We assessed the in vitro susceptibility of five echinocandin-susceptible Candida glabrata isolates after exposure to micafungin. The direct exposure to plates at different micafungin concentrations resulted in the inhibition of growth at 0.062 g/ml. The progressive exposure was performed on plates using 0.031 g/ml of micafungin and sequential propagation on plates containing the next 2-fold concentration; the MICs of micafungin and anidulafungin increased sequentially, and all the isolates became echinocandin resistant, showing fks2 mutations.

We report the mutant prevention concentration (MPC) and mutant selection window (MSW) for micafungin and anidulafungin administered to treat We also determine the mutation frequency. We studied 20 echinocandin-susceptible, fluconazole-intermediate, and wild-type 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 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 ( model) of the resulting individual 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 and 2.8 × 10, 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, 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 mutant colonies. Echinocandin concentrations lower than 2 mg/liter can lead to selection of resistance mutations in isolates.

The high rates of antifungal resistance inCandida glabratamay be facilitated by the presence of alterations in theMSH2gene. We aimed to study the sequence of theMSH2gene in 124 invasiveC. glabrataisolates causing incident episodes of candidemia (n= 81), subsequent candidemia episodes (n= 9), endocarditis (n= 2), andin vitro-generated echinocandin-resistant isolates (n= 32) and assessed its relationship with genotypes, acquisition of antifungal resistancein vivoandin vitro, and patient prognosis. TheMSH2gene 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).MSH2gene mutations were present in 44.4% of the incident isolates, the most common being V239L. The presence ofMSH2mutations was not correlated within vitroorin vivoantifungal resistance. Microsatellite and MLST revealed 27 genotypes and 17 sequence types, respectively. Fluconazole-resistant isolates were unrelated. MostMSH2mutations 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-typeMSH2gene isolates and isolates with any point mutation. The presence ofMSH2gene mutations inC. glabrataisolates causing candidemia is not correlated with specific genotypes, the promotion of antifungal resistance, or the clinical outcome.

We aim to assess intra- and interspecies differences in the virulence of Candida spp. strains causing candidemia using the invertebrate Galleria mellonella model. We studied 739 Candida spp. isolates (C. albicans [n = 373], C. parapsilosis [n = 203], C. glabrata [n = 92], C. tropicalis [n = 53], and C. krusei [n = 18]) collected from patients with candidemia admitted to Gregorio Marañon Hospital (Madrid, Spain). Species-specific infecting inocula (yeast cells/larva) were adjusted (5 × 105 [C. albicans, and C. tropicalis], 2 × 106–5 × 106 [C. parapsilosis, C. glabrata, and C. krusei]) and used to infect 10 larvae per isolate; percentage of survival and median survival per isolate were calculated. According to the interquartile range of the median survival, isolates with a median survival under P25 were classified as of high-virulence and isolates with a median survival over P75 as of low virulence. The median survival of larvae infected with different species was variable: C. albicans (n = 2 days, IQR <1−3 days), C. tropicalis (n = 2 days, IQR 1.5−4 days), C. parapsilosis (n = 2 days, IQR 2−3.5 days), C. glabrata (n = 3 days, IQR 2−3 days), and C. krusei (n = 7 days, 6.5−>8 days) (P < .001). Differences in virulence among species were validated by histological examination (day +1 post-infection) in the larvae infected by the isolates of each virulence category and species. Virulence-related gene expression in C. albicans isolates did not reach statistical significance. We report species-specific virulence patterns of Candida spp. and show that isolates within a given species have different degrees of virulence in the animal model.

We examined the rapid evaluation of susceptibility to echinocandins in 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 spp. ( = 60) and echinocandin-intermediate ( = 20) from patients with candidemia. Additionally, blood culture bottles of nonfungemic/bacteremic patients were spiked with 35 echinocandin-resistant 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) and Sabouraud agar plates containing 2 mg/liter of anidulafungin. The isolates were tested according to the EUCAST method and Etest standard (ET). Essential and categorical agreement between the methods was calculated. The essential agreement and categorical agreement between the EUCAST method and ET and ET were both >97.4%. The essential agreement between ET and the EUCAST method for both echinocandins was >97%. The categorical agreement between the sequence and ET was 97.4%. The ET MICs of anidulafungin and micafungin (≥0.19 mg/liter and ≥0.064 mg/liter, respectively) effectively separated all susceptible wild-type isolates from the resistant 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 (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 species isolates.