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.

No ethical approval was required for this study.
1. Subculture and inoculum preparation for Candida glabrata
<ol>
	<li>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.</li>
	<li>Subculture an isolate by touching a single colony using a sterile disposable plastic loop and streaking onto a Sabouraud&#39;s dextrose agar (SDA) plate8.</li>
	<li>Incubate the SDA plate for 24-48 h at 35 &#176;C for pure culture of isolate with good growth.</li>
</ol>
2. Determination of Antifungal Susceptibility
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Perform susceptibility testing using commercial assay (see Table of Materials and Reagents) on all C. glabrata isolates following manufacturer&#39;s instructions. Interpret the susceptibility of isolates based on resultant MICs of antifungal drugs according to CLSI guidelines and prepare report (Table 1)8.</li>
</ol>
3. Genomic DNA extraction for sequencing
<ol>
	<li>Resuspend a loopful of colonies from a freshly grown SDA plate in 300 &#181;L of 50 mM EDTA in a 1.5 mL tube.</li>
	<li>Add 40 &#181;L of zymolyase (10 mg/mL) to the suspension, and gently pipet 5 times until suspension is uniform.</li>
	<li>Incubate the sample at 37 &#176;C for 1-2 h to digest the cell wall. Cool at room temperature for 5 min.</li>
	<li>Centrifuge the suspension at 14,000 &#215; g for 2 min and then carefully remove the supernatant.</li>
	<li>Extract genomic DNA following DNA extraction kit guidelines (see Table of Materials).</li>
	<li>Resuspend extracted DNA pellet in 50 &#181;L of 10 mM Tris Buffer (pH 7.5-8.5) instead of the elution buffer provided in the kit.</li>
	<li>Check the purity of DNA by measurement of optical density (O.D) at 260/280 nm25.</li>
</ol>
4. Genomic DNA quantification
<ol>
	<li>Prepare a 1x Tris EDTA (TE) buffer provided in the assay kit based on manufacturer&#39;s guidelines for the fluorescence assay (see Table of Materials).</li>
	<li>Dilute the DNA sample by adding 2 &#181;L to 98 &#181;L of 1x TE assay buffer (final volume of 100 &#181;L, 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.</li>
	<li>Prepare the range of standards by diluting the lambda DNA (100 &#181;g/mL) provided in the fluorescence assay kit (Table 2) and include for measurement along with samples.</li>
	<li>Add 100 &#181;L of the fluorescent dye to 100 &#181;L diluted DNA samples and standards for the reaction. Incubate for 5 min at room temperature, protected from light.</li>
	<li>Measure the fluorescence of all samples based on manufacturer&#39;s guidelines.</li>
	<li>Plot the standard curve using the fluorescence readings and calculate the original concentration of the DNA samples.</li>
	<li>Determine the volume of DNA and 10 mM Tris buffer (pH 8) to be added to adjust the DNA concentration to 0.2 ng/&#181;L.</li>
	<li>Perform the DNA dilutions using automated liquid handling workstation. This step can be also achieved by manual pipetting.</li>
</ol>
5. DNA Library Preparation
Note: Library preparation and sequencing was performed following manufacturer&#39;s protocols and guidelines provided by company (Figure 1A) (see Table of Materials).
<ol>
	<li>Tagmentation and PCR amplification
	<ol>
		<li>Label a new 96-well hard-shell thin wall plate.</li>
		<li>Add 5 &#181;L of quantified input DNA at 0.2 ng/&#181;L (1 ng in total) to each sample well of the plate.</li>
		<li>Add 10 &#181;L of tagmentation buffer and 5 &#181;L of amplification buffer to each well containing DNA and gently pipette to mix. Seal the plate with an adhesive plate seal.</li>
		<li>Place the plate in a thermal cycler and run the following PCR program: 55 &#176;C for 5 min, and hold at 10 &#176;C. When the sample reaches 10 &#176;C, proceed immediately to neutralize.</li>
		<li>Add 5 &#181;L 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.</li>
