Antifungal drug susceptibility, molecular basis of resistance to echinocandins and molecular epidemiology of fluconazole resistance among clinical Candida glabrata isolates in Kuwait. molecular basis of resistance to echinocandins and fluconazole

Candida glabrata is the second/third most common cause of invasive candidiasis in at-risk patients. It is less susceptible to azoles and also readily develops resistance to echinocandins. This study performed molecular identification, antifungal susceptibility testing (AST) and determined molecular basis of resistance of C. glabrata isolates to echinocandins in Kuwait. Clinical C. glabrata isolates (n=75) identified by Vitek2 were tested by multiplex PCR and/or PCR-sequencing of rDNA. AST to fluconazole, caspofungin, micafungin and amphotericin B was determined by Etest and to micafungin by reference broth microdilution (BMD). Mutations in hotspot-1 and hotspot-2 of FKS1 and FKS2 were detected by PCR-sequencing. PCR-sequencing of ERG11 was also performed for fluconazole-resistant C. glabrata. All 75 Vitek2-characterized C. glabrata isolates were identified as C. glabrata sensu stricto by mPCR/rDNA sequencing. Based on EUCAST breakpoints, 70 (93.3%) isolates were susceptible (MIC = 0.032 μg/ml) to micafungin by Etest and BMD (essential agreement, 93%; categorical agreement, 100%). Three micafungin-resistant isolates were resistant and two were susceptible dose-dependent to caspofungin. Four and one micafungin-resistant isolate contained S663P and ∆659F mutation, respectively, in hotspot-1 of FKS2. No nonsynonymous mutation was detected in 70 micafungin-susceptible isolates. Micafungin-resistant isolates were genotypically distinct strains. Fluconazole, amphotericin B and multidrug resistance was detected in 36, four and one isolate, respectively. Only one of 36 fluconazole-resistant isolate harbored nonsynonymous mutations in ERG11. Multiple loci-based fingerprinting studies showed that 34 of 36 fluconazole-resistant isolates were genotypically distinct strains. Our data show that micafungin susceptibility reliably identifies echinocandin-resistant isolates and may serve as a surrogate marker for predicting susceptibility/resistance of C. glabrata to caspofungin. All micafungin-resistant isolates harbored a nonsynonymous/deletion mutation in hotspot-1 of FKS2 and were genotypically distinct strains. Fingerprinting data also showed that fluconazole resistance development in C. glabrata is not clonal in Kuwait.