Echinocandin persistence directly impacts the evolution of resistance and survival of the pathogenic fungus Candida glabrata

Abstract

Recent epidemiological studies documented an alarming increase in the prevalence of echinocandin-resistant (ECR) Candida glabrata blood isolates. ECR isolates are known to arise from a minor subpopulation of a clonal population, termed echinocandin persisters. Although it is believed that isolates with a higher echinocandin persistence (ECP) are more likely to develop ECR, the implication of ECP needs to be better understood. Moreover, replacing laborious and time-consuming traditional approaches to determine ECP levels with rapid, convenient, and reliable tools is imperative to advance our understanding of this emerging concept in clinical practice. Herein, using extensive ex vivo and in vivo systemic infection models, we showed that high ECP isolates are less effectively cleared by micafungin treatment and exclusively give rise to ECR colonies. Additionally, we developed a flow cytometry-based tool that takes advantage of a SYTOX-based assay for the stratification of ECP levels. Once challenged with various collections of echinocandin-susceptible blood isolates, our assay reliably differentiated ECP levels in vitro and predicted ECP levels in real time under ex vivo and in vivo conditions when compared to traditional methods relying on colony-forming unit counting. Given the high and low ECP predictive values of 92.3% and 82.3%, respectively, our assay showed a high agreement with traditional approach. Collectively, our study supports the concept of ECP level determination in clinical settings and provides a robust tool scalable for high-throughput settings. Application of this tool facilitates the interrogation of mutant and drug libraries to further our understanding of persister biology and designing anti-persister therapeutics.

Importance: Candida glabrata is a prevalent fungal pathogen able to replicate inside macrophages and rapidly develop resistance against frontline antifungal echinocandins. Multiple studies have shown that echinocandin resistance is fueled by the survival of a small subpopulation of susceptible cells surviving lethal concentrations of echinocandins. Importantly, bacterial pathogens that exhibit high antibiotic persistence also impose a high burden and generate more antibiotic-resistant colonies. Nonetheless, the implications of echinocandin persistence (ECP) among the clinical isolates of C. glabrata have not been defined. Additionally, ECP level determination relies on a laborious and time-consuming method, which is prone to high variation. By exploiting in vivo systemic infection and ex vivo models, we showed that C. glabrata isolates with a higher ECP are associated with a higher burden and more likely develop echinocandin resistance upon micafungin treatment. Additionally, we developed an assay that reliably determines ECP levels in real time. Therefore, our study identified C. glabrata isolates displaying high ECP levels as important entities and provided a reliable and convenient tool for measuring echinocandin persistence, which is extendable to other fungal and bacterial pathogens.

Keywords: Candida glabrata; echinocandin; ex vivo; in vivo; persistence; tolerance.

Fig 1

High-persister C. glabrata isolates exhibited increased micafungin tolerance both in vitro and ex vivo (21 and 25 as high persisters, 36 and 44 as unstable high persisters, whereas CBS138 and 35 were noted as low persisters). Overnight-grown C. glabrata isolates were incubated in cRPMI medium for 2 h, followed by exposure to micafungin (0.125 µg/mL), and survival was evaluated at different timepoints during the course of 24 h of exposure (A). The in vitro killing dynamics of various echinocandin-susceptible C. glabrata isolates treated with micafungin (0.125 µg/mL) revealed variation in echinocandin persistence (ECP) (B). Individual comparisons between CBS138 (a low persister) and other isolates and pertinent statistical analysis are presented in Fig. S1. The micafungin tolerance of C. glabrata isolates with varying ECP levels was equal after 2 h of internalization by macrophages (denoted as M), whereas the same isolates actively growing in cRPMI show significantly lower ECP levels (denoted as P) (C). Mature human primary macrophages were obtained from the primary human monocytes of three healthy donors. After maturation by incubation in macrophage colony stimulating factor, primary human macrophages were infected with the various C. glabrata isolates. The macrophages were extensively washed 2 h post infection and treated with fresh cRPMI with or without micafungin (0.125 µg/mL), after which the killing dynamics were monitored for 5 days. The burden was calculated by normalizing the CFU of treated wells against the intracellular untreated control at 1 h post infection. The emergence of echinocandin-resistant colonies was determined by plating treated samples on agar plates containing micafungin (0.125 µg/mL), followed by FKS mutation characterization (D). Macrophages infected with high persister isolates generally had a higher burden than did their low-persister counterparts (E). Notably echinocandin resistance was only noted for two of the high-persister isolates (21 and 25), not low persisters. At each timepoint, at least three biological replicates were included in panels B and C, and panel D shows the macrophage data of at least three healthy donors. Two-tailed t-test was used for statistical analysis, and values ≤0.05 were considered to indicate statistical significance. Symbols *, **, and *** indicate P-values ≤0.05, <0.01, and <0.001, respectively.

