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We have been monitoring the antifungal resistance in Candida parapsilosis isolates collected from inpatients at Madrid metropolitan area hospitals for the last 3 years. The study aimed to elucidate the presence of fluconazole-resistant C. parapsilosis genotypes in Madrid. From January 2019 to December 2021, a total of 354 C. parapsilosis isolates ( n  = 346 patients) from blood (76.6%) or intraabdominal samples were collected and genotyped using species-specific microsatellite markers. Antifungal susceptibilities to amphotericin B, the triazoles, micafungin, anidulafungin, and ibrexafungerp were performed according to EUCAST E.Def 7.3.2; the ERG11 gene was sequenced in fluconazole-resistant isolates. A total of 13.6% ( n  = 48/354) isolates (one per patient) were found to be resistant to fluconazole and non-wild-type to voriconazole but fully susceptible to ibrexafungerp. Resistant isolates were mostly sourced from blood ( n  = 45/48, 93.8%) and were detected in five hospitals. Two hospitals accounted for a high proportion of resistant isolates ( n  = 41/48). Resistant isolates harbored either the Y132F ERG11p amino acid substitution ( n  = 43) or the G458S substitution ( n  = 5). Isolates harboring the Y132F substitution clustered into a clonal complex involving three genotypes (one genotype accounted for n  = 39/43 isolates) that were found in four hospitals. Isolates harboring the G458S substitution clustered into another genotype found in a fifth hospital. C. parapsilosis genotypes demonstrating resistance to fluconazole have been spreading across hospitals in Madrid, Spain. Over the last 3 years, the frequency of isolation of such isolates and the number of hospitals affected is on the rise.

To identify unrecognized niches of resistant Candida isolates and compartmentalization, we retrospectively studied the antifungal susceptibility of 1,103 Candida spp. isolates from blood cultures, nonblood sterile samples, and nonsterile samples. Antifungal susceptibility was assessed by EUCAST E.Def 7.3.2; sequencing and genotyping of the fks1-2 and erg11 genes were carried out for non-wild-type isolates. Resistance compartmentalization (presence of resistant and susceptible isogenic isolates in different anatomical sites of a given patient) was studied. Clinical charts of patients carrying non-wild-type isolates were reviewed. Most isolates (63%) were Candida albicans, regardless the clinical source; Candida glabrata (27%) was the second most frequently found species in abdominal cavity samples. Fluconazole and echinocandin resistance rates were 1.5 and 1.3%, respectively, and were highest in C. glabrata. We found 22 genotypes among non-wild-type isolates, none of them widespread across the hospital. Fluconazole/echinocandin resistance rates of isolates from the abdominal cavity (3.2%/3.2%) tended to be higher than those from blood cultures (0.7%/1.3%). Overall, 15 patients with different forms of candidiasis were infected by resistant isolates, 80% of whom had received antifungals before or at the time of isolate collection; resistance compartmentalization was found in six patients, mainly due to C. glabrata. The highest antifungal resistance rate was detected in isolates from the abdominal cavity, mostly C. glabrata. Resistance was not caused by the spread of resistant clones but because of antifungal treatment. Resistance compartmentalization illustrates how resistance might be overlooked if susceptibility testing is restricted to bloodstream isolates.

We conducted an updated analysis on yeast isolates causing fungemia in patients admitted to a tertiary hospital in Madrid, Spain, over a 13-year period. We studied 896 isolates associated with 872 episodes of fungemia in 857 hospitalized patients between January 2007 and December 2019. Antifungal susceptibility was assessed by EUCAST EDef 7.3.2. Mutations conferring azole and echinocandin resistance were further studied, and genotyping of resistant clones was performed with species-specific microsatellite markers. Candida albicans (45.8%) was the most frequently identified species, followed by the Candida parapsilosis complex (26.4%), Candida glabrata (12.3%), Candida tropicalis (7.3%), Candida krusei (2.3%), other Candida spp. (3.1%), and non- Candida yeasts (2.8%). The rate of fluconazole resistance in Candida spp. was 4.7%, ranging from 0% ( C. parapsilosis ) to 9.1% ( C. glabrata ). The overall rate of echinocandin resistance was 3.1%. Resistance was highly influenced by the presence of intrinsically resistant species. Although the number of isolates between 2007 and 2013 was almost 2-fold higher than that in the period from 2014 to 2019 (566 versus 330), fluconazole resistance in Candida spp. was greater in the second period (3.5% versus 6.8%; P <  0.05), while overall resistance to echinocandins remained stable (3.5% versus 2.4%; P >  0.05). Resistant clones were collected from different wards and/or time points, suggesting that there were no epidemiological links. The number of fungemia episodes has been decreasing over the last 13 years, with a slight increase in the rate of fluconazole resistance and stable echinocandin resistance. Antifungal resistance is not the cause of the spread of resistant clones., (Copyright © 2021 American Society for Microbiology.)

