Nosocomial transmission of fluconazole-resistant Candida glabrata bloodstream isolates revealed by whole-genome sequencing

Abstract

The clonal transmission of fluconazole-resistant Candida glabrata isolates within hospitals has seldom been analyzed by whole-genome sequencing (WGS). We performed WGS on 79 C. glabrata isolates, comprising 31 isolates from three premature infants with persistent C. glabrata bloodstream infection despite antifungal treatment in the same neonatal intensive care unit (NICU) in 2022 and 48 (27 fluconazole-resistant and 21 fluconazole-susceptible dose-dependent) bloodstream isolates from 48 patients in 15 South Korean hospitals from 2010 to 2022. Phylogenetic analysis based on WGS single-nucleotide polymorphisms (SNPs) distinguished the 79 isolates according to multilocus sequence typing (MLST) (17 sequence type [ST]3, 13 ST7, two ST22, 41 ST26, four ST55, and two ST59 isolates) and unveiled two possible clusters of nosocomial transmission among ST26 isolates. One cluster from two premature infants with overlapping NICU hospitalizations in 2022 encompassed 15 fluconazole-resistant isolates harboring pleiotropic drug-resistance transcription factor (Pdr1p) P258L (13 isolates) or N1086I (two isolates), together with 10 fluconazole-susceptible dose-dependent isolates lacking Pdr1p SNPs. The other cluster indicated unforeseen clonal transmission of fluconazole-resistant bloodstream isolates among five patients (four post-lung transplantation and one with diffuse interstitial lung disease) in the same hospital over 8 months. Among these five isolates, four obtained after exposure to azole antifungals harbored distinct Pdr1p SNPs (N1091D, E388Q, K365E, and R376Q). The findings reveal the transmission patterns of clonal bloodstream isolates of C. glabrata among patients undergoing antifungal treatment, exhibiting different levels of fluconazole susceptibility or distinct Pdr1p SNP profiles.

Importance: The prevalence of fluconazole-resistant bloodstream infections caused by Candida glabrata is increasing globally, but the transmission of these resistant strains within hospitals has rarely been documented. Through whole-genome sequencing and epidemiological analyses, this study identified two potential clusters of C. glabrata bloodstream infections within the same hospital, revealing the transmission of clonal C. glabrata strains with different levels of fluconazole susceptibility or distinct transcription factor pleiotropic drug resistance protein 1 (Pdr1p) single-nucleotide polymorphism profiles among patients receiving antifungal therapy.

Keywords: Candida glabrata; PDR1; clonal transmission; fluconazole resistance; multilocus sequence typing; whole-genome sequencing.

Fig 1

Three phylogenetic trees constructed to analyze the genetic relatedness among C. glabrata isolates. The first tree (A) was constructed using the average nucleotide identity-based neighbor-joining method of 12,338,304 nucleotide positions from whole-genome sequencing (WGS) analysis of 79 C. glabrata isolates from 51 patients (Patients 1–51). The second tree (B) was based on single-nucleotide polymorphisms (SNPs) from six multilocus sequencing typing (MLST) loci for the same 79 isolates. Finally, a WGS-based phylogenetic tree (C) was generated to show the genetic relatedness among 41 C. glabrata isolates from 18 patients with ST26. Information is shown in the format isolate number (No), isolation year, hospital (Hos A–O), MLST sequence type (ST), and phenotype (FR, fluconazole-resistant; F-SDD, fluconazole-susceptible dose-dependent). The isolate numbers of three premature patients (Patients 1, 2, and 3) in the same ICU include the patient number and the serial number of the source (B, blood; C, central venous catheter tip; S, stool). Two blue boxes with dotted lines in ST26 indicate the two nosocomial clusters, and the green box with dotted lines in ST3 indicates serial isolates from the same patient (Patient 3). Lower bars indicate genetic distances. See Tables 1 and 2 for information on the isolates.

Fig 2

Single-nucleotide polymorphism (SNP) differences for each pairwise comparison among related isolates, indicating serial isolates from the same patients (Patients 1, 2, and 3) and each clonal cluster (Clusters 1 and 2). The median SNP differences between isolates for each clonal cluster (Cluster 1 and 2) were significantly lower than the median SNP differences between pairs of six serial isolates of ST3 from Patient 3 (P  <  0.0001). Horizontal bars indicate median SNP levels of each group. See Fig. S1 for SNP difference data in detail.

Fig 3

Fluconazole resistance and Pdr1p single-nucleotide polymorphism (SNP) profiles of 31 isolates of C. glabrata from three premature infants (Patients 1, 2, and 3) who were admitted to the same NICU. Two patients (Patients 1 and 2) had overlapping hospitalization periods. The boxes contain the isolate numbers with corresponding Pdr1p SNPs for 15 fluconazole-resistant (red boxes) and 16 F-SDD (green boxes) isolates. The isolate numbers of three patients (Patients 1, 2, and 3) include the patient number and the serial number of the source (B, blood; C, central venous catheter tip; S, stool). Table 1 shows information on the isolates. Abbreviations: NICU, neonatal intensive care unit; M, male; w, week.

Fig 4

Schematic of the epidemiological status, antifungal treatment regimens, and outcomes of five patients (Patients 21 to 25) with clonal fluconazole-resistant bloodstream isolates of C. glabrata obtained between August 2017 and March 2018. Boxes contain isolate numbers with fluconazole minimum inhibitory concentrations (MICs, mg/L) and corresponding Pdr1p single-nucleotide polymorphisms (SNPs). See Table 2 for information on the isolates. Abbreviations: y, year; F, female; M, male; S/P, status post; DILD, diffuse interstitial lung disease; FLU, fluconazole.

Fig 5

Single-nucleotide polymorphisms (SNPs) in genes potentially linked to azole resistance in fluconazole-resistant bloodstream isolates of C. glabrata by whole-genome sequencing (WGS). Three isolates resistant to fluconazole (Isolates 15, 23, and 26) that did not have the Pdr1p SNP and their associated nucleotide polymorphisms in other potential azole-resistant genes are in blue text or blue boxes. Five isolates (Isolates 21 to 25) that formed a clonal cluster in a phylogenetic tree based on WGS SNP analysis are in the large black box. Abbreviations: MLST, multilocus sequence typing; ST, strain type; FLU, fluconazole; VRC, voriconazole; POS, posaconazole, ITR, itraconazole.