Azole uptake in Candida auris is strongly correlated with drug resistance

Analyses of fluconazole uptake in clinical isolates of C. auris, with wide ranging drug resistance profiles, has revealed interesting differences within the species as well as major distinctions from other yeast species. We previously proposed that prevention of drug uptake is a potential mechanism of drug resistance and our C. auris data provide further support for this. We developed an assay using radio-labeled fluconazole to measure intracellular azole accumulation in fungal cells. The assay is performed under glucose-replete conditions to inhibit ATP-dependent efflux. A comparative study measuring fluconazole uptake in 63 C. auris isolates as well as a panel of other species such as C. albicans, S. cerevisiae, C. glabrata, C. krusie, C. lusitanea, C. tropicalis, and C. dublinienses revealed striking C. auris phenotypes that we have not seen in other fungal species. There is a strong correlation between fluconazole resistance and reduced drug uptake in C. auris. Fluconazole-resistant C. auris isolates had reduced levels of intracellular fluconazole accumulation compared to susceptible isolates. Drug-resistant C. auris isolates had the lowest drug accumulation of any of the yeast species tested. Fluconazole-susceptible C. auris isolates had dramatically increased fluconazole accumulation compared to the resistant isolates as well as when compared to other yeast species. Of the 63 C. auris isolates, 28 of 32 (∼88%) resistant isolates had extremely low fluconazole uptake, whereas 15 of 18 (∼83%) susceptible isolates had high fluconazole uptake. This association between reduced drug uptake and resistance could be a C. auris-distinctive mechanism of drug resistance.

Investigation of the MFS drug transporter family in Candida parapsilosis using CRISPR-Cas9

The function of specific transporters is a key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS) transporters. Some members of the drug/H (+) antiporter (DHA1) of the MFS superfamily have been shown to function as multidrug transporters. We targeted 16 genes belonging to five families that compose one branch of the DHA1 transporter group. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (3 members); TPO4, associated with polyamine transport (1 member); NAG3/4, associated with transport of N-acetyl glucosamine (2 members); TPO2/3, associated with polyamine transport (1 member); and TPO1/FLU1, possibly associated with fluconazole resistance (9 members). We used CRISPR-Ca9 based gene editing to explore the function of of the five families in C. parapsilosis. All 16 members were individually disrupted by introducing stop codons in the first third of the open reading frames (editing), or by deleting the whole gene. In addition, members of each family were disrupted together, including all 9 members of the TPO1/FLU1 family. CPAR2_603010, CPAR2_207540, and CPAR2_301760 all belonged to the MDR1 family. Editing CPAR2_603010 conferred sensitivity to fluconazole and voriconazole, though disrupting the other two genes had no effect. The azole sensitivity of the CPAR2_603010 edited strain was reverted by introducing the wild type sequence. Disrupting CPAR2_603010 or CPAR2_301760 individually did not affect sensitivity to 4-nitroquinoline 1-oxide. However, the double disruptant was sensitive. Disrupting CPAR2_300760, a member of the TPO1/FLU1 family, resulted in sensitivity to mycophenolic acid. Whole genome sequencing analysis of a strain in which all nine TPO1 genes were disrupted revealed that few off-target effects introduced by the CRISPR-Cas9 system, as few unexpected changes were found after eight rounds of transformation.

Precise editing using CRISPR-Cas9 to explore the contribution of clinically-derived mutations to antifungal resistance in the pathogenic yeast Candida parapsilosis

Introduction Candida parapsilosis is both a commensal/saprophytic yeast of the human skin and an opportunistic pathogen which can be responsible for life-threatening infections. The increasing reports of clonal outbreaks involving azole-resistant C. parapsilosis in the clinical setting is worrisome and urges for a better understanding of antifungal resistance in this species. Previous studies have identified mutations in key genes which can explain acquired fluconazole resistance. Reverse genetics approaches are now warranted to confirm their involvement and to determine whether they can affect other clinically-licensed antifungals. Here, we used a CRISPR-Cas9 technique to study the relative contributions of clinically-derived mutations to antifungal resistance and provide answers to these questions. Materials and Methods Six clinically-derived mutations were selected (ERG11Y132F, ERG11K143R,ERG11R398I, TAC1G650E, MRR1G583R, ERG3G111R) to be engineered in two C. parapsilosis fluconazole-susceptible backgrounds (ATCC22019, STZ5) using a previously described CRISPR-Cas9 method. In vitro susceptibility of the transformants to fluconazole, voriconazole, posaconazole, isavuconazole and micafungin was determined by Etest®. Results/Discussion The impact on fluconazole susceptibility was highly variable depending on the residue/gene involved, but roughly similar between the two genetic backgrounds. All but two(ERG11R398I, ERG3G111R) conferred fluconazole resistance, though the highest MIC increase was observed for MRR1G583R (≥650 fold). As expected in a diploid species, we noted an impact of allelic dosage. Some kind of cross-resistance to the other azoles was noted from some mutations, although the impact was lower for posaconazole and isavuconazole, except for MRR1G583R which led to multi-azole resistance. Finally, ERG3G111R increased tolerance to both azoles and echinocandins.

Epidemiology of Candidemia in Children over 7 Years in a Medical Center in Turkey

In our study, the changing epidemiology of Candida species in candidemia in children was evaluated. The dominance of Candida parapsilosis species in the changing epidemiology was remarkable. We found that fluconazole resistance was high in both parapsilosis and nonparapsilosis groups. Updating local epidemiologic data at certain intervals in candidemia cases is important in determining both the changing epidemiology and empirical antifungal agents.

The natural occurring Y129F polymorphism in Rhizopus oryzae (R. arrhizus) Cyp51Ap accounts for its intrinsic voriconazole resistance

Rhizopus oryzae (heterotypic synonym: R. arrhizus) intrinsic voriconazole and fluconazole resistance has been linked to its CYP51A gene. However, the amino acid residues involved in this phenotype have not yet been established. A comparison between R. oryzae and Aspergillus fumigatus Cyp51Ap sequences showed differences in several amino acid residues. Some of them were already linked with voriconazole resistance in A. fumigatus. The objective of this work was to analyze the role of two natural polymorphisms in the intrinsic voriconazole resistance phenotype of R. oryzae (Y129F and T290A, equivalent to Y121F and T289A seen in triazole-resistant A. fumigatus). We have generated A. fumigatus chimeric strains harboring different R. oryzae CYP51A genes (wild-type and mutants). These mutant R. oryzae CYP51A genes were designed to carry nucleotide changes that produce mutations at Cyp51Ap residues 129 and 290 (emulating the Cyp51Ap protein of azole susceptible A. fumigatus). Antifungal susceptibilities were evaluated for all the obtained mutants. The polymorphism T290A (alone or in combination with Y129F) had no impact on triazole MIC. On the other hand, a > 8-fold decrease in voriconazole MICs was observed in A. fumigatus chimeric strains harboring the RoCYP51Ap-F129Y. This phenotype supports the assumption that the naturally occurring polymorphism Y129F at R. oryzae Cyp51Ap is responsible for its voriconazole resistance phenotype. In addition, these chimeric mutants were posaconazole hypersusceptible. Thus, our experimental data demonstrate that the RoCYP51Ap-F129 residue strongly impacts VRC susceptibility and that it would be related with posaconazole-RoCYP51Ap interaction. Lay summary Rhizopus oryzae is intrinsically resistant to voriconazole, a commonly used antifungal agent. In this work, we analyze the role of two natural polymorphisms present in the target of azole drugs. We established that F129 residue is responsible of the intrinsic voriconazole resistance in this species.

Participation of the ABC Transporter CDR1 in Azole Resistance of Candida lusitaniae

Candida lusitaniae is an opportunistic pathogen in humans that causes infrequent but difficult-to-treat diseases. Antifungal drugs are used in the clinic to treat C. lusitaniae infections, however, this fungus can rapidly acquire antifungal resistance to all known antifungal drugs (multidrug resistance). C. lusitaniae acquires azole resistance by gain-of-function (GOF) mutations in the transcriptional regulator MRR1. MRR1 controls the expression of a major facilitator transporter (MFS7) that is important for fluconazole resistance. Here, we addressed the role of the ATP Binding Cassette (ABC) transporter CDR1 as additional mediator of azole resistance in C. lusitaniae. CDR1 expression in isolates with GOF MRR1 mutations was higher compared to wild types, which suggests that CDR1 is an additional (direct or indirect) target of MRR1. CDR1 deletion in the azole-resistant isolate P3 (V688G GOF) revealed that MICs of long-tailed azoles, itraconazole and posaconazole, were decreased compared to P3, which is consistent with the role of this ABC transporter in the efflux of these azoles. Fluconazole MIC was only decreased when CDR1 was deleted in the background of an mfs7Δ mutant from P3, which underpins the dominant role of MFS7 in the resistance of the short-tailed azole fluconazole. With R6G efflux readout as Cdr1 efflux capacity, our data showed that R6G efflux was increased in P3 compared to an azole-susceptible wild type parent, and diminished to background levels in mutant strains lacking CDR1. Milbemycin oxim A3, a known inhibitor of fungal ABC transporters, mimicked efflux phenotypes of cdr1Δ mutants. We therefore provided evidence that CDR1 is an additional mediator of azole resistance in C. lusitaniae, and that CDR1 regulation is dependent on MRR1 and associated GOF mutations.

Epidemiology of Candidemia in Kuwait: A Nationwide, Population-Based Study

The Candida species cause a majority of invasive fungal infections. In this article, we describe the nationwide epidemiology of candidemia in Kuwait in 2018. Yeast bloodstream isolates submitted from all major hospitals and identified by phenotypic MALDI-TOF MS and/or by molecular methods were studied. Susceptibility testing was performed by Etest. Out of 313 bloodstream yeasts, 239 Candida spp. isolates (excluding duplicate isolates) were obtained during 234 candidemic episodes among 223 patients. Mixed-species candidemia and re-infection occurred in 5 and 11 patients, respectively. C. albicans (n = 74), C. parapsilosis (n = 54), C. tropicalis (n = 35), C. auris (n = 33), C. glabrata (n = 32), other Candida spp. (n = 11), and other yeasts (n = 9) caused fungemia. Nearly 50% of patients were in intensive care units. Candida spp. isolates (except C. glabrata) were susceptible to caspofungin and 27% of C. auris were amphotericin B-resistant. Resistance to fluconazole was 100% in C. auris, 17% in C. parapsilosis, 12% in C. glabrata, and 1% in C. albicans. Mortality was 47% for other Candida/yeast infections. Nationwide candidemia incidence in 2018 was 5.29 cases/100,000 inhabitants. Changes in species spectrum, increasing fluconazole resistance in C. parapsilosis, and the emergence of C. auris as a major pathogen in Kuwait are noteworthy findings. The data could be of help in informing decisions regarding planning, in the allocation of resources, and in antimicrobial stewardship.

Genetic and Phenotypic Diversities in Experimental Populations of Diploid Inter-Lineage Hybrids in the Human Pathogenic Cryptococcus

To better understand the potential factors contributing to genome instability and phenotypic diversity, we conducted mutation accumulation (MA) experiments for 120 days for 7 diploid cryptococcal hybrids under fluconazole (10 MA lines each) and non-fluconazole conditions (10 MA lines each). The genomic DNA content, loss of heterozygosity (LOH) rate, growth ability, and fluconazole susceptibility were determined for all 140 evolved cultures. Compared to that of their ancestral clones, the evolved clones showed: (i) genomic DNA content changes ranging from ~22% less to ~27% more, and (ii) reduced, similar, and increased phenotypic values for each tested trait, with most evolved clones displaying increased growth at 40 °C and increased fluconazole resistance. Aside from the ancestral multi-locus genotypes (MLGs) and heterozygosity patterns (MHPs), 77 unique MLGs and 70 unique MPHs were identified among the 140 evolved cultures at day 120. The average LOH rates of the MA lines in the absence and presence of fluconazole were similar at 1.27 × 10−4 and 1.38 × 10−4 LOH events per MA line per mitotic division, respectively. While LOH rates varied among MA lines from different ancestors, there was no apparent correlation between the genetic divergence of the parental haploid genomes within ancestral clones and LOH rates. Together, our results suggest that hybrids between diverse lineages of the human pathogenic Cryptococcus can generate significant genotypic and phenotypic diversities during asexual reproduction.

Dynamics and Predictors of Mortality Due to Candidemia Caused by Different Candida Species: Comparison of Intensive Care Unit-Associated Candidemia (ICUAC) and Non-ICUAC

We investigated mortality and predictors of mortality due to intensive care unit-associated candidemia (ICUAC) versus non-ICUAC by Candida species. This study included all candidemia cases in 11 hospitals from 2017 to 2018 in South Korea. The all-cause mortality rates in all 370 patients with ICUAC were approximately twofold higher than those in all 437 patients with non-ICUAC at 7 days (2.3-fold, 31.1%/13.3%), 30 days (1.9-fold, 49.5%/25.4%), and 90 days (1.9-fold, 57.8%/30.9%). Significant species-specific associations with 7- and 30-day ICUAC-associated mortality were not observed. Multivariate analysis revealed that ICU admission was an independent predictor of Candida glabrata (OR, 2.07–2.48) and Candida parapsilosis-associated mortality (OR, 6.06–11.54). Fluconazole resistance was a predictor of C. glabrata-associated mortality (OR, 2.80–5.14). Lack (less than 3 days) of antifungal therapy was the strongest predictor of 7-day mortality due to ICUAC caused by Candida albicans (OR, 18.33), Candida tropicalis (OR, 10.52), and C. glabrata (OR, 21.30) compared with 30- and 90-day mortality (OR, 2.72–6.90). C. glabrata ICUAC had a stronger association with lack of antifungal therapy (55.2%) than ICUAC caused by other species (30.6–36.7%, all p < 0.05). Most predictors of mortality associated with ICUAC were distinct from those associated with non-ICUAC and were mediated by Candida species.