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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 echinocandins1,2,3. 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 therapy as well as in the context of antifungal stewardship to limit drivers of antimicrobial resistance1,5,6. Establishing an efficient workflow to rapidly detect the presence of confirmatory mutations linked to resistance biomarkers in resistant isolates will also help to improve prescribing decisions and clinical outcomes.

Antifungal susceptibility is usually assessed by measuring minimum inhibitory concentration (MIC) which is defined as the lowest drug concentration that results in a significant reduction in growth of a microorganism compared with that of a drug-free growth control. The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) have standardized susceptibility testing methods in order to make MIC determination more accurate and consistent7,8. However, the utility of antifungal MIC remains limited especially for the echinocandins, in particular with regards to inter-laboratory comparisons where varying methodologies and conditions are used9. There is also uncertain correlation of MICs with response to echinocandin treatment and inability to distinguish WT (or susceptible) isolates from those harboring FKS mutations (echinocandin-resistant strains)10,11. Despite the availability of confirmatory single-gene PCRs and Sanger sequencing of antifungal resistance markers, realization of results is often delayed due to lack of simultaneous detection of multiple resistance markers5,12. Hence, concurrent detection of resistance-conferring mutations in different locations in the genome, enabled by whole genome sequencing-based analysis, offers significant advantages over current approaches.

Whole genome sequencing (WGS) has been successfully implemented to track disease transmission during outbreaks as well as an approach for genome-wide risk assessment and drug resistance testing in bacteria and viruses13. Recent advances in nucleic acid sequencing technology have made the whole genome sequencing (WGS) of pathogens in a clinically actionable turn-around-time both technically and economically feasible. DNA sequencing offers important advantages over other methods of pathogen identification and characterization employed in microbiology laboratories14,15,16. First, it provides a universal solution with high throughput, speed and quality. Sequencing can be applied to any of microorganisms and allows economies of scale at local or regional laboratories. Second, it produces data in a 'future-proof' format amenable to comparison at national and international levels. Finally, the potential utility of WGS in medicine has been augmented by the rapid growth of public data bases containing reference genomes, which can be linked to equivalent data bases that contain additional clinical and epidemiological metadata17,18.

Recent studies have demonstrated the utility of WGS for identification of antifungal resistance markers from clinical isolates of Candida spp.10,19,20. This is mostly due to the availability of high-throughput benchtop sequencers, established bioinformatics pipelines and decreasing cost of sequencing21,22. The advantage of fungal WGS over Sanger sequencing is that WGS allows sequencing of multiple genomes on a single run. In addition, WGS of Candida genomes can identify novel mutations in drug targets, track genetic evolution, and emergence of clinically relevant sequence-types20,22,23. Most importantly, in cases of intrinsic multidrug resistance, WGS can assist in early detection of resistance-conferring mutations prior to treatment selection22,24.

Here, we examined the feasibility of WGS-enabled screening for mutations associated with drug resistance to different classes of antifungal agents. We present a methodology for the implementation of WGS from end-user and diagnostic mycology laboratory perspectives. We included in this analysis three isolate pairs cultured from three separate clinical cases in which in vitro resistance to the echinocandins and 5-flucytosine developed over time following antifungal treatment.

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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.

2. Determination of Antifungal Susceptibility

  1. Use a sterile disposable plastic loop to pick 4-5 colonies of approximately 1 mm diameter from a freshly subcultured C. glabrata isolate on SDA plate. Resuspend in 3 mL of sterile distilled water. Mix well by gentle pipetting to obtain uniform suspension.
  2. Adjust the cell density to 0.5 McFarland which is equivalent to 1 x 106 to 5 x 106 cells/mL using a densitometer 8.
  3. Perform susceptibility testing using commercial assay (see Table of Materials and Reagents) on all C. glabrata isolates following manufacturer's instructions. Interpret the susceptibility of isolates based on resultant MICs of antifungal drugs according to CLSI guidelines and prepare report (Table 1)8.

3. Genomic DNA extraction for sequencing

  1. Resuspend a loopful of colonies from a freshly grown SDA plate in 300 µL of 50 mM EDTA in a 1.5 mL tube.
  2. Add 40 µL of zymolyase (10 mg/mL) to the suspension, and gently pipet 5 times until suspension is uniform.
  3. Incubate the sample at 37 °C for 1-2 h to digest the cell wall. Cool at room temperature for 5 min.
  4. Centrifuge the suspension at 14,000 × g for 2 min and then carefully remove the supernatant.
  5. Extract genomic DNA following DNA extraction kit guidelines (see Table of Materials).
  6. Resuspend extracted DNA pellet in 50 µL of 10 mM Tris Buffer (pH 7.5-8.5) instead of the elution buffer provided in the kit.
  7. Check the purity of DNA by measurement of optical density (O.D) at 260/280 nm25.

