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Front Microbiol. 2018 Dec 3;9:2946. doi: 10.3389/fmicb.2018.02946. eCollection 2018.

Whole Genome Sequencing of Australian Candida glabrata Isolates Reveals Genetic Diversity and Novel Sequence Types.

Author information

1
Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, NSW, Australia.
2
Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
3
Centre for Infectious Diseases and Microbiology, Westmead Institute for Medical Research, Westmead, NSW, Australia.
4
Marie Bashir Institute for Emerging Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia.
5
Department of Infectious Diseases and Microbiology, New South Wales Health Pathology, Royal Prince Alfred Hospital, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
6
Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, New South Wales Health Pathology, Westmead Hospital, Sydney, NSW, Australia.
7
National Mycology Reference Centre, SA Pathology, Adelaide, SA, Australia.
8
Department of Microbiology and Infectious Diseases, Canberra Hospital & Health Services, Australian National University Medical School, Canberra, ACT, Australia.
9
Department of Microbiology and Infectious Diseases, St Vincent's Hospital, Sydney, NSW, Australia.
10
Department of Infectious Diseases, Alfred Health and Monash University, Melbourne, VIC, Australia.
11
Department of Microbiology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Perth, WA, Australia.
12
Department of Microbiology and Infectious Diseases, Royal North Shore Hospital, Sydney, NSW, Australia.
13
National Centre for Infections in Cancer, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Abstract

Candida glabrata is a pathogen with reduced susceptibility to azoles and echinocandins. Analysis by traditional multilocus sequence typing (MLST) has recognized an increasing number of sequence types (STs), which vary with geography. Little is known about STs of C. glabrata in Australia. Here, we utilized whole genome sequencing (WGS) to study the genetic diversity of 51 Australian C. glabrata isolates and sought associations between STs over two time periods (2002-2004, 2010-2017), and with susceptibility to fluconazole by principal component analysis (PCA). Antifungal susceptibility was determined using Sensititre YeastOneTM Y010 methodology and WGS performed on the NextSeq 500 platform (Illumina) with in silico MLST STs inferred by WGS data. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-fluorocytosine resistance were analyzed. Of 51 isolates, WGS identified 18 distinct STs including four novel STs (ST123, ST124, ST126, and ST127). Four STs accounted for 49% of isolates (ST3, 15.7%; ST83, 13.7%; ST7, 9.8%; ST26, 9.8%). Split-tree network analysis resolved isolates to terminal branches; many of these comprised multiple isolates from disparate geographic settings but four branches contained Australian isolates only. ST3 isolates were common in Europe, United States and now Australia, whilst ST8 and ST19, relatively frequent in the United States, were rare/absent amongst our isolates. There was no association between ST distribution (genomic similarity) and the two time periods or with fluconazole susceptibility. WGS identified mutations in the FKS1 (S629P) and FKS2 (S663P) genes in three, and one, echinocandin-resistant isolate(s), respectively. Both mutations confer phenotypic drug resistance. Twenty-five percent (13/51) of isolates were fluconazole-resistant (MIC ≥ 64 μg/ml) of which 9 (18%) had non wild-type MICs to voriconazole and posaconazole. Multiple SNPs were present in genes linked to azole resistance such as CgPDR1 and CgCDR1, as well as several in MSH2; however, SNPs occurred in both azole-susceptible and azole-resistant isolates. Although no particular SNP in these genes was definitively associated with resistance, azole-resistant/non-wild type isolates had a propensity to harbor SNPs resulting in amino acid substitutions in Pdr1 beyond the first 250 amino acid positions. The presence of SNPs may be markers of STs. Our study shows the value of WGS for high-resolution sequence typing of C. glabrata, discovery of novel STs and potential to monitor trends in genetic diversity. WGS assessment for echinocandin resistance augments phenotypic susceptibility testing.

KEYWORDS:

Australia; Candida glabrata; MLST; sequence type; whole genome sequencing

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