Name: whole genome sequencing of candida glabrata for detection of markers of antifungal drug resistance
Uploaded: 2017-12-28T15:00:00
Description: Watch this Scientific Journal Video about Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance at JoVE.com

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) plate<sup class...

<ol>
	<li>Beyda, N. 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=FKS+mutant+Candida+glabrata:+risk+factors+and+outcomes+in+patients+with+candidemia.">FKS mutant Candida glabrata: risk factors and outcomes in patients with candidemia.</a> Clin Infect Dis. 59 (6), 819-825 (2014).</li><li>Chapeland-Leclerc, F., 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=Acquisition+of+flucytosine,+azole,+and+caspofungin+resistance+in+Candida+glabrata.+bloodstream+isolates+serially+obtained+from+a+hematopoietic+stem+cell+transplant+recipient.">Acquisition of flucytosine, azole, and caspofungin resistance in Candida glabrata. bloodstream isolates serially obtained from a hematopoietic stem cell transplant recipient.</a> Antimicrob Agents Chemother. 54 (3), 1360-1362 (2010).</li><li>Glockner, A., Cornely, O. A. <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-unique+features+and+challenges+in+the+clinical+management+of+invasive+infections.">Candida glabrata-unique features and challenges in the clinical management of invasive infections.</a> Mycoses. 58 (8), 445-450 (2015).</li><li>Pfaller, M. 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=Frequency+of+decreased+susceptibility+and+resistance+to+echinocandins+among+fluconazole-resistant+bloodstream+isolates+of+Candida+glabrata.">Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata.</a> J Clin Microbiol. 50 (4), 1199-1203 (2012).</li><li>Lewis, J. S., Wiederhold, N. P., Wickes, B. L., Patterson, T. F., Jorgensen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+emergence+of+echinocandin+resistance+in+Candida+glabrata+resulting+in+clinical+and+microbiologic+failure.">Rapid emergence of echinocandin resistance in Candida glabrata resulting in clinical and microbiologic failure.</a> Antimicrob Agents Chemother. 57 (9), 4559-4561 (2013).</li><li>Klevay, M. J., 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=Therapy+and+outcome+of+Candida+glabrata+versus+Candida+albicans+bloodstream+infection.">Therapy and outcome of Candida glabrata versus Candida albicans bloodstream infection.</a> Diagn Microbiol Infect Dis. 60 (3), 273-277 (2008).</li><li>Arendrup, M. C., Cuenca-Estrella, M., Lass-Florl, C., Hope, W., Eucast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCAST+technical+note+on+the+EUCAST+definitive+document+EDef+7.2:+method+for+the+determination+of+broth+dilution+minimum+inhibitory+concentrations+of+antifungal+agents+for+yeasts+EDef+7.2+(EUCAST-AFST).">EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST).</a> Clin Microbiol Infect. 18 (7), 246-247 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+Method+for+Broth+Dilution+Antifungal+Susceptibility+Testing+of+Yeasts.">Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts.</a> Clinical and Laboratory Standards Institute. , (2012).</li><li>Arendrup, M. C., Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danish+Fungaemia+Study,+G.+Caspofungin+Etest+susceptibility+testing+of+Candida+species:+risk+of+misclassification+of+susceptible+isolates+of+C.+glabrata+and+C.+krusei+when+adopting+the+revised+CLSI+caspofungin+breakpoints.">Danish Fungaemia Study, G. Caspofungin Etest susceptibility testing of Candida species: risk of misclassification of susceptible isolates of C. glabrata and C. krusei when adopting the revised CLSI caspofungin breakpoints.</a> Antimicrob Agents Chemother. 56 (7), 3965-3968 (2012).</li><li>Singh-Babak, S. 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=Global+analysis+of+the+evolution+and+mechanism+of+echinocandin+resistance+in+Candida+glabrata.">Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata.</a> PLoS Pathog. 8 (5), 1002718 (2012).</li><li>Shields, R. K., Nguyen, M. H., Clancy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+perspectives+on+echinocandin+resistance+among+Candida+species.">Clinical perspectives on echinocandin resistance among Candida species.</a> Curr Opin Infect Dis. 28 (6), 514-522 (2015).</li><li>Dudiuk, 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=Set+of+classical+PCRs+for+detection+of+mutations+in+Candida+glabrata+FKS.