4. Genomic DNA quantification

  1. Prepare a 1x Tris EDTA (TE) buffer provided in the assay kit based on manufacturer's guidelines for the fluorescence assay (see Table of Materials).
  2. Dilute the DNA sample by adding 2 µL to 98 µL of 1x TE assay buffer (final volume of 100 µL, dilution factor 1:50) in a disposable 96 well plate. This dilution step can be performed either manually using a multichannel pipette or by automated liquid handling workstation.
  3. Prepare the range of standards by diluting the lambda DNA (100 µg/mL) provided in the fluorescence assay kit (Table 2) and include for measurement along with samples.
  4. Add 100 µL of the fluorescent dye to 100 µL diluted DNA samples and standards for the reaction. Incubate for 5 min at room temperature, protected from light.
  5. Measure the fluorescence of all samples based on manufacturer's guidelines.
  6. Plot the standard curve using the fluorescence readings and calculate the original concentration of the DNA samples.
  7. Determine the volume of DNA and 10 mM Tris buffer (pH 8) to be added to adjust the DNA concentration to 0.2 ng/µL.
  8. Perform the DNA dilutions using automated liquid handling workstation. This step can be also achieved by manual pipetting.

5. DNA Library Preparation

Note: Library preparation and sequencing was performed following manufacturer's protocols and guidelines provided by company (Figure 1A) (see Table of Materials).

