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.
Highlights
The opportunistic yeast Candida glabrata is the second most common cause of candidemia and invasive candidiasis (IC) in many countries (Arendrup et al, 2013; Giunea, 2014; Pfaller et al, 2014; Chapman et al, 2017)
The results indicate that there is no evidence of genomic similarity or sequence types (STs) distribution among the sequences isolated in the two timeframes studied (Figure 2A), suggesting that there have been no marked shifts in the present STs
We further found no association between whole genome sequencing (WGS) STs and susceptibility or /resistance to fluconazole (Figure 2B), consistent with that reported by traditional multilocus sequence typing (MLST) studies (Dodgson et al, 2003; Amanloo et al, 2018)
Summary
The opportunistic yeast Candida glabrata is the second most common cause of candidemia and invasive candidiasis (IC) in many countries (Arendrup et al, 2013; Giunea, 2014; Pfaller et al, 2014; Chapman et al, 2017). Its clinical importance as a species lies in its reduced susceptibility to azole antifungal agents; more recently, resistance to the echinocandins as well as resistance to both these drug classes (Pfaller et al, 2012; Wisplinghoff et al, 2014; Shields et al, 2015) This has prompted much investigation of the epidemiology and biological properties of C. glabrata infections (Vale-Silva and Sanglard, 2015). Because the prevalence of C. glabrata candidiasis and drug resistance rates varies both between and within geographical region, local epidemiological data are essential to inform management (Pfaller et al, 2012; Giunea, 2014) The reasons for this variation are uncertain but likely include prior exposure to azoles, patient factors and geographic locationspecific determinants. Relatively little is known about the genomic variation between isolates from different regions or the clinical significance of such differences
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