Correction: Jiménez-Ortigosa et al. Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins. J. Fungi 2021, 7, 120.

The authors wish to update the article title to "Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins" [...].

ERG11 Analysis among Clinical Isolates of Trichosporon asahii with Different Azole Susceptibility Profiles.

We analyzed a cohort of Trichosporon asahii strains with different MICs of fluconazole and voriconazole and evaluated the presence of ERG11 mutations. ERG11 mutation conferring an amino acid change was found and its resistance potential was evaluated by cloning into Saccharomyces cerevisiae susceptible host strain. Transformants were not resistant to either fluconazole nor voriconazole. Our results suggest that ERG11 variants exist among T. asahii isolates, but are not responsible for resistance phenotypes.

Multifactorial Role of Mitochondria in Echinocandin Tolerance Revealed by Transcriptome Analysis of Drug-Tolerant Cells.

Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target ß-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida sp., a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single-cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, of which the latter was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypotolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance. IMPORTANCE Echinocandin drugs are a first-line therapy to treat invasive candidiasis, which is a major source of morbidity and mortality worldwide. The opportunistic fungal pathogen Candida glabrata is a prominent bloodstream fungal pathogen, and it is notable for rapidly developing echinocandin-resistant strains associated with clinical failure. Echinocandin resistance is thought to emerge within a small echinocandin-tolerant subset of C. glabrata cells that are not killed by drug exposure, but mechanisms underlying echinocandin tolerance are still unknown. Here, we describe the unique transcriptional signature of echinocandin-tolerant cells and the results of follow-up analyses, which reveal a multifactorial role of mitochondria in C. glabrata echinocandin tolerance. In particular, although chemical inhibition of respiratory chain enzymes increased echinocandin tolerance, deletion of multiple mitochondrial components made C. glabrata cells hypotolerant to echinocandins. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal the involvement of mitochondria in echinocandin tolerance.

Antifungal Drug Susceptibility and Genetic Characterization of Fungi Recovered from COVID-19 Patients.

Fungal infections are common complications of respiratory viral infections and are associated with the increased need for intensive care and elevated mortality. Data regarding microbiological and molecular characteristics of such infections in COVID-19 patients are scarce. Here, we performed a comprehensive analysis, including species identification, antifungal susceptibility testing, molecular resistance determinants analysis, typing, and retrospective clinical data review, of fungal isolates recovered from 19 COVID-19 patients, who were hospitalized at the Hackensack University Medical Center (HUMC) in Hackensack, New Jersey, USA, in the initial phase of the pandemic from April-May 2020. In total, 17 Candida albicans, two C. parapsilosis, and two Aspergillus fumigatus were analyzed. All Candida spp. isolates were susceptible to micafungin and azole drugs (fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole). A. fumigatus isolates were susceptible to micafungin and all triazole drugs except fluconazole (intrinsic resistance). Multilocus sequence typing (MLST) of C. albicans isolates revealed 15 different sequence types (STs), which clustered below the clade-defining limit of p-distance < 0.04. Pulsed-field gel electrophoresis (PFGE) karyotyping revealed no chromosomal rearrangements in these isolates. A. fumigatus isolates were of different, non-related genotypes. We speculate that virus- and drug-induced immunosuppression (94.7% of the patients received corticosteroids), together with prolonged hospital stay (median duration of 29 days) and mechanical ventilation (median duration of 24 days) likely increased the susceptibility to secondary respiratory and bloodstream infections in the studied patient population. The presence of fungi in blood or respiratory tract fluid was a prognosticator for poor clinical outcome, which presented as an 89.5% 30-day mortality in our patient cohort.

Preliminary Structural Elucidation of ß-(1,3)-glucan Synthase from Candida glabrata Using Cryo-Electron Tomography.

