gms | German Medical Science

Due to the rising number of immunocompromised patients in recent decades, the risk of fungal opportunistic infections has increased. Candida is the fifth most common pathogen in nosocomial sepsis, which is associated with a high mortality rate. Additionally, an increase in antifungal-resistant Candida strains has been reported in recent years. Next Generation Sequencing (NGS) allows for the investigation of genetic variations in different genes in large populations. The aim of this study was to detect mutations in resistance genes using NGS technology and to determine the impact of the underlying mechanisms of azole and echinocandin resistance.

45 Candida strains were examined consisting of four susceptible control strains, one azole-resistant control strain ATCC 64124, 19 azole-resistant, four borderline echinocandin-resistant, nine echinocandin-resistant and eight multi-resistant clinical isolates. The MICs of anidulafungin, micafungin, caspofungin, fluconazole, posaconazole, voriconazole, itraconazole and isavuconazole were determined using the EUCAST reference method. The genes ERG11, ERG3, TAC1 and GSC1 in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced using the MiSeq^® Platform (Illumina California).

140 different mutations were identified, of which 53 were missense mutations. Out of these, 42 mutations were found to be presumably causal, 13 of which have not been reported before. 10 target mutations in FKS genes in echinocandin-resistant isolates were detected that could lead to reduced binding affinity of echinocandins. Seven point mutations in ERG11 were determined in azole-resistant C. albicans. In azole-resistant C. glabrata, no ERG11 mutations were found. In 10 out of 13 azole-resistant isolates, 11 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified. In C. albicans, five potential gain-of-function mutations in the transcription factor TAC1 were found. In ERG3, five potential loss-of-function mutations and two homozygous premature stop codons were identified that could lead to a metabolic by-pass.

In conclusion all echinocandin-resistant Candida isolates showed a mutation in the hotspot regions of FKS1/2 or GSC1. In contrast to C. albicans strains no ERG11 mutations were found in azole-resistant C. glabrata strains. Thus, point mutations in ERG11 are not involved in the development of azole resistance in these C. glabrata strains. Genetic variations in the transcription factor CgPDR1, which are associated with an overexpression of the efflux pumps CDR1/2, seem to be a more important cause for azole resistance. These results provide a better insight into the underlying mechanisms of resistance. Next Generation Sequencing is a very good method to detect resistance mutations in many potential resistance genes in a large population.