Candida auris Pan-Drug-Resistant to Four Classes of Antifungal Agents

Abstract

Candida auris is an urgent antimicrobial resistance threat due to its global emergence, high mortality, and persistent transmissions. Nearly half of C. auris clinical and surveillance cases in the United States are from the New York and New Jersey Metropolitan area. We performed genome, and drug-resistance analysis of C. auris isolates from a patient who underwent multi-visceral transplantation. Whole-genome comparisons of 19 isolates, collected over 72 days, revealed closed similarity (Average Nucleotide Identity > 0.9996; Aligned Percentage > 0.9764) and a distinct subcluster of NY C. auris South Asia Clade I. All isolates had azole-linked resistance in ERG11(K143R) and CDR1(V704L). Echinocandin resistance first appeared with FKS1(S639Y) mutation and then a unique FKS1(F635C) mutation. Flucytosine-resistant isolates had mutations in FCY1, FUR1, and ADE17. Two pan-drug-resistant C. auris isolates had uracil phosphoribosyltransferase deletion (FUR1[1Δ33]) and the elimination of FUR1 expression, confirmed by a qPCR test developed in this study. Besides ERG11 mutations, four amphotericin B-resistant isolates showed no distinct nonsynonymous variants suggesting unknown genetic elements driving the resistance. Pan-drug-resistant C. auris isolates were not susceptible to two-drug antifungal combinations tested by checkerboard, Etest, and time-kill methods. The fungal population pattern, discerned from SNP phylogenetic analysis, was consistent with in-hospital or inpatient evolution of C. auris isolates circulating locally and not indicative of a recent introduction from elsewhere. The emergence of pan-drug-resistance to four major classes of antifungals in C. auris is alarming. Patients at high risk for drug-resistant C. auris might require novel therapeutic strategies and targeted pre-and/or posttransplant surveillance.

Keywords: Candida auris; New York; amphotericin B; antifungal combination; caspofungin; echinocandins; fluconazole; flucytosine; gene deletion; gene mutation; multidrug-resistant; pan-drug-resistant; real-time PCR; transplant patient; whole-genome sequencing.

FIG 1

A timeline of the clinical course of the patient. Candida auris isolations at different intervals, including pan-drug-resistant isolates recovered on hospital day 72 are shown. Also highlighted are antimicrobial drugs and duration, major complications, and other pathogens encountered in the patient.

FIG 2

Candida auris population analysis. SNPs scored from whole-genome assemblies were analyzed by the Maximum Likelihood (ML) method to show the placement of patient C. auris isolates vis-à-vis representative C. auris South Asia Clade I isolates and three other clades. All C. auris patient isolates are in a distinct subcluster among NY isolates.

FIG 3

Deletion in C. auris FUR1. A set of primers/probe anchored to amplify full-length FUR1 using real-time PCR test with positive Ct value (average from two readings). Another set of primers/probe, designed to only amplify deleted portion of FUR1, did not yield positive Ct values in pan-drug-resistant isolates of C. auris consistent with in silico prediction of a truncated FUR1. Finally, no transcript was present in four C. auris Isolates with deleted FUR1. *, a select number of isolates tested with no deletion in FUR1; **, all four isolates with FUR1 deletion.

FIG 4

Time-kill assays for two-drug combinations against C. auris. Fixed concentration of two drug combinations were prepared and inoculated with 10³ CFU of echinocandins-susceptible (20–34) and pan-drug-resistant (20–32) C. auris isolates. Plates were incubated at 35°C for 24 h followed by enumeration of CFU. Significant killing of C. auris 20–34 with micafungin combination was observed (A) while other combinations were not that significant (B–D). No significant killing of C. auris 20–32 observed against any of the drug combinations tested (E–H).