Echinocandin drugs have become a front-line therapy against Candida spp. infections due to the increased incidence of infections by species with elevated azole resistance, such as Candida glabrata. Echinocandins target the fungal-specific enzyme ß-(1,3)-glucan synthase (GS), which is located in the plasma membrane and catalyzes the biosynthesis of ß-(1,3)-glucan, the major component of the fungal cell wall. However, resistance to echinocandin drugs, which results from hotspot mutations in the catalytic subunits of GS, is an emerging problem. Little structural information on GS is currently available because, thus far, the GS enzyme complex has resisted homogenous purification, limiting our understanding of GS as a major biosynthetic apparatus for cell wall assembly and an important therapeutic drug target. Here, by applying cryo-electron tomography (cryo-ET) and subtomogram analysis, we provide a preliminary structure of the putative C. glabrata GS complex as clusters of hexamers, each subunit with two notable cytosolic domains, the N-terminal and central catalytic domains. This study lays the foundation for structural and functional studies of this elusive protein complex, which will provide insight into fungal cell wall synthesis and the development of more efficacious antifungal therapeutics.

Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1.

Invasive infections caused by the opportunistic pathogen Candida glabrata are treated with echinocandin antifungals that target ß-1,3-glucan synthase, an enzyme critical for fungal cell wall biosynthesis. Echinocandin resistance develops upon mutation of genes (FKS1 or FKS2) that encode the glucan synthase catalytic subunits. We have analyzed cellular factors that influence echinocandin susceptibility and here describe effects of the post-transcriptional regulator Ssd1, which in S. cerevisiae, can bind cell wall related gene transcripts. The SSD1 homolog in C. glabrata was disrupted in isogenic wild type and equivalent FKS1 and FKS2 mutant strains that demonstrate echinocandin resistance (MICs ˃ 0.5 µg/mL). A reversal of resistance (8- to 128-fold decrease in MICs) was observed in FKS1 mutants, but not in FKS2 mutants, following SSD1 deletion. Additionally, this phenotype was complemented upon expression of SSD1 from plasmid (pSSD1). All SSD1 disruptants displayed susceptibility to the calcineurin inhibitor FK506, similar to fks1∆. Decreases in relative gene expression ratios of FKS1 to FKS2 (2.6- to 4.5-fold) and in protein ratios of Fks1 to Fks2 (2.7- and 8.4-fold) were observed in FKS mutants upon SSD1 disruption. Additionally, a complementary increase in protein ratio was observed in the pSSD1 expressing strain. Overall, we describe a cellular factor that influences Fks1-specific mediated resistance and demonstrates further differential regulation of FKS1 and FKS2 in C. glabrata.

Breakthrough Bloodstream Infections Caused by Echinocandin-Resistant Candida tropicalis: An Emerging Threat to Immunocompromised Patients with Hematological Malignancies.

BACKGROUND: Candida tropicalis is a virulent fungal pathogen for which echinocandins are the primary therapy. Emergence of resistance to echinocandins of C. tropicalis carries potentially ominous therapeutic implications. METHODS: We describe herein two patients with breakthrough C. tropicalis fungemia during echinocandin therapy, characterize their molecular mechanism of resistance, and systematically review 13 previously reported cases of echinocandin-resistant C. tropicalis bloodstream infections (BSIs) and other diseases. RESULTS: Among these 15 patients with echinocandin-resistant C. tropicalis infections, the median age was 61 years (ages 28-84 years) and 13 (86%) were immunocompromised. Thirteen (86%) of all patients had a history of pervious or concurrent exposure to echinocandins. Isolates of C. tropicalis from 11 cases, including the two index cases, underwent DNA sequencing of the FKS1 gene for mutations known to confer echinocandin resistance. The amino acid substitution Ser654Pro was shown in four cases, while other FKS1 mutations encoded Ser80S/Pro, Phe641Leu, Phe641Ser, Ser80S/Pro substitutions. These mutational events were not associated with collateral increases in minimum inhibitory concentrations to antifungal triazoles and amphotericin B. Overall mortality in patients with echinocandin-resistant C. tropicalis infections was 40%. Among those six patients who died, two received monotherapy with voriconazole, one was treated with fluconazole, one remained on caspofungin, and two were switched to liposomal amphotericin B. Nine patients (60%) survived after being treated with an antifungal agent other than an echinocandin. CONCLUSIONS: Emergence of resistance to echinocandins by C. tropicalis, occurs during antifungal therapy, is associated with high mortality, is mediated by a diverse range of FKS1 mutations, retains in vitro susceptibility to triazoles and amphotericin B, and constitutes an emerging threat to patients with hematological malignancies.

Stress-Induced Changes in the Lipid Microenvironment of ß-(1,3)-d-Glucan Synthase Cause Clinically Important Echinocandin Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in ß-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain fks1 mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins.IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.

