Identification and Elimination of Antifungal Tolerance in Candida auris

Abstract

Antimicrobial resistance is a global health crisis to which pathogenic fungi make a substantial contribution. The human fungal pathogen C. auris is of particular concern due to its rapid spread across the world and its evolution of multidrug resistance. Fluconazole failure in C. auris has been recently attributed to antifungal "tolerance". Tolerance is a phenomenon whereby a slow-growing subpopulation of tolerant cells, which are genetically identical to susceptible cells, emerges during drug treatment. We use microbroth dilution and disk diffusion assays, together with image analysis, to investigate antifungal tolerance in C. auris to all three classes of antifungal drugs used to treat invasive candidiasis. We find that (1) C. auris is tolerant to several common fungistatic and fungicidal drugs, which in some cases can be detected after 24 h, as well as after 48 h, of antifungal drug exposure; (2) the tolerant phenotype reverts to the susceptible phenotype in C. auris; and (3) combining azole, polyene, and echinocandin antifungal drugs with the adjuvant chloroquine in some cases reduces or eliminates tolerance and resistance in patient-derived C. auris isolates. These results suggest that tolerance contributes to treatment failure in C. auris infections for a broad range of antifungal drugs, and that antifungal adjuvants may improve treatment outcomes for patients infected with antifungal-tolerant or antifungal-resistant fungal pathogens.

Keywords: Candida auris; adjuvant; antifungal tolerance/resistance; broth microdilution assay; disk diffusion assay; diskImageR; human fungal pathogen.

Figure 1

Minimum inhibitory concentration (MIC) for clinical C. auris isolates (A–E) growing in antifungal microwell plates to determine susceptibility/resistance to antifungal drugs. Mean MICs of five clinical C. auris isolates measured after 24 and 48 h for four fungistatic drugs (fluconazole, itraconazole, posaconazole, and voriconazole) and two fungicidal drugs (amphotericin B and caspofungin). Different symbols denote C. auris isolates with the same MIC.

Figure 2

Radius of the zone of inhibition (RAD) (A–E) and fraction of growth in the zone of inhibition (FoG₂₀) (F–J) for C. auris isolates treated with antifungal drugs. Mean RAD where 20% of growth is inhibited (RAD₂₀) at 24 and 48 h. (B) Mean FoG where 20% of growth is inhibited (FoG₂₀) 24 and 48 h. C. auris isolate 2 treated with caspofungin at 48 h is not plotted in (B), as it exhibited FoG in the entire ZOI (i.e., a “NA” data point was generated by diskImageR [29]); the reduction in RAD and FoG₂₀ for C. auris isolate 3 in (C) and (H), respectively, is due to the exclusion of FoG₂₀ within the ZOI by diskImageR (see Section 2.5 for details). C. auris isolate 5 exhibited resistances to fluconazole and caspofungin.

Figure 3

Tolerance from supra-MIC growth (SMG) for clinical C. auris isolates grown in antifungal microwell plates. (A–E) Mean SMG of tolerant isolates after 24 and 48 h. (F–I) Mean SMG of non-tolerant isolates after 24 and 48 h. C. auris 5 was resistant to fluconazole and caspofungin hence tolerance/non-tolerance could not be determined for these isolate–antifungal combinations.

Figure 4

Correlation analysis for mean supra-MIC growth (SMG) and mean pixel intensity measured by ImageJ [51] to determine tolerance. (A) Analysis performed after 24 h of growth (R² = 0.3128; Pearson correlation test, p = 0.0469). (B) Analysis performed after 48 h of growth (R² = 0.2862; Pearson correlation test, p = 0.0085).

Figure 5

Radius of the zone of inhibition (RAD) and fraction of growth in the zone of inhibition (FoG₂₀) measurements for C. auris isolates for adjuvant antifungal disk diffusion assays. (A–E) Mean RAD measured for the C. auris isolates at 24 h against antifungal drugs with and without the adjuvant chloroquine. (F–J) Mean RAD measured for the C. auris isolate at 48 h against antifungal drugs with and without chloroquine. (K–O) Mean FoG₂₀ measured using diskImageR [29] for all C. auris isolates at 24 h against antifungal drugs with and without chloroquine. (P–T) Mean FoG₂₀ measured using diskImageR for the C. auris isolates at 48 h against antifungal drugs with and without chloroquine. Note that the single data points in (L), (Q), and (T) at 48 h are due to the mitigation of resistance in presence of chloroquine, as FoG₂₀ could not be measured for these isolates at 24 h because of their resistance to the corresponding antifungal drugs.

Figure 5

Radius of the zone of inhibition (RAD) and fraction of growth in the zone of inhibition (FoG₂₀) measurements for C. auris isolates for adjuvant antifungal disk diffusion assays. (A–E) Mean RAD measured for the C. auris isolates at 24 h against antifungal drugs with and without the adjuvant chloroquine. (F–J) Mean RAD measured for the C. auris isolate at 48 h against antifungal drugs with and without chloroquine. (K–O) Mean FoG₂₀ measured using diskImageR [29] for all C. auris isolates at 24 h against antifungal drugs with and without chloroquine. (P–T) Mean FoG₂₀ measured using diskImageR for the C. auris isolates at 48 h against antifungal drugs with and without chloroquine. Note that the single data points in (L), (Q), and (T) at 48 h are due to the mitigation of resistance in presence of chloroquine, as FoG₂₀ could not be measured for these isolates at 24 h because of their resistance to the corresponding antifungal drugs.

Figure 6

Supra-MIC growth (SMG) of C. auris isolates 1 to 5 (A–E) for adjuvant antifungal broth microdilution assays. Chloroquine did not show any adjuvant effect on C. auris isolate 1 when combined with itraconazole, nor for C. auris isolate 2 when combined with itraconazole or voriconazole at 48 h compared to 24 h. Similarly, no adjuvant effect was noted for C. auris isolate 4 against fluconazole nor for C. auris isolate 5 against posaconazole and amphotericin B. Since C. auris isolate 2 is resistant to fluconazole and C. auris isolate 5 is resistant to fluconazole and caspofungin, the SMGs were not calculated for these isolate–adjuvant–antifungal combinations.