		<li>Add 15 &#181;L of indexing PCR mastermix to the samples.</li>
		<li>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).</li>
		<li>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.</li>
		<li>Place the tagmentation plate with added PCR mastermix on the index plate rack with the index tubes in order.</li>
		<li>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 &#181;L of index primers to each sample.</li>
		<li>Replace old caps of index tubes with new caps to avoid cross-contamination between indices.</li>
		<li>Seal the plate using 96-well clear plate sealers and perform the second PCR as follows: 95 &#176;C for 30 s, 12 cycles of 95 &#176;C for 10 s, 55 &#176;C for 30 s, 72 &#176;C for 30 s and 72 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>PCR Cleanup
	<ol>
		<li>Transfer the PCR product for PCR-cleanup, from the tagmentation plate to a deep-well plate (see Table of Materials).</li>
		<li>Vortex the commercial magnetic beads solution (see Table of Materials) based on manufacturer&#39;s instructions and add 30 &#181;L of beads to each PCR product in deep-well plate.</li>
		<li>Shake the plate on a microplate shaker at 1800 rpm for 2 min and incubate at room temperature without shaking for 5 min.</li>
		<li>Place the plate on a magnetic stand for 2 min until the supernatant has cleared.</li>
		<li>Discard the supernatant carefully with the plate still on the magnetic stand.</li>
		<li>Add 200 &#181;L of freshly prepared 80% ethanol with the plate on the magnetic stand.</li>
		<li>Incubate the plate on the magnetic stand for 30 s and carefully remove and discard the supernatant without disturbing the beads.</li>
		<li>Repeat the wash step again and allow the beads to air-dry for 15 min. Remove excess ethanol if any.</li>
		<li>Remove the plate from the magnetic stand and add 52.5 &#181;L of resuspension buffer to the beads.</li>
		<li>Shake the plate on a microplate shaker at 1800 rpm for 2 min and incubate at room temperature for 2 min without shaking.</li>
		<li>Place the plate on the magnetic stand and allow the supernatant to clear.</li>
		<li>Using a multichannel pipette, carefully transfer 50 &#181;L of the supernatant from the cleanup plate to a new hard-shell plate.</li>
	</ol>
	</li>
	<li>Library Normalization
	<ol>
		<li>Thaw library normalization reagents according to manufacturer&#39;s guidelines (see Table of Materials).</li>
		<li>Transfer 20 &#181;L of the supernatant from the cleanup plate to a new deep-well plate.</li>
		<li>Add 45 &#181;L of magnetic bead suspension and seal the plate with a plate sealer.</li>
		<li>Shake the plate on a microplate shaker at 1800 rpm for 30 min. This incubation time is critical and should not be exceeded.</li>
		<li>Place the plate on a magnetic stand for 2 min and confirm that the supernatant has cleared (Figure 1B).</li>
		<li>Remove and discard the supernatant in an appropriate hazardous waste container with the plate still on the magnetic stand.</li>
		<li>Remove the plate from the magnetic stand and wash the beads with 45 &#181;L wash buffer.</li>
		<li>Shake the plate with wash buffer on a microplate shaker at 1800 rpm for 5 min.</li>
		<li>Place plate on magnetic stand for 2 min and discard supernatant when it turns clear.</li>
		<li>Remove the plate from the magnetic stand and repeat the wash with wash buffer again.</li>
		<li>Remove the plate from the magnetic stand and add 30 &#181;L of 0.1 N NaOH.</li>
		<li>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.</li>
		<li>Add 30 &#181;L of elution buffer to each well of a new 96-well hard-shell thin wall final normalized library plate.</li>
		<li>Transfer 30 &#181;L of supernatant from normalization plate to final normalized library plate to make final volume 60 &#181;L. The libraries are now ready to be sequenced.</li>
	</ol>
	</li>
	<li>Determination of DNA Library Concentration by qPCR
	<ol>