Rapid Detection of ERG11-Associated Azole Resistance and FKS-Associated Echinocandin Resistance in Candida auris.

Candida auris is an emerging multidrug-resistant yeast that can cause serious invasive infections. The accurate and rapid assessment of antifungal resistance is important for effective patient management. A novel and highly accurate diagnostic platform was established for the rapid identification of ERG11 mutations conferring azole resistance and FKS1 mutations associated with echinocandin resistance in C. auris Using allele-specific molecular beacons and DNA melting curve analysis following asymmetric PCR, a duplex ERG11 assay and a simplex FKS1 HS1 assay were developed to identify the most prominent resistance-associated mutations (Y132F and K143R in ERG11; S639F in FKS1 HS1) within 2 h. Assays were validated by testing a panel of 94 C. auris clinical isolates in a blind manner. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify azole- and/or echinocandin-resistant C. auris, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

Limited ERG11 Mutations Identified in Isolates of Candida auris Directly Contribute to Reduced Azole Susceptibility.

Multiple Erg11 amino acid substitutions were identified in clinical isolates of Candida auris originating from India and Colombia. Elevated azole MICs were detected in Saccharomyces cerevisiae upon heterologous expression of C. aurisERG11 alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to other Candida species, specific C. aurisERG11 mutations resulted directly in reduced azole susceptibility.

Significantly Improved Pharmacokinetics Enhances In Vivo Efficacy of APX001 against Echinocandin- and Multidrug-Resistant Candida Isolates in a Mouse Model of Invasive Candidiasis.

APX001 is a first-in-class, intravenous and orally available, broad-spectrum antifungal agent in clinical development for the treatment of life-threatening invasive fungal infections. The half-life of APX001A, the active moiety of APX001, is significantly shorter in mice than in humans (1.4 to 2.75 h in mice versus 2 to 2.5 days in humans), making the exploration of efficacy in mouse models difficult. After pretreatment with 1-aminobenzotriazole (ABT), a nonspecific cytochrome P450 inhibitor, greatly increased plasma APX001A exposure was observed in mice of different strains and of both genders. As a consequence, 26 mg/kg APX001 plus ABT sterilized kidneys in mice infected with Candida albicans, while APX001 alone at the same dose resulted in a modest burden reduction of only 0.2 log10 CFU/g, relative to the vehicle control. In the presence of ABT, 2 days of once-daily dosing with APX001 at 26 mg/kg also demonstrated significant in vivo efficacy in the treatment of Candida glabrata infections in mice. Potent kidney burden reduction was achieved in mice infected with susceptible, echinocandin-resistant, or multidrug-resistant strains. In contrast, the standard of care (micafungin) was ineffective in treating infections caused by the resistant C. glabrata isolates.

Comparative study of Candida spp. isolates: Identification and echinocandin susceptibility in isolates obtained from blood cultures in 15 hospitals in Medellín, Colombia.

OBJECTIVES: Invasive candidiasis has a high impact on morbidity and mortality in hospitalised patients. Accurate and timely methods for identification of Candida spp. and determination of echinocandin susceptibility have become a priority for clinical microbiology laboratories. METHODS: This study was performed to compare matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) identification with sequencing of the D1/D2 region of the rRNA gene complex 28 subunit in 147 Candida spp. isolates obtained from patients with candidaemia. Antimicrobial susceptibility testing was performed by broth microdilution (BMD) and Etest. Sequencing of the FKS1 and FKS2 genes was performed. RESULTS: The most common species isolated were Candida albicans (40.8%), followed by Candida parapsilosis (23.1%) and Candida tropicalis (17.0%). Overall agreement between the results of identification by MALDI-TOF/MS and molecular identification was 99.3%. Anidulafungin and caspofungin susceptibility by the BMD method was 98.0% and 88.4%, respectively. Susceptibility to anidulafungin and caspofungin by Etest was 93.9% and 98.6%, respectively. Categorical agreement between Etest and BMD was 91.8% for anidulafungin and 89.8% for caspofungin, with lower agreements in C. parapsilosis for anidulafungin (76.5%) and C. glabrata for caspofungin (40.0%). No mutations related to resistance were found in the FKS genes, although 54 isolates presented synonymous polymorphisms in the hotspots sequenced. CONCLUSIONS: MALDI-TOF/MS is a good alternative for routine identification of Candida spp. isolates. DNA sequencing of the FKS genes suggested that the isolates analysed were susceptible to echinocandins; alternatively, unknown resistance mechanisms or limitations related to antifungal susceptibility tests may explain the resistance found in a few isolates.

