composition did affect assay outcomes. In general, inoculum density affected the ability to distinguish between the levels of drug susceptibility and tolerance in different strains. Of note, fluconazole tolerance measurements were much more sensitive than susceptibility measurements to the medium properties. Finally, we recommend conditions that accurately and reproducibly measure fluconazole susceptibility and tolerance levels in C. albicans isolates.IMPORTANCEDisk diffusion assays are a popular method for testing antimicrobial responses because they are inexpensive and easy to perform. However, experimental factors that influence assay measurements, especially factors that affect antifungal drug tolerance, have not been standardized; antifungal drug tolerance is defined as the ability of a subpopulation of cells to grow slowly at drug concentrations that inhibit growth of the remaining population. Here, we examined how modifying details of the drug format, media properties, and the initial cell number affects the ability to measure resistance and tolerance levels. We provide specific recommendations for performing reliable and robust disk diffusion assays that report both drug susceptibility and tolerance levels.