		<li>Thaw the mastermix, primers and standards provided in the qPCR kit according to manufacturer&#39;s guidelines (see Table of Materials).</li>
		<li>Combine the PCR reagent mastermix and primer provided in the qPCR kit following manufacturer&#39;s instructions and aliquots can be stored at -20 &#176;C.</li>
		<li>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 &#181;L DNA library to 148.5 &#181;L Tris buffer) followed by a 1:80 dilution (2 &#181;L from 1:100 to 158 &#181;L Tris buffer).</li>
		<li>Shake the dilution plate at 700 rpm for at least 1 min and then centrifuge at 14000 &#215; g for 1 min.</li>
		<li>Prepare 20 &#181;L of final PCR reaction by mixing 4 &#181;L of diluted DNA library or DNA standards and 16 &#181;L of mastermix.</li>
		<li>Perform PCR following manufacturer&#39;s settings in thermocycler: 95 &#176;C for 5 min, 35 cycles of 95 &#176;C for 30 s and 60 &#176;C for 45 s, and final 65 &#176;C to 95 &#176;C for melt curve analysis.</li>
		<li>Obtain the Ct values of the sample DNA libraries and standards from the qPCR thermocycler.</li>
		<li>Generate a standard curve from the Ct value of the standards (Figure 2). Define the upper and lower QC range by &#177; 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.</li>
		<li>Determine the individual and average library concentrations (ALC) from the standard curve and Ct values.</li>
		<li>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 &#181;L of library is added to 835 &#181;L of buffer, then from this dilution (Dil 1) 195 is added to a total volume of 1300 &#181;L: (65/900) * dnPAL = Dil1 
		Dil1 * (195/1300) = Final Concentration (should be between 1.4-1.8 pM)</li>
	</ol>
	</li>
</ol>
6. Library pooling and Initiating Sequencing in Benchtop Sequencer
<ol>
	<li>Thaw reagent cartridge according to manufacturer&#39;s guidelines. Take out a new flow cell from its package from 4 &#176;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).</li>
	<li>Prepare a control library by mixing 5 &#181;L of library (1 nM) and 5 &#181;L of 0.2 N NaOH. Vortex briefly and incubate for 5 min at room temperature to denature the control library into single strands.</li>
	<li>Add 5 &#181;L of 200 mM Tris-HCl, pH 7 and vortex. Add 235 &#181;L prechilled sequencing buffer and mix gently. The total volume is 250 &#181;L with control library final concentration at 20 pM.</li>
	<li>Pool DNA libraries by transferring 5 &#181;L of each sample library to be sequenced from the final normalized library plate into a single low-bind 1.5 mL tube.</li>
	<li>Add 30 &#181;L of pooled library and 30 &#181;L of 0.2 N NaOH to denature libraries in another low bind tube.</li>
	<li>Vortex the low-bind tube and incubate for 5 min at room temperature to denature libraries into single strands.</li>
	<li>Add 30 &#181;L of 200 mM Tris-HCl, pH 7 to tube with denatured libraries to neutralize reaction.</li>
	<li>Add 65 &#181;L of neutralized denatured libraries suspension and 835 &#181;L of pre-chilled sequencing buffer and vortex to mix well.</li>
	<li>In a final low bind tube combine the following: 195 &#181;L from neutralized denatured libraries, 1.30 &#181;L of control library and 1103.70 &#181;L of sequencing buffer. Mix properly.</li>
	<li>Load the final library mix (1300 &#181;L) into the designated spot on reagent cartridge.</li>
	<li>Set-up the sequencing run by entering project and sample details in the Sequencer designated website following guidelines.</li>
	<li>Initiate sequencing following guidelines. Load flow cell, reagent cartridge with libraries and buffer cartridge in benchtop sequencer.</li>
	<li>Record batch numbers of all reagent kits and cartridges used in the sequencing.</li>
</ol>
7. Data Download from Sequencing Website
<ol>
	<li>Download FASTQ files following manufacturer&#39;s instructions provided on website.</li>