  1. Tagmentation and PCR amplification
    1. Label a new 96-well hard-shell thin wall plate.
    2. Add 5 µL of quantified input DNA at 0.2 ng/µL (1 ng in total) to each sample well of the plate.
    3. Add 10 µL of tagmentation buffer and 5 µL of amplification buffer to each well containing DNA and gently pipette to mix. Seal the plate with an adhesive plate seal.
    4. Place the plate in a thermal cycler and run the following PCR program: 55 °C for 5 min, and hold at 10 °C. When the sample reaches 10 °C, proceed immediately to neutralize.
    5. Add 5 µL of neutralization buffer to the plate to neutralize the amplicon reaction, and gently pipette mix. Seal the plate and incubate at room temperature for 5 min.
    6. Add 15 µL of indexing PCR mastermix to the samples.
    7. Use a box of index primer tubes available for a setup of 96-well plate format so that each sample gets a unique combination of indices based on index template ( Table 3).
    8. Arrange the index primer tubes in the index plate rack (Figure 1B) using the order provided in the template on Table 3 and record the position of the indices on the template.
    9. Place the tagmentation plate with added PCR mastermix on the index plate rack with the index tubes in order.
    10. Put index primer 1 tubes in vertical arrangement, and index primer 2 tubes in horizontal arrangement on the index rack. Using a multichannel pipette, carefully add 5 µL of index primers to each sample.
    11. Replace old caps of index tubes with new caps to avoid cross-contamination between indices.
    12. Seal the plate using 96-well clear plate sealers and perform the second PCR as follows: 95 °C for 30 s, 12 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s and 72 °C for 5 min.
  2. PCR Cleanup
    1. Transfer the PCR product for PCR-cleanup, from the tagmentation plate to a deep-well plate (see Table of Materials).
    2. Vortex the commercial magnetic beads solution (see Table of Materials) based on manufacturer's instructions and add 30 µL of beads to each PCR product in deep-well plate.
    3. Shake the plate on a microplate shaker at 1800 rpm for 2 min and incubate at room temperature without shaking for 5 min.
    4. Place the plate on a magnetic stand for 2 min until the supernatant has cleared.
    5. Discard the supernatant carefully with the plate still on the magnetic stand.
    6. Add 200 µL of freshly prepared 80% ethanol with the plate on the magnetic stand.
    7. Incubate the plate on the magnetic stand for 30 s and carefully remove and discard the supernatant without disturbing the beads.
    8. Repeat the wash step again and allow the beads to air-dry for 15 min. Remove excess ethanol if any.
    9. Remove the plate from the magnetic stand and add 52.5 µL of resuspension buffer to the beads.
    10. Shake the plate on a microplate shaker at 1800 rpm for 2 min and incubate at room temperature for 2 min without shaking.
    11. Place the plate on the magnetic stand and allow the supernatant to clear.
    12. Using a multichannel pipette, carefully transfer 50 µL of the supernatant from the cleanup plate to a new hard-shell plate.
  3. Library Normalization
    1. Thaw library normalization reagents according to manufacturer's guidelines (see Table of Materials).
    2. Transfer 20 µL of the supernatant from the cleanup plate to a new deep-well plate.
    3. Add 45 µL of magnetic bead suspension and seal the plate with a plate sealer.
    4. Shake the plate on a microplate shaker at 1800 rpm for 30 min. This incubation time is critical and should not be exceeded.
    5. Place the plate on a magnetic stand for 2 min and confirm that the supernatant has cleared (Figure 1B).
    6. Remove and discard the supernatant in an appropriate hazardous waste container with the plate still on the magnetic stand.
    7. Remove the plate from the magnetic stand and wash the beads with 45 µL wash buffer.
    8. Shake the plate with wash buffer on a microplate shaker at 1800 rpm for 5 min.
    9. Place plate on magnetic stand for 2 min and discard supernatant when it turns clear.
    10. Remove the plate from the magnetic stand and repeat the wash with wash buffer again.
    11. Remove the plate from the magnetic stand and add 30 µL of 0.1 N NaOH.
    12. Shake the plate with 0.1 N NaOH on a microplate shaker at 1800 rpm for 5 min and place the plate on the magnetic stand for 2 min or until the liquid is clear.
    13. Add 30 µL of elution buffer to each well of a new 96-well hard-shell thin wall final normalized library plate.
    14. Transfer 30 µL of supernatant from normalization plate to final normalized library plate to make final volume 60 µL. The libraries are now ready to be sequenced.
  4. Determination of DNA Library Concentration by qPCR
    1. Thaw the mastermix, primers and standards provided in the qPCR kit according to manufacturer's guidelines (see Table of Materials).
    2. Combine the PCR reagent mastermix and primer provided in the qPCR kit following manufacturer's instructions and aliquots can be stored at -20 °C.
    3. To determine the DNA library concentration, make a 1/8000 dilution of the DNA libraries using 10 mM Tris buffer (pH 8) by performing a 1:100 dilution (1.5 µL DNA library to 148.5 µL Tris buffer) followed by a 1:80 dilution (2 µL from 1:100 to 158 µL Tris buffer).
    4. Shake the dilution plate at 700 rpm for at least 1 min and then centrifuge at 14000 × g for 1 min.
    5. Prepare 20 µL of final PCR reaction by mixing 4 µL of diluted DNA library or DNA standards and 16 µL of mastermix.
    6. Perform PCR following manufacturer's settings in thermocycler: 95 °C for 5 min, 35 cycles of 95 °C for 30 s and 60 °C for 45 s, and final 65 °C to 95 °C for melt curve analysis.
    7. Obtain the Ct values of the sample DNA libraries and standards from the qPCR thermocycler.
    8. Generate a standard curve from the Ct value of the standards (Figure 2). Define the upper and lower QC range by ± 3 cycles from the mean. For example, if the mean Ct value is 13 cycles then the QC range is between 16 and 10 cycles.
    9. Determine the individual and average library concentrations (ALC) from the standard curve and Ct values.
    10. Determine volume of total pooled libraries (PAL) to be used based on calculation given below for final sequencing considering that target library concentration is between 1.4-1.8 pM.
      Note: Average library concentration obtained from qPCR= ALC; Total Pooled Libraries (PAL) = ALC / 2; Denatured PAL (DAL) = PAL * 0.666
      Depending on the volume of DAL that is mixed with buffer, is the concentration of library to be added to the flow cell. For example, if 65 µL of library is added to 835 µL of buffer, then from this dilution (Dil 1) 195 is added to a total volume of 1300 µL: (65/900) * dnPAL = Dil1
      Dil1 * (195/1300) = Final Concentration (should be between 1.4-1.8 pM)

6. Library pooling and Initiating Sequencing in Benchtop Sequencer

  1. Thaw reagent cartridge according to manufacturer's guidelines. Take out a new flow cell from its package from 4 °C storage and bring to room temperature atleast 30 min prior to sequencing. Take out buffer cartridge and prechill sequencing buffer before use (see Table of Materials).
  2. Prepare a control library by mixing 5 µL of library (1 nM) and 5 µL of 0.2 N NaOH. Vortex briefly and incubate for 5 min at room temperature to denature the control library into single strands.
  3. Add 5 µL of 200 mM Tris-HCl, pH 7 and vortex. Add 235 µL prechilled sequencing buffer and mix gently. The total volume is 250 µL with control library final concentration at 20 pM.
  4. Pool DNA libraries by transferring 5 µL of each sample library to be sequenced from the final normalized library plate into a single low-bind 1.5 mL tube.
  5. Add 30 µL of pooled library and 30 µL of 0.2 N NaOH to denature libraries in another low bind tube.
  6. Vortex the low-bind tube and incubate for 5 min at room temperature to denature libraries into single strands.
  7. Add 30 µL of 200 mM Tris-HCl, pH 7 to tube with denatured libraries to neutralize reaction.
  8. Add 65 µL of neutralized denatured libraries suspension and 835 µL of pre-chilled sequencing buffer and vortex to mix well.
  9. In a final low bind tube combine the following: 195 µL from neutralized denatured libraries, 1.30 µL of control library and 1103.70 µL of sequencing buffer. Mix properly.
  10. Load the final library mix (1300 µL) into the designated spot on reagent cartridge.
  11. Set-up the sequencing run by entering project and sample details in the Sequencer designated website following guidelines.
  12. Initiate sequencing following guidelines. Load flow cell, reagent cartridge with libraries and buffer cartridge in benchtop sequencer.
  13. Record batch numbers of all reagent kits and cartridges used in the sequencing.