+genes+linked+with+echinocandin+resistance.">Set of classical PCRs for detection of mutations in Candida glabrata FKS. genes linked with echinocandin resistance.</a> J Clin Microbiol. 52 (7), 2609-2614 (2014).</li><li>Koboldt, D. C., Steinberg, K. M., Larson, D. E., Wilson, R. K., Mardis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next-generation+sequencing+revolution+and+its+impact+on+genomics.">The next-generation sequencing revolution and its impact on genomics.</a> Cell. 155 (1), 27-38 (2013).</li><li>Barzon, L., 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=Next-generation+sequencing+technologies+in+diagnostic+virology.">Next-generation sequencing technologies in diagnostic virology.</a> J Clin Virol. 58 (2), 346-350 (2013).</li><li>Didelot, X., Bowden, R., Wilson, D. J., Peto, T. E. A., Crook, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+clinical+microbiology+with+bacterial+genome+sequencing.">Transforming clinical microbiology with bacterial genome sequencing.</a> Nat Rev Gen. 13 (9), 601-612 (2012).</li><li>Koser, C. U., 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=Routine+use+of+microbial+whole+genome+sequencing+in+diagnostic+and+public+health+microbiology.">Routine use of microbial whole genome sequencing in diagnostic and public health microbiology.</a> PLoS Pathog. 8 (8), 1002824 (2012).</li><li>Lipkin, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+face+of+pathogen+discovery+and+surveillance.">The changing face of pathogen discovery and surveillance.</a> Nat Rev Microbiol. 11 (2), 133-141 (2013).</li><li>Sintchenko, V., Holmes, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pathogen+genomics+in+assessing+disease+transmission.">The role of pathogen genomics in assessing disease transmission.</a> BMJ. 350, 1314 (2015).</li><li>Garnaud, 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=Next-generation+sequencing+offers+new+insights+into+the+resistance+of+Candida+spp.+to+echinocandins+and+azoles.">Next-generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles.</a> J Antimicrob Chemother. 70 (9), 2556-2565 (2015).</li><li>Sanmiguel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+and+potential+applications+in+fungal+genomics.">Next-generation sequencing and potential applications in fungal genomics.</a> Methods Mol Biol. 722, 51-60 (2011).</li><li>Mardis, E. 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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. 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&#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.

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		<tr height="21">
			<td height="21" style="height:21px;width:275px;">DensiCHECK Plus</td>
			<td style="width:183px;">BioM&#233;rieux Inc</td>
			<td style="width:151px;">K083536</td>
			<td style="width:383px;">Densitometer used for McFarland readings</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Sensititre YeastOne</td>
			<td style="width:183px;">TREK Diagnostic Systems, Thermo Scientific</td>
			<td style="width:151px;">YO10</td>
			<td style="width:383px;">Commercial susceptibility assay plate with standard antifungal drugs.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Fisherbrand Disposable Inoculating Loops and Needles</td>
			<td style="width:183px;">Fisher Scientific, Thermo Fisher Scientific</td>
			<td style="width:151px;">22-363-605</td>
			<td style="width:383px;">Disposable plastic loops can be used directly from package. No flaming required.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Eppendorf Safe-Lock microcentrifuge tubes</td>
			<td style="width:183px;">Sigma Aldrich, Merck</td>
			<td style="width:151px;">T2795&#160;&#160;&#160;</td>
			<td style="width:383px;">Volume 2.0 mL, natural 
			&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">ZYMOLYASE 20T from Arthrobacter luteus</td>
			<td style="width:183px;">MP Biomedicals, LLC</td>
			<td style="width:151px;">8320921</td>
			<td style="width:383px;">Used for cell wall lysis of fungal isolate before 
			DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Wizard Genomic DNA Purification Kit&#160;</td>
			<td style="width:183px;">Promega&#160;</td>
			<td style="width:151px;">A1120</td>
			<td>Does 100 DNA extractions</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:275px;">Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td style="width:183px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">P7589</td>
			<td style="width:383px;">&#160;Picogreen reagent referred to as fluorescent dye in the protocol. 