	<li>For a good quality run check that the percentage Q30 is &#8805; 75% and cluster density is between 170-280 K/mm2 with optimal at 200-210 K/mm2 (Table 4).</li>
</ol>
8. Sequencing Data analysis
<ol>
	<li>Import FASTQ files of sequenced samples into data analysis integrated software package (see Table of Materials).</li>
	<li>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.</li>
	<li>Run workflow by selecting a single sample or a batch of sample FASTQ files and save output files in designated sample folders.</li>
	<li>Generate report for sequence coverage depth, mapped regions and list of structural variants in genome (Figure 3B).</li>
	<li>Use list of structural variants to search for non-synonymous single nucleotide polymorphisms (SNPs) in genes conferring resistance and virulence.</li>
	<li>Prepare report by listing SNP location, gene, and number of resistant or susceptible isolates (Table 6).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+nucleic+acid+purities+monitored+by+260nm/280nm+absorbance+ratios.">Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios.</a> BioTechniques. 18 (1), 62-63 (1995).</li><li>Cannon, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efflux-mediated+antifungal+drug+resistance.">Efflux-mediated antifungal drug resistance.</a> Clin Microbiol Rev. 22 (2), 291-321 (2009).</li><li>Ferrari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+function+mutations+in+CgPDR1.+of+Candida+glabrata+not+only+mediate+antifungal+resistance+but+also+enhance+virulence.">Gain of function mutations in CgPDR1. of Candida glabrata not only mediate antifungal resistance but also enhance virulence.</a> PLoS Pathog. 5 (1), 1000268 (2009).</li><li>Rodrigues, C. F., Silva, S., Henriques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+glabrata:+a+review+of+its+features+and+resistance.">Candida glabrata: a review of its features and resistance.</a> Eur J Clin Microbiol Infect Dis. 33 (5), 673-688 (2014).</li><li>Garcia-Effron, G., Lee, S., Park, S., Cleary, J. D., Perlin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Candida+glabrata+FKS1+and+FKS2+mutations+on+echinocandin+sensitivity+and+kinetics+of+1,3-beta-D-glucan+synthase:+implication+for+the+existing+susceptibility+breakpoint.">Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-beta-D-glucan synthase: implication for the existing susceptibility breakpoint.</a> Antimicrob Agents Chemother. 53 (9), 3690-3699 (2009).</li><li>Arendrup, M. C., Perlin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echinocandin+resistance:+an+emerging+clinical+problem.">Echinocandin resistance: an emerging clinical problem.</a> Curr Opin Infect Dis. 27 (6), 484-492 (2014).</li><li>Costa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Mechanisms+of+Flucytosine+Resistance+in+C.+glabrata+Unveiled+by+a+Chemogenomics+Analysis+in+S.+cerevisiae.">New Mechanisms of Flucytosine Resistance in C. glabrata Unveiled by a Chemogenomics Analysis in S. cerevisiae.</a> PLoS One. 10 (8), 0135110 (2015).</li><li>de Groot, P. W., Bader, O., de Boer, A. D., Weig, M., Chauhan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesins+in+human+fungal+pathogens:+glue+with+plenty+of+stick.">Adhesins in human fungal pathogens: glue with plenty of stick.</a> Eukaryot Cell. 12 (4), 470-481 (2013).</li><li>de Groot, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+wall+of+the+human+pathogen+Candida+glabrata:+differential+incorporation+of+novel+adhesin-like+wall+proteins.">The cell wall of the human pathogen Candida glabrata: differential incorporation of novel adhesin-like wall proteins.</a> Eukaryot Cell. 7 (11), 1951-1964 (2008).</li><li>Vale-Silva, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+the+adhesin+gene+EPA1+mediated+by+PDR1+in+Candida+glabrata+leads+to+enhanced+host+colonization.">Upregulation of the adhesin gene EPA1 mediated by PDR1 in Candida glabrata leads to enhanced host colonization.</a> mSphere. 1 (2), (2016).</li></ol>

