Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Summary

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

Abstract

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing.

Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing. Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing.

In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Introduction

Candida glabrata is an increasingly encountered pathogen with importance as a species that exhibits resistance to the azoles as well as more recently, to the echinocandins^1,2,3. Unlike the diploid C. albicans, the haploid genome of C. glabrata may allow it to acquire mutations and develop multi-drug resistance more easily. Co-resistance to both drug classes has also been reported⁴. Hence, early evaluation of antifungal susceptibility and detection of drug resistance in C. glabrata is crucial for correct, targeted the....

Protocol

No ethical approval was required for this study.

1. Subculture and inoculum preparation for Candida glabrata

1. Select a panel of C.glabrata isolates to be studied which should also include at least one C. glabrata American Type Culture Collection (ATCC) with known susceptibility pattern.
2. Subculture an isolate by touching a single colony using a sterile disposable plastic loop and streaking onto a Sabouraud's dextrose agar (SDA) plate⁸.
3. Incubate the SDA plate for 24-48 h at 35 °C for pure culture of isolate with good growth.

Results

Thirteen C. glabrata comprising C. glabrata ATCC 90030 and 12 isolates from the Clinical Mycology Reference laboratory (isolates CMRL1 to CMRL12), Westmead Hospital, Sydney were studied (Table 1). These included three pairs of isolates CMRL-1/CMRL-2, CMRL-3/CMRL-4 and CMRL-5/CMRL-6 obtained before and after antifungal therapy with no epidemiological links between them ²⁴ (Table 1).

The M.......

Discussion

   This study determined feasibility, approximate timelines and precision of WGS-guided detection of drug resistance in C. glabrata. The turnaround time (TAT) for the library preparation and sequencing was four days and reporting of analyzed results one-two days. This compares with at least a similar amount TAT for susceptibility assays from culture plates and Sanger sequencing with significantly higher number of samples. Around 30-90 C. glabrata genomes can be sequenced based on sequencing fl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DensiCHECK Plus	BioMérieux Inc	K083536	Densitometer used for McFarland readings
Sensititre YeastOne	TREK Diagnostic Systems, Thermo Scientific	YO10	Commercial susceptibility assay plate with standard antifungal drugs.
Fisherbrand Disposable Inoculating Loops and Needles	Fisher Scientific, Thermo Fisher Scientific	22-363-605	Disposable plastic loops can be used directly from package. No flaming required.
Eppendorf Safe-Lock microcentrifuge tubes	Sigma Aldrich, Merck	T2795	Volume 2.0 mL, natural
ZYMOLYASE 20T from Arthrobacter luteus	MP Biomedicals, LLC	8320921	Used for cell wall lysis of fungal isolate before DNA extraction
Wizard Genomic DNA Purification Kit	Promega	A1120	Does 100 DNA extractions
Quant-iT PicoGreen dsDNA Assay Kit	Thermo Fisher Scientific	P7589	Picogreen reagent referred to as fluorescent dye in the protocol. Includes Lambda DNA standard and picogreen reagent for assay.
Nextera XT DNA Sample Preparation Kit	Illumina	FC-131-1096	Includes Box 1 and Box 2  reagents for 96 samples
Nextera XT Index Kit v2	Illumina	FC-131-2001, FC-131-2002, FC-131-2003, FC-131-2004	Index set A Index set B Index set C Index set D
NextSeq 500/550 High Output Kit v2	Illumina	FC-404-2004	300 cycles, More than 250 samples per kit
NextSeq 500 Mid Output v2 Kit	Illumina	FC-404-2003	300 cycles, More than 130 samples per kit
PhiX Control Kit	Illumina	FC-110-3001	To arrange indices from Index kit in order
TruSeq Index Plate Fixture Kit		FC-130-1005	2 Fixtures
KAPA Library Quantification Kit for Next-Generation Sequencing	KAPA Biosystems	KK4824	Includes premade standards, primers and MasterMix
Janus NGS Express Liquid handling system	PerkinElmer	YJS4NGS	Used for DNA dilutions during sequencing
0.8 mL Storage Plate	Thermo Scientific	AB0765B	MIDI Plate for DNA Library cleanup and normalisation
Agencourt AMPure XP	Beckman Coulter	A63881	Magnetic beads in solution for library purification
Magnetic Stand-96	Thermo Fisher Scientific	AM10027	Used for magnetic bead based DNA purification
OrbiShaker MP	Benchmark Scientific	BT1502	96-well plate shaker with 4 platforms
Hard Shell PCR Plate	BioRad	HSP9601	Thin Wall, 96 Well
LightCycler 480 Instrument II	Roche	5015278001	Accomodates 96 well plate
Microseal 'B' PCR Plate Sealing Film, adhesive, optical	BioRad	MSB1001	Clear 96-well plate sealers
CLC Genomics Workbench	Qiagen	CLCBio	Software for data analysis, Version 8
NextSeq500 instrument	Illumina	Illumina	Benchtop Sequencer used for next generation sequencing