7. Data Download from Sequencing Website

  1. Download FASTQ files following manufacturer's instructions provided on website.
  2. For a good quality run check that the percentage Q30 is ≥ 75% and cluster density is between 170-280 K/mm2 with optimal at 200-210 K/mm2 (Table 4).

8. Sequencing Data analysis

  1. Import FASTQ files of sequenced samples into data analysis integrated software package (see Table of Materials).
  2. Create a sequencing workflow in software by adding features from list namely trimming, mapping to reference (select reference genome), local realignment and variant analysis (Figure 3A) using settings listed in Table 5.
  3. Run workflow by selecting a single sample or a batch of sample FASTQ files and save output files in designated sample folders.
  4. Generate report for sequence coverage depth, mapped regions and list of structural variants in genome (Figure 3B).
  5. Use list of structural variants to search for non-synonymous single nucleotide polymorphisms (SNPs) in genes conferring resistance and virulence.
  6. Prepare report by listing SNP location, gene, and number of resistant or susceptible isolates (Table 6).

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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...

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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...

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Disclosures

The authors have no competing financial interests and no conflict of interest to disclose.

Acknowledgements

This work was supported by the Centre for Infectious Diseases and Microbiology, Public Health. The authors have not received any other funding for this study. The authors thank Drs Alicia Arnott, Nathan Bachmann and Ranjeeta Menon for their expert advice and assistance with the whole genome sequencing experiment.

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Materials

NameCompanyCatalog NumberComments
DensiCHECK PlusBioMérieux IncK083536Densitometer used for McFarland readings
Sensititre YeastOneTREK Diagnostic Systems, Thermo ScientificYO10Commercial susceptibility assay plate with standard antifungal drugs.
Fisherbrand Disposable Inoculating Loops and NeedlesFisher Scientific, Thermo Fisher Scientific22-363-605Disposable plastic loops can be used directly from package. No flaming required.
Eppendorf Safe-Lock microcentrifuge tubesSigma Aldrich, MerckT2795   Volume 2.0 mL, natural
 
ZYMOLYASE 20T from Arthrobacter luteusMP Biomedicals, LLC8320921Used for cell wall lysis of fungal isolate before
DNA extraction
Wizard Genomic DNA Purification Kit Promega A1120Does 100 DNA extractions
Quant-iT PicoGreen dsDNA Assay KitThermo Fisher ScientificP7589 Picogreen reagent referred to as fluorescent dye in the protocol.
Includes Lambda DNA standard and picogreen reagent for assay.
Nextera XT DNA Sample Preparation KitIlluminaFC-131-1096Includes Box 1 and Box 2  reagents for 96 samples
Nextera XT Index Kit v2IlluminaFC-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 IlluminaFC-404-2004300 cycles, More than 250 samples per kit
NextSeq 500 Mid Output v2 KitIlluminaFC-404-2003300 cycles, More than 130 samples per kit
PhiX Control KitIlluminaFC-110-3001To arrange indices from Index kit in order
TruSeq Index Plate Fixture KitFC-130-1005 2 Fixtures
KAPA Library Quantification Kit
for Next-Generation Sequencing		KAPA BiosystemsKK4824Includes premade standards, primers and MasterMix
Janus NGS Express Liquid handling system PerkinElmerYJS4NGSUsed for DNA dilutions during sequencing
 0.8 mL Storage PlateThermo ScientificAB0765BMIDI Plate for DNA Library cleanup and
normalisation
Agencourt AMPure XPBeckman CoulterA63881 Magnetic beads in solution for library purification
Magnetic Stand-96Thermo Fisher ScientificAM10027Used for magnetic bead based DNA purification
OrbiShaker MP Benchmark ScientificBT150296-well plate shaker with 4 platforms
Hard Shell PCR PlateBioRadHSP9601Thin Wall, 96 Well
LightCycler 480 Instrument II Roche 5015278001Accomodates 96 well plate
Microseal 'B' PCR Plate Sealing Film, adhesive, optical BioRad MSB1001Clear 96-well plate sealers
CLC Genomics WorkbenchQiagenCLCBioSoftware for data analysis, Version 8
NextSeq500 instrumentIllumina Illumina Benchtop Sequencer used for next generation sequencing

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