Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates

Abstract

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

Keywords: Candida glabrata; WGS; antifungal; epidemiology; genomic; resistance.

Fig 1

Inclusion chart

Fig 2

Neighbor-joining phylogeny of C. glabrata. The final data set included a total of 243,415 nucleotides for 82 isolates. Each leaf node color represents a patient, and the external squares indicate susceptible/resistant phenotype. Box: zoom in shows examples of patients with isolates that acquired resistance to echinocandins (Patient 24), azoles (Patient 18), or both (Patient 1). Branch length indicates the number of SNPs.

Fig 3

Genetic diversity of C. glabrata isolates. Left: SNP differences among several isolates collected from the same patient in 33 patients. Right: SNP differences for each pairwise comparison among all isolates from different patients in 106 patients. Y-axis shows the log 10 of SNP distances. Boxplot R version 4.0.3

Fig 4

FSK2 and FKS1 common mutations and susceptible/resistant phenotype (graphic representation of Table 1). Red and green boxes represent resistant and susceptible isolates, respectively. Black dots represent the presence of the mutation indicated on the top of each panel. Collection dates are displayed on the right side of the panels and Patient ID on the left. cas: caspofungin, anid: anidulafungin.*echinocandin-resistant isolate from Patient 24 carried the mutation Phe659Try in the FKS2 gene, not shown in the figure

Fig 5

Left: SNP differences for each pairwise comparison among isolates from the same patient, where the resistant pattern did not change (S->S and R->R). Right: SNP differences for each pairwise comparison among isolates from the same patient where the resistant pattern changed (S->R). Boxplot R version 4.0.3