			Includes Lambda DNA standard and picogreen reagent for assay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Nextera XT DNA Sample Preparation Kit</td>
			<td style="width:183px;">Illumina</td>
			<td style="width:151px;">FC-131-1096</td>
			<td style="width:383px;">Includes Box 1 and Box 2&#160; reagents for 96 samples</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:275px;">Nextera XT Index Kit v2</td>
			<td style="width:183px;">Illumina</td>
			<td style="width:151px;">FC-131-2001, 
			FC-131-2002, 
			FC-131-2003, 
			FC-131-2004</td>
			<td style="width:383px;">Index set A 
			Index set B 
			Index set C 
			Index set D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">NextSeq 500/550 High Output Kit v2&#160;</td>
			<td style="width:183px;">Illumina</td>
			<td style="width:151px;">FC-404-2004</td>
			<td>300 cycles, More than 250 samples per kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">NextSeq 500 Mid Output v2 Kit</td>
			<td style="width:183px;">Illumina</td>
			<td style="width:151px;">FC-404-2003</td>
			<td>300 cycles, More than 130 samples per kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">PhiX Control Kit</td>
			<td style="width:183px;">Illumina</td>
			<td style="width:151px;">FC-110-3001</td>
			<td style="width:383px;">To arrange indices from Index kit in order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">TruSeq Index Plate Fixture Kit</td>
			<td style="width:183px;"></td>
			<td style="width:151px;">FC-130-1005</td>
			<td>&#160;2 Fixtures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">KAPA Library Quantification Kit 
			for Next-Generation Sequencing</td>
			<td style="width:183px;">KAPA Biosystems</td>
			<td style="width:151px;">KK4824</td>
			<td>Includes premade standards, primers and MasterMix</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Janus NGS Express Liquid handling system&#160;</td>
			<td style="width:183px;">PerkinElmer</td>
			<td style="width:151px;">YJS4NGS</td>
			<td>Used for DNA dilutions during sequencing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">&#160;0.8 mL Storage Plate</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:151px;">AB0765B</td>
			<td style="width:383px;">MIDI Plate for DNA Library cleanup and 
			normalisation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agencourt AMPure XP</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:151px;">A63881</td>
			<td>&#160;Magnetic beads in solution for library purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Magnetic Stand-96</td>
			<td style="width:183px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">AM10027</td>
			<td>Used for magnetic bead based DNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">OrbiShaker MP&#160;</td>
			<td style="width:183px;">Benchmark Scientific</td>
			<td style="width:151px;">BT1502</td>
			<td>96-well plate shaker with 4 platforms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hard Shell PCR Plate</td>
			<td style="width:183px;">BioRad</td>
			<td style="width:151px;">HSP9601</td>
			<td>Thin Wall, 96 Well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Instrument II&#160;</td>
			<td style="width:183px;">Roche&#160;</td>
			<td style="width:151px;">5015278001</td>
			<td>Accomodates 96 well plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical&#160;</td>
			<td style="width:183px;">BioRad</td>
			<td style="width:151px;">&#160;MSB1001</td>
			<td>Clear 96-well plate sealers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">CLC Genomics Workbench</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:151px;">CLCBio</td>
			<td>Software for data analysis, Version 8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">NextSeq500 instrument</td>
			<td style="width:183px;">Illumina&#160;</td>
			<td style="width:151px;">Illumina&#160;</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 ...

The overall goal of this study is to implement whole genome sequencing for identification of mutations in genes that confer antifungal drug resistance in Candida glabrata in a diagnostic laboratory. Whole genome sequencing of fungi can make significant contributions in the field of antifungal drug resistance. It allows simultaneous detection of genome-wide mutations in drug-resistant fungi to important antifungal agents.Whole genome sequencing of medically important fungi can also enable the detection and monitoring of outbreaks, of fungal disease, and transmission events in health care settings, as well as emergence of fungal species and clones that clinicians would need to know about. Demonstrating this procedure will be Dr.Rebecca Rockett and Dr.Verlaine Timms both doctoral research fellows from our laboratory. To begin, prepare genomic DNA samples from clinical and commercial strains of Candida glabrata.Then add five microliters of quantified DNA to each well of previously-labeled fresh 96-well hard-shell thin-wall plate. Add 10 microliters of tagmentation buffer and five microliters of amplification buffer to each well. Then use a pipette to gently mix the samples and seal the plate with an adhesive seal, put the plate inside a thermal cycler, and run the appropriate PCR program.When the sample has reached 10 degrees Celsius, immediately add five microliters of neutralization buffer and use a pipette to gently mix the samples. Then seal the plate, and incubate at room temperature for five minutes. After the incubation period, add 15 microliters of Indexing PCR Master Mix to each well.Arrange primer tools in an index plate rack and record the position of the indices on the template. Then place the tagmentation plate containing PCR Master Mix on the index plate rack with the index tubes in the correct order. It is important to arrange the index primer one tubes in horizontal orientation and the index primer two tubes in vertical orientation.It is based on the order and position provided in the index template. Remove the old caps from the index primer tubes and discard them. With a multi-channel pipette, add five microliters of index primers to each well.It is important to perform this step carefully in order to avoid cross contamination between samples and indices. To prevent cross contamination between indices, replace each of the old index tube caps. Finally, seal the plate and run the second PCR program.First transfer the PCR product from the tagmentation plate to a deep-well plate. Vortex a magnetic bead solution and add 30 microliters of beads to each well. Then agitate the plate on a microplate shaker and incubate at room temperature for five minutes.Place the plate on a magnetic stand for two minutes or until the supernatant is clear. Then, without moving the plate, carefully discard the supernatant and add 200 microliters of 80%ethanol. With the plate still on the magnetic stand, incubate for 30 