Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study.
OBJECTIVE
Multi-antifungal drug resistance in Candida glabrata is increasing. We examined the feasibility of next-generation sequencing (NGS) to investigate the presence of antifungal drug resistance markers in C. glabrata.


METHODS
The antifungal susceptibility of 12 clinical isolates and one ATCC strain of C. glabrata was determined using the Sensititre YeastOne®YO10 assay. These included three isolate pairs where the second isolate of each pair had developed a rise in drug MICs. Single nucleotide polymorphisms (SNPs) in genes known to be linked to echinocandin, azole and 5-fluorocytosine resistance were analysed in all isolates through NGS.


RESULTS
High-quality non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgCDR1, CgPDR1 and FCY2 were identified. For two of three isolate pairs, there was a >60-fold rise in MICs to all echinocandins in the second isolate from each pair; one echinocandin-resistant isolate harboured a mutation in FKS1 (S629P) and the other in FKS2 (S663P). Of the third pair, both the 5-fluorocytosine-susceptible, and resistant isolates had a mutation in FCY2 (A237T). SNPs in CgPDR1 were found in pan-azole-resistant isolates. SNPs in other genes linked to azole resistance (CgCDR1, ERG9 and CgFLR1) were present in both azole-susceptible and azole-resistant isolates. SNPs were also identified in Candida adhesin genes EPA1, EPA6, PWP2 and PWP5 but their presence was not associated with higher drug MICs.


CONCLUSIONS
Genome-wide analysis of antifungal resistance markers was feasible and simultaneously revealed mutation patterns of genes implicated in resistance to different antifungal drug classes.
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, Australia; Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, Australia. Electronic address: chayanika_biswas@yahoo.com.
C
C
Biswas
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, Australia; Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia.
S C-A
SC
Chen
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, Australia.
C
C
Halliday
Department of Infectious Diseases and Microbiology, Canberra Hospital, Australian National University Medical School, Canberra, Australia.
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K
Kennedy
Infection Management Services, Princess Alexandra Hospital, Brisbane, Australia.
E G
EG
Playford
Department of Microbiology and Infectious Diseases, St Vincent's Hospital, Sydney, Australia.
D J
DJ
Marriott
Department of Infectious Diseases, Peter MacCallum Cancer Centre, Melbourne, Australia.
M A
MA
Slavin
Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia.
T C
TC
Sorrell
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, Australia; Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia.
V
V
Sintchenko
D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D041221:Candida glabrata / Q000187:drug effects / Q000235:genetics; D002177:Candidiasis / Q000382:microbiology; D025141:Drug Resistance, Fungal / Q000235:genetics*; D054714:Echinocandins / Q000494:pharmacology*; D005240:Feasibility Studies; D005437:Flucytosine / Q000494:pharmacology*; D005819:Genetic Markers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008828:Microbiological Techniques; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
D000935:Antifungal Agents; D001393:Azoles; D054714:Echinocandins; D005819:Genetic Markers; D005437:Flucytosine
Antifungal resistance; Azoles; Candida glabrata; Echinocandins; Next-generation sequencing; Single nucleotide polymorphisms