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

&#160; &#160;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...

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 &#39;future-proof&#39;&#160;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.

<table><tbody><tr><td>DensiCHECK Plus</td><td>BioM&amp;eacute;rieux Inc</td><td>K083536</td><td>Densitometer used for McFarland readings</td></tr><tr><td>Sensititre YeastOne</td><td>TREK Diagnostic Systems, Thermo Scientific</td><td>YO10</td><td>Commercial susceptibility assay plate with standard antifungal drugs.</td></tr><tr><td>Fisherbrand Disposable Inoculating Loops and Needles</td><td>Fisher Scientific, Thermo Fisher Scientific</td><td>22-363-605</td><td>Disposable plastic loops can be used directly from package. No flaming required.</td></tr><tr><td>Eppendorf Safe-Lock microcentrifuge tubes</td><td>Sigma Aldrich, Merck</td><td>T2795&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Volume 2.0 mL, natural&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>ZYMOLYASE 20T from Arthrobacter luteus</td><td>MP Biomedicals, LLC</td><td>8320921</td><td>Used for cell wall lysis of fungal isolate before&lt;br/&gt; DNA extraction</td></tr><tr><td>Wizard Genomic DNA Purification Kit&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>A1120</td><td>Does 100 DNA extractions</td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Thermo Fisher Scientific</td><td>P7589</td><td>&amp;nbsp;Picogreen reagent referred to as fluorescent dye in the protocol.&lt;br/&gt; Includes Lambda DNA standard and picogreen reagent for assay.</td></tr><tr><td>Nextera XT DNA Sample Preparation Kit</td><td>Illumina</td><td>FC-131-1096</td><td>Includes Box 1 and Box 2&amp;nbsp; reagents for 96 samples</td></tr><tr><td>Nextera XT Index Kit v2</td><td>Illumina</td><td>FC-131-2001,&lt;br/&gt; FC-131-2002,&lt;br/&gt; FC-131-2003,&lt;br/&gt; FC-131-2004</td><td>Index set A&lt;br/&gt; Index set B&lt;br/&gt; Index set C&lt;br/&gt; Index set D</td></tr><tr><td>NextSeq 500/550 High Output Kit v2&amp;nbsp;</td><td>Illumina</td><td>FC-404-2004</td><td>300 cycles, More than 250 samples per kit</td></tr><tr><td>NextSeq 500 Mid Output v2 Kit</td><td>Illumina</td><td>FC-404-2003</td><td>300 cycles, More than 130 samples per kit</td></tr><tr><td>PhiX Control Kit</td><td>Illumina</td><td>FC-110-3001</td><td>To arrange indices from Index kit in order</td></tr><tr><td>TruSeq Index Plate Fixture Kit</td><td></td><td>FC-130-1005</td><td>&amp;nbsp;2 Fixtures</td></tr><tr><td>KAPA Library Quantification Kit&lt;br/&gt; for Next-Generation Sequencing</td><td>KAPA Biosystems</td><td>KK4824</td><td>Includes premade standards, primers and MasterMix</td></tr><tr><td>Janus NGS Express Liquid handling system&amp;nbsp;</td><td>PerkinElmer</td><td>YJS4NGS</td><td>Used for DNA dilutions during sequencing</td></tr><tr><td>&amp;nbsp;0.8 mL Storage Plate</td><td>Thermo Scientific</td><td>AB0765B</td><td>MIDI Plate for DNA Library cleanup and&lt;br/&gt; normalisation</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>&amp;nbsp;Magnetic beads in solution for library purification</td></tr><tr><td>Magnetic Stand-96</td><td>Thermo Fisher Scientific</td><td>AM10027</td><td>Used for magnetic bead based DNA purification</td></tr><tr><td>OrbiShaker MP&amp;nbsp;</td><td>Benchmark Scientific</td><td>BT1502</td><td>96-well plate shaker with 4 platforms</td></tr><tr><td>Hard Shell PCR Plate</td><td>BioRad</td><td>HSP9601</td><td>Thin Wall, 96 Well</td></tr><tr><td>LightCycler 480 Instrument II&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>5015278001</td><td>Accomodates 96 well plate</td></tr><tr><td>Microseal &#039;B&#039; PCR Plate Sealing Film, adhesive, optical&amp;nbsp;</td><td>BioRad</td><td>&amp;nbsp;MSB1001</td><td>Clear 96-well plate sealers</td></tr><tr><td>CLC Genomics Workbench</td><td>Qiagen</td><td>CLCBio</td><td>Software for data analysis, Version 8</td></tr><tr><td>NextSeq500 instrument</td><td>Illumina&amp;nbsp;</td><td>Illumina&amp;nbsp;</td><td>Benchtop Sequencer used for next generation sequencing</td></tr></tbody></table>

whole genome sequencing of candida glabrata for detection of markers of antifungal drug resistance

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.&#160;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.

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...

Watch this Scientific Journal Video about Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance at JoVE.com

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

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.

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

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

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Research

JoVE Journal

Medicine

13.7K Views.  Westmead Hospital. Deze studie uitgevoerd hele genoom sequencing p.a. van mutaties in genen antischimmel medicamenteuse resistentie bij Candida glabratatoekennen. C. glabrata isolaten resistent tegen echinocandins, azoles en 5-flucytosine, werden sequenced ter illustratie van de methodologie. De profielen van de gevoeligheid van de isolaten gecorreleerd met aanwezigheid of afwezigheid van bepaalde mutatie patronen in de genen.

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Immunology and Infection

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Genetics

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Biology

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&amp;RUN Method and Data Analysis Workflow

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&amp;RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&amp;RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&amp;RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&amp;RUN data analysis.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow

This protocol describes an experimental method and data analysis workflow for cleavage under targets and release using nuclease (CUT&amp;RUN) in the human fungal pathogen Candida albicans.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations ...

Analyzing Petite Colony Formation and Drug Resistance in Candida glabrata

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Author Spotlight: Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug&#45;Resistant Candida for Antifungal Research

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

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

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