Whole-Genome Sequence Analysis of Candida glabrata Isolates from a Patient with Persistent Fungemia and Determination of the Molecular Mechanisms of Multidrug Resistance

Abstract

Whole-genome sequencing (WGS) was used to determine the molecular mechanisms of multidrug resistance for 10 serial Candida glabrata bloodstream isolates obtained from a neutropenic patient during 82 days of amphotericin B (AMB) or echinocandin therapy. For WGS, a library was prepared and sequenced using a Nextera DNA Flex Kit (Illumina) and the MiseqDx (Illumina) instrument. All isolates harbored the same Msh2p substitution, V239L, associated with multilocus sequence type 7 and a Pdr1p substitution, L825P, that caused azole resistance. Of six isolates with increased AMB MICs (≥2 mg/L), three harboring the Erg6p A158fs mutation had AMB MICs ≥ 8 mg/L, and three harboring the Erg6p R314K, Erg3p G236D, or Erg3p F226fs mutation had AMB MICs of 2–3 mg/L. Four isolates harboring the Erg6p A158fs or R314K mutation had fluconazole MICs of 4–8 mg/L while the remaining six had fluconazole MICs ≥ 256 mg/L. Two isolates with micafungin MICs > 8 mg/L harbored Fks2p (I661_L662insF) and Fks1p (C499fs) mutations, while six isolates with micafungin MICs of 0.25–2 mg/L harbored an Fks2p K1357E substitution. Using WGS, we detected novel mechanisms of AMB and echinocandin resistance; we explored mechanisms that may explain the complex relationship between AMB and azole resistance.

Adhesive and biofilm‐forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins

“…Among all mutations detected in genes involved in azole resistance, seven were previously documented but seem to be uninvolved in azole resistance: H58Y in Cdr1, E839D and T1530K in Pdh1 37 and S76P, V91I, L98S and T143P in Pdr1 39,40 (Table 4). However, two documented amino acid substitutions were potentially involved in azole resistance: V239L in Msh2 and S343F in Pdr1 (Table 4).…”

“…Among all mutations detected in genes implicated in virulence, only 11 were previously reported of which nine did not have any clear documented role in increased virulence: F1335L in Fks1, 33 E78D and T926P in Fks2 34–36 and I3T, A42G, K206E, N865S, R1472Q and F1768I in Fks3 37 (Table 3). The two other previously reported amino substitutions D679E and I739N in the subtelomeric silencer Sir3 (Table 4) showed increased C. glabrata adhesion to surfaces 38 (Figure 6), which is an important step in biofilm formation 16 …”

Implication of genotypes for prognosis of Candida glabrata bloodstream infections

“…It might be due to acquired antifungal resistance by a specific loss-of-function mutation in the mismatch repair ( MSH2 ) gene with V239L substitution in ST7. 12 , 21 , 23 The MSH2 gene regulates correction of DNA errors during replication or exposure to toxicants and studies analysing a series of C. glabrata isolates collected from in vitro evolution experiments or clinical sequential isolates illustrated V239L substitution has an increased mutation rate, promotes antifungal resistance, and causes persistent infections and death. 23–25 …”