Infections caused by the coexistence of Candida glabrata echinocandin-resistant and echinocandin-susceptible cells may be possible, and the detection of FKS mutants when the proportions of FKS mutants are underrepresented poses a problem. We assessed the role of EUCAST and methods directly performed on positive blood cultures-Etest (ET DIR ) and anidulafungin-containing agar plate assays-for detecting resistance in C. glabrata isolates containing different amounts of echinocandin-susceptible and -resistant Candida glabrata isolates. We studied 10 pairs of C. glabrata isolates involving parental echinocandin-susceptible isolates and isogenic echinocandin-resistant FKS mutant isolates. Three inocula per pair (1 ×   10 3 to 5   ×   10 3 , 1 ×   10 2 to 5   ×   10 2 , and 10 to 50 CFU/ml) spanning suspensions with different amounts of susceptible/resistant isolates (9/1, 5/5, and 1/9 proportions for each the three inocula) were prepared. The suspensions were spiked in Bactec bottles and incubated until they were positive, and the three methods were compared. The EUCAST method showed echinocandin resistance when the bottles were spiked with susceptible/resistant isolates at 5/5 and 1/9 proportions; the results for the suspensions with a 9/1 proportion of susceptible/resistant isolates were susceptible for three pairs. We observed with the ET DIR resistance to both echinocandins in all pairs (resistance to micafungin and anidulafungin; MICs, ≥0.064   mg/liter and ≥0.125   mg/liter, respectively) and a double ring of growth inhibition in two pairs. The anidulafungin-containing plates showed fungal growth in the 90 spiked blood cultures at 48 h. Testing of echinocandin susceptibility with the ET DIR directly on the positive blood culture bottles is a reliable and rapid method to detect echinocandin resistance in C. glabrata On the other hand, resistance can be missed with the EUCAST method when resistant isolates are underrepresented., (Copyright © 2019 American Society for Microbiology.)

The high rates of antifungal resistance in Candida glabrata may be facilitated by the presence of alterations in the MSH2 gene. We aimed to study the sequence of the MSH2 gene in 124 invasive C. glabrata isolates causing incident episodes of candidemia ( n = 81), subsequent candidemia episodes ( n = 9), endocarditis ( n = 2), and in vitro -generated echinocandin-resistant isolates ( n = 32) and assessed its relationship with genotypes, acquisition of antifungal resistance in vivo and in vitro , and patient prognosis. The MSH2 gene was sequenced, and isolates were genotyped using six microsatellite markers and multilocus sequence typing (MLST) based on six housekeeping genes. According to EUCAST, isolates causing candidemia ( n = 90) were echinocandin susceptible, and four of them were fluconazole resistant (MIC ≥64 mg/liter). One isolate obtained from a heart valve was resistant to micafungin and anidulafungin (MICs, 2 mg/liter and 1 mg/liter, respectively). MSH2 gene mutations were present in 44.4% of the incident isolates, the most common being V239L. The presence of MSH2 mutations was not correlated with in vitro or in vivo antifungal resistance. Microsatellite and MLST revealed 27 genotypes and 17 sequence types, respectively. Fluconazole-resistant isolates were unrelated. Most MSH2 mutations were found in cluster isolates; conversely, some mutations were found in more than one genotype. No clinical differences, including previous antifungal use, were found between patients infected by wild-type MSH2 gene isolates and isolates with any point mutation. The presence of MSH2 gene mutations in C. glabrata isolates causing candidemia is not correlated with specific genotypes, the promotion of antifungal resistance, or the clinical outcome., (Copyright © 2018 American Society for Microbiology.)

