Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study

C. Biswas; S. C.A. Chen; C. Halliday; K. Kennedy; E. G. Playford; D. J. Marriott; M. A. Slavin; T. C. Sorrell; V. Sintchenko

doi:10.1016/j.cmi.2017.03.014

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study

C. Biswas^*, S. C.A. Chen, C. Halliday, K. Kennedy, E. G. Playford, D. J. Marriott, M. A. Slavin, T. C. Sorrell, V. Sintchenko

^*Corresponding author for this work

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Abstract

Objectives 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.

Original language	English
Pages (from-to)	676.e7-676.e10
Journal	Clinical Microbiology and Infection
Volume	23
Issue number	9
DOIs	https://doi.org/10.1016/j.cmi.2017.03.014
Publication status	Published - Sept 2017

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Biswas, C., Chen, S. C. A., Halliday, C., Kennedy, K., Playford, E. G., Marriott, D. J., Slavin, M. A., Sorrell, T. C., & Sintchenko, V. (2017). Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study. Clinical Microbiology and Infection, 23(9), 676.e7-676.e10. https://doi.org/10.1016/j.cmi.2017.03.014

@article{695fcf12b1514d9da345a3335828ec5b,

title = "Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study",

abstract = "Objectives 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{\textregistered} 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.",

keywords = "Antifungal resistance, Azoles, Candida glabrata, Echinocandins, Next-generation sequencing, Single nucleotide polymorphisms",

author = "C. Biswas and Chen, {S. C.A.} and C. Halliday and K. Kennedy and Playford, {E. G.} and Marriott, {D. J.} and Slavin, {M. A.} and Sorrell, {T. C.} and V. Sintchenko",

note = "Publisher Copyright: {\textcopyright} 2017 European Society of Clinical Microbiology and Infectious Diseases",

year = "2017",

month = sep,

doi = "10.1016/j.cmi.2017.03.014",

language = "English",

volume = "23",

pages = "676.e7--676.e10",

journal = "Clinical Microbiology and Infection",

issn = "1198-743X",

publisher = "Elsevier B.V.",

number = "9",

}

Biswas, C, Chen, SCA, Halliday, C, Kennedy, K, Playford, EG, Marriott, DJ, Slavin, MA, Sorrell, TC & Sintchenko, V 2017, 'Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study', Clinical Microbiology and Infection, vol. 23, no. 9, pp. 676.e7-676.e10. https://doi.org/10.1016/j.cmi.2017.03.014

TY - JOUR

T1 - Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing

T2 - a feasibility study

AU - Biswas, C.

AU - Chen, S. C.A.

AU - Halliday, C.

AU - Kennedy, K.

AU - Playford, E. G.

AU - Marriott, D. J.

AU - Slavin, M. A.

AU - Sorrell, T. C.

AU - Sintchenko, V.

PY - 2017/9

Y1 - 2017/9

N2 - Objectives 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.

AB - Objectives 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.

KW - Antifungal resistance

KW - Azoles

KW - Candida glabrata

KW - Echinocandins

KW - Next-generation sequencing

KW - Single nucleotide polymorphisms

UR - http://www.scopus.com/inward/record.url?scp=85018751780&partnerID=8YFLogxK

U2 - 10.1016/j.cmi.2017.03.014

DO - 10.1016/j.cmi.2017.03.014

M3 - Article

SN - 1198-743X

VL - 23

SP - 676.e7-676.e10

JO - Clinical Microbiology and Infection

JF - Clinical Microbiology and Infection

IS - 9

ER -

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study

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