Fig 2

Mice infected with a high-persister isolate (21) had a significantly higher burden and exclusively harbored echinocandin-resistant colonies after treatment with humanized dose of micafungin (5 mg/kg). Mice were grouped into two major groups (26 mice in total, 13 mice per group); infected with high (isolate 21) or low (CBS138) persister C. glabrata isolates treated (10 mice for each isolate, 3 mice for days 5, 3 mice for 10 days, or 4 mice for 15 days) or untreated (only three mice for 5 days per isolate) subgroups. Micafungin treatment (5 mg/kg) was initiated 1 day after infection and continued every other day throughout the end of the experiment. Mice were sacrificed at designated timepoints, and the kidneys, liver, and spleen were homogenized and plated on agar plates with or without micafungin to determine the echinocandin resistance rate and burden, respectively. The burden was defined by normalizing the CFU of treated mice against that of untreated mice (A). Mice infected with the high-persister (isolate 21) isolate had significantly higher burdens in the liver (B), kidney (C), and spleen (D). Two-tailed t-tests were used for statistical analysis; values ≤0.05 were considered to indicate statistical significance. Symbols *, **, and *** indicate P-values ≤0.05, <0.01, and <0.001, respectively.

Fig 3

The echinocandin persistence (ECP) level can be precisely determined by a SYTOX-based flow cytometry assay. High and low echinocandin persister isolates were exposed to a cidal concentration of micafungin (0.125 µg/mL), stained with SYTOX, and subjected to fluorescent activated cell sorting. A total of 20,000 SYTOX− and SYTOX⁺ CFUs were collected and plated to determine the viability at each timepoint (A). The viability of SYTOX⁺ plants was significantly lower than that of SYTOX⁻ counterparts, and the viability dramatically decreased at a later timepoint. Moreover, the SYTOX⁻ events collected from high persisters had significantly higher viability compared to the lower persister ones (B). The gating strategy used to differentiate the SYTOX⁻ fraction of C. glabrata isolates (C). The schematic workflow for distinguishing the ECP level using SYTOX-based flow cytometry (D). The ECP level was more discriminatory when the incubation with the drug was carried out in MOPS-treated RPMI and cRPMI (E). Subjecting the initial collection of C. glabrata isolates to micafungin in cRPMI supplemented with micafungin (0.125 µg/mL) readily differentiated the high and low micafungin persisters (F). Two-tailed t-test was used for statistical analysis, and values ≤0.05 were considered to indicate statistical significance. Symbols *, **, and *** indicate P-values ≤0.05, <0.01, and <0.001, respectively.

Fig 4

The SYTOX-based assay better predicts micafungin tolerance ex vivo and in vivo. A collection of echinocandin-susceptible (n = 31) and echinocandin-resistant isolates harboring various FKS mutations were subjected to micafungin (1 µg/mL) for 24 h, and their ECP levels were determined by our assay (21 and CBS138 were used as high- and low-persister control isolates, respectively). After setting a threshold, high and low persisters were readily differentiated. Five isolates were high persisters (A). The time-kill curve of Austrian high persisters resolutely distinguished high (3, 10, 11, 12, and 14) and low persisters (18 and 20) (B), whereas extensive CFU counting showed that some of the high persisters lost their tolerance at later timepoints and, therefore, grouped with low persisters (C). All the experiments included at least three biological replicates. All the high persisters (3, 10, 11, 12, and 14) and two randomly selected low persisters (18 and 20) were subjected to ex vivo burden measurements following micafungin treatment at 24 and 48 h. Consistent with the time-kill curve of SYTOX-based assay, all the high persisters had a significantly higher intracellular burden (D) (Mann-Whitney U test). Mice were grouped into two major groups (36 mice in total, 9 mice per group) and infected with high- or low-persister C. glabrata isolates, and each group had treated (three mice only for 5 days and three mice for 10 days) or untreated (three mice for only 5 days per isolate) subgroups. Like in the ex vivo assay, all the high persisters defined by our SYTOX-based assay, including the unstable persisters defined by CFU counting, were bona fide high persisters in the context of the systemic mouse infection model. High Austrian persisters generally had a higher burden in all organs tested, especially at later timepoints (E and F) (for all in vivo analyses, analysis of variance was performed, except for day 5 liver, which was carried out with the Kruskal-Wallis test). Values ≤0.05 were considered to indicate deemed significant. Symbols *, **, and *** indicate P-values ≤0.05, <0.01, and <0.001, respectively.

Fig 5

The SYTOX-based assay and 3-h CFU count could reliably predict the in vivo burden in different organs. In vitro (24-h SYTOX^Neg fraction and 3-h CFU counting) and in vivo burdens of the HP and LP isolates were used to determine the correlation and regression. Both SYTOX-based assay (A) and 3-h CFU counting (B) were correlated with in vivo burden in all organs tested. Pearson coefficient correlation and simple regression analysis were used. Two-tailed t-tests were used for statistical analysis, and values ≤0.05 were considered to indicate statistical significance. The echinocandin persistence of 30 randomly selected C. glabrata isolates fully susceptible to all the antifungals was determined using SYTOX-based and 3-h CFU counting assays. Isolates displaying survival ≥1% at 3 h following micafungin exposure (1 µg/mL) (C) and SYTOX^Neg fractions ≥28% (D) were considered HPs. Three independent biological replicates were used for each isolate.