Approaches for identifying and measuring heteroresistance in azole-susceptible Candida isolates

Abstract

Heteroresistance to antifungal agents poses a significant challenge in the treatment of fungal infections. Currently, the absence of established methods for detecting and measuring heteroresistance impedes progress in understanding this phenomenon in fungal pathogens. In response to this gap, we present a comprehensive set of new and optimized methods designed to detect and quantify azole heteroresistance in Candida albicans. Here, we define two primary assays for measuring heteroresistance: population analysis profiling, based on growth on solid medium, and single-cell assays, based on growth in liquid culture. We observe good correlations between the measurements obtained with liquid and solid assays, validating their utility for studying azole heteroresistance. We also highlight that disk diffusion assays could serve as an additional tool for the rapid detection of heteroresistance. These methods collectively provide a versatile toolkit for researchers seeking to assess heteroresistance in C. albicans. They also serve as a critical step forward in the characterization of antifungal heteroresistance, providing a framework for investigating this phenomenon in diverse fungal species and in the context of other antifungal agents. Ultimately, these advancements will enhance our ability to effectively measure antifungal drug responses and combat fungal infections.IMPORTANCEHeteroresistance involves varying antimicrobial susceptibility within a clonal population. This phenomenon allows the survival of rare resistant subpopulations during drug treatment, significantly complicating the effective management of infections. However, the absence of established detection methods hampers progress in understanding this phenomenon in human fungal pathogens. We propose a comprehensive toolkit to address this gap in the yeast Candida albicans, encompassing population analysis profiling, single-cell assays, and disk diffusion assays. By providing robust and correlated measurements through both solid and liquid assays, this work will provide a framework for broader applications across clinically relevant Candida species. These methods will enhance our ability to understand this phenomenon and the failure of antifungal therapy.

Keywords: Candida albicans; azoles; drug responses; heteroresistance; mycology.

Fig 1

Disk diffusion assays can detect the presence of heteroresistant cells. (A) Images show cross-sections of disk diffusion assays of C. albicans isolates displaying different numbers of heteroresistant colonies in the zone of inhibition (FLC drug disk, 25 µg). Isolates CAY8847 and CAY8856 show a clear area of inhibition without any colonies present, while isolates CAY8762 and P60002 were fully resistant and grew to the edge of the FLC disk. Red arrows indicate potential heteroresistant colonies detected in the other eight isolates. FLC susceptibility (B, MIC₅₀) and tolerance (C, SMG determined from MIC assays) levels of parent isolates (black) and five randomly selected large colonies (salmon) from each of the eight isolates. Error bars show the standard error of the mean (S.E.M.) of three biological replicates.

Fig 2

PAPs can accurately quantify azole heteroresistance. (A) C. albicans cultures were serially diluted and plated on YPD plates without antifungal or supplemented with a gradient of FLC concentrations (0.5–128 µg/mL in two-fold dilutions). The plates were incubated for 48 h and imaged at 24 and 48 h for CFU determination. The fraction of cells growing at each concentration was determined by counting the number of colonies growing on FLC relative to the number of colonies present on plates without FLC. (B) PAP profiles of the 12 strains examined in this study. Heteroresistance levels were calculated by taking the average growth on PAP at 48 h for FLC concentrations equal to or greater than 10-fold the MIC₅₀ of the parent isolate (Avg HR). The dotted lines indicate the detection limit of the assay (0.04% of the population), and the red lines show MIC₅₀ levels. Error bars show the standard error of the mean (S.E.M.) of three biological replicates. (C) Susceptibility measurements of up to five randomly selected colonies (salmon) recovered from FLC 128 µg/mL plates. Susceptibility levels of parent isolates (black) are included for reference. Susceptibility was determined by assessing cell growth at FLC concentrations 8- and 16-fold higher than the MIC₅₀ of the corresponding parent isolate.

Fig 3

Short PAP assays represent an efficient method for measuring azole heteroresistance. (A) C. albicans cultures were serially diluted and plated on YPD plates without antifungal or supplemented with 128 µg/mL FLC. The plates were incubated for 48 h and imaged for CFU determination. The fraction of cells growing on FLC was determined relative to the growth on plates without drug. (B) Short PAP profiles of the 12 strains examined in this study. The dotted lines indicate the detection limit of the assay (0.067% of the population). Conventional PAP assays are included for reference. Avg HR, average heteroresistance rates measured with short PAP assays. Red lines show MIC₅₀ levels, and error bars show the standard error of the mean (S.E.M.) of three biological replicates.

Fig 4

Single-cell assays can measure the frequency of heteroresistance in liquid culture. (A) Schematic of single-cell assays, whereby individual cells were grown in FLC concentrations 10-fold higher than the MIC₅₀ of the parent isolate. A subset of cells recovered from wells showing robust growth were further tested in susceptibility assays. Heteroresistance rates were calculated by multiplying the fraction of wells with proficient growth with the rate of recovery of resistant isolates from the tested wells. Histograms show the fraction of wells with proficient growth (OD₆₀₀ ≥ 0.5, B), the fraction of heteroresistant isolates recovered from the tested wells (C), and the rate of heteroresistance for each isolate (D). Experiments were performed with three to four biological replicates, each with at least 384 cells tested. Lines show average values.

Fig 5

PAP profiles of a collection of 30 isolates from other yeast species. Assays were performed as described in Fig. 2. Black lines show average heteroresistance rates determined after 48 h incubation. Heteroresistance rates were calculated by taking the average growth on PAP at 48 h for FLC concentrations equal to or greater than 10-fold the MIC₅₀ of the parent isolate (Avg HR). When such concentrations were outside of the dynamic range of the assay, the rate observed at 128 µg/mL FLC was used instead. The dotted lines indicate the detection limit of the assay (0.04%), and the red lines show MIC₅₀ levels. Error bars show the standard error of the mean (S.E.M.) from three biological replicates. Graphs are color-coded according to the species.