Emergence of Echinocandin Resistance Due to a Point Mutation in the fks1 Gene of Aspergillus fumigatus in a Patient with Chronic Pulmonary Aspergillosis.

We have identified the first case of an fks1 hot spot 1 point mutation causing echinocandin resistance in a clinical Aspergillus fumigatus isolate recovered from a chronic pulmonary aspergillosis patient with an aspergilloma who first failed azole and polyene therapy and subsequently failed micafungin treatment.

De Novo Acquisition of Resistance to SCY-078 in Candida glabrata Involves FKS Mutations That both Overlap and Are Distinct from Those Conferring Echinocandin Resistance.

SCY-078 is an orally active antifungal whose target is the ß-(1,3)-d-glucan synthase (GS). We evaluated the spontaneous emergence of SCY-078-resistant Candida glabrata isolates following drug exposure in vitro Resistant isolates were analyzed using broth microdilution methodology and FKS sequencing. The kinetic inhibition parameter IC50 (50% inhibitory concentration) was also determined from GS complexes. The spectrum of resistance mutations found suggested a partially overlapping but independent binding site for SCY-078 relative to echinocandins on GS.

Changes in In Vitro Susceptibility Patterns of Aspergillus to Triazoles and Correlation With Aspergillosis Outcome in a Tertiary Care Cancer Center, 1999-2015.

BACKGROUND: Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern. METHODS: We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999-2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003-2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality. RESULTS: Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5-173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9-48.5; P = .006) were associated with azole resistance. CONCLUSIONS: Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort.

Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling.

Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidy-independent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

Genetic Drivers of Multidrug Resistance in Candida glabrata.

Both the incidence of invasive fungal infections and rates of multidrug resistance associated with fungal pathogen Candida glabrata have increased in recent years. In this perspective, we will discuss the mechanisms underlying the capacity of C. glabrata to rapidly develop resistance to multiple drug classes, including triazoles and echinocandins. We will focus on the extensive genetic diversity among clinical isolates of C. glabrata, which likely enables this yeast to survive multiple stressors, such as immune pressure and antifungal exposure. In particular, over half of C. glabrata clinical strains collected from U.S. and non-U.S. sites have mutations in the DNA mismatch repair gene MSH2, leading to a mutator phenotype and increased frequencies of drug-resistant mutants in vitro. Furthermore, recent studies and data presented here document extensive chromosomal rearrangements among C. glabrata strains, resulting in a large number of distinct karyotypes within a single species. By analyzing clonal, serial isolates derived from individual patients treated with antifungal drugs, we were able to document chromosomal changes occurring in C. glabrata in vivo during the course of antifungal treatment. Interestingly, we also show that both MSH2 genotypes and chromosomal patterns cluster consistently into specific strain types, indicating that C. glabrata has a complex population structure where genomic variants arise, perhaps during the process of adaptation to environmental changes, and persist over time.

CD101: a novel long-acting echinocandin.

CD101 is a novel echinocandin drug being developed to treat severe fungal infections including invasive candidiasis. We have performed a series of studies to evaluate the antifungal properties of CD101 against both echinocandin-susceptible and -resistant Candida strains. Antifungal susceptibility testing performed on a collection of 95 Candida strains including 30 caspofungin-resistant isolates containing fks mutations demonstrated comparable antifungal potency of CD101 relative to micafungin (MCF) across different Candida species. Comparable kinetic inhibition of glucan synthase activity was also observed for CD101 and MCF on both wild-type (WT) and resistant fks mutant Candida strains. Similarly, both drugs yielded nearly identical values for a mutant prevention concentration. In a murine model of invasive candidiasis, CD101 displayed better or at least comparable efficacy relative to MCF in treating WT or fks mutant Candida albicans. An exceptional long-lived pharmacokinetic profile was observed in mice following a single dose of CD101. Collectively, CD101 has great potential not only in treating invasive Candida infections but also in preventing emergence of resistance to currently approved echinocandin drugs.

Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance.

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.