We examined the rapid evaluation of susceptibility to echinocandins in Candida spp. using the Etest performed directly on positive blood cultures and anidulafungin-containing agar plates. We prospectively collected 80 positive blood cultures (Bactec-FX system, Becton-Dickinson, Cockeysville, MD, USA) with echinocandin-susceptible Candida spp. ( n = 60) and echinocandin-intermediate Candida parapsilosis ( n = 20) from patients with candidemia. Additionally, blood culture bottles of nonfungemic/bacteremic patients were spiked with 35 echinocandin-resistant Candida species isolates. A total of 2 to 4 drops of medium from each bottle were stroked directly onto both RPMI 1640 agar plates with micafungin and anidulafungin Etest strips (ET DIR ) and Sabouraud agar plates containing 2 mg/liter of anidulafungin. The isolates were tested according to the EUCAST method and Etest standard (ET SD ). Essential and categorical agreement between the methods was calculated. The essential agreement and categorical agreement between the EUCAST method and ET DIR and ET SD were both >97.4%. The essential agreement between ET DIR and the EUCAST method for both echinocandins was >97%. The categorical agreement between the FKS sequence and ET DIR was 97.4%. The ET DIR MICs of anidulafungin and micafungin (≥0.19 mg/liter and ≥0.064 mg/liter, respectively) effectively separated all susceptible FKS wild-type isolates from the resistant FKS mutant isolates. The categorical agreement (62.6%) between the EUCAST method and growth on anidulafungin-containing plates was poor, with the best agreement observed for Candida glabrata (94.2%). When performed directly on positive blood cultures from patients with candidemia, the Etest with micafungin and anidulafungin is a reliable procedure for the rapid testing of susceptibility to echinocandins in Candida species isolates., (Copyright © 2018 American Society for Microbiology.)

We report the mutant prevention concentration (MPC) and mutant selection window (MSW) for micafungin and anidulafungin administered to treat Candida glabrata We also determine the mutation frequency. We studied 20 echinocandin-susceptible, fluconazole-intermediate, and FKS wild-type C. glabrata isolates. Adjusted inocula were stroked directly onto Sabouraud agar plates containing different concentrations of micafungin or anidulafungin and visually inspected daily for up to 5 days of incubation. Individual colonies growing on the plates containing echinocandins at 1 mg/liter were selected for antifungal susceptibility testing. The FKS genes of the resulting individual phenotypically resistant colonies were sequenced, and the MPC, MSW, and mutation frequency were determined. Biofilm was quantified, and the growth kinetics and virulence ( Galleria mellonella model) of the resulting individual FKS mutant colonies were studied. For micafungin and anidulafungin, we found similar results for the MPC (0.06 to 2 mg/liter and 0.25 to 2 mg/liter, respectively), MSW (0.015 to 2 mg/liter for both echinocandins), and mutation frequency (3.7 × 10 -8 and 2.8 × 10 -8 , respectively). A total of 12 isolates were able to grow at 1 mg/liter on echinocandin-containing plates, yielding a total of 32 phenotypically resistant colonies; however, FKS2 mutations (ΔF658, S663P, W715L, and E655A) were observed only in 21 colonies. We did not find differences in biofilm formation, the kinetic parameters studied, or the median survival of larvae infected by wild-type isolates and the resulting individual FKS2 mutant colonies. Echinocandin concentrations lower than 2 mg/liter can lead to selection of resistance mutations in C. glabrata isolates in vitro ., (Copyright © 2018 American Society for Microbiology.)

The objectives of our study were to describe the characteristics of patients with Candida guilliermondii candidemia and to perform an in-depth microbiological characterization of isolates and compare them with those of patients with C. albicans candidemia. We described the risk factors and outcomes of 22 patients with candidemia caused by the C. guilliermondii complex. Incident isolates were identified using molecular techniques, and susceptibility to fluconazole, anidulafungin, and micafungin was studied. Biofilm formation was measured using the crystal violet assay (biomass production) and the XTT reduction assay (metabolic activity), and virulence was studied using the Galleria mellonella model. Biofilm formation was compared with that observed for C. albicans The main conditions predisposing to infection were malignancy (68%), immunosuppressive therapy (59%), and neutropenia (18%). Clinical presentation of candidemia was less severe in patients infected by the C. guilliermondii complex than in patients infected by C. albicans , and 30-day mortality was lower in C. guilliermondii patients (13.6% versus 33.9%, respectively; P = 0.049). Isolates were identified as C. guilliermondii sensu stricto ( n = 17) and Candida fermentati ( n = 5). The isolates produced biofilms with low metabolic activity and moderate biomass. The G. mellonella model showed that C. guilliermondii was less virulent than C. albicans (mean of 6 days versus 1 day of survival, respectively; P < 0.001). Patients with candidemia caused by the C. guilliermondii complex had severe and debilitating underlying conditions. Overall, the isolates showed diminished susceptibility to fluconazole and echinocandins, although poor biofilm formation and the low virulence were associated with a favorable outcome., (Copyright © 2017 American Society for Microbiology.)

We report data on the frequency of the paradoxical effect of echinocandins against Candida spp. (n = 602 incident isolates) using the EUCAST definitive document EDef 7.2 procedure. The paradoxical effect for one or more echinocandins was observed in 16% of the isolates. However, differences between species were found, and the paradoxical effect was more common in Candida tropicalis (P < 0.001). Caspofungin was the drug in which the paradoxical effect was most common, followed by anidulafungin and micafungin (P < 0.001)., (Copyright © 2016 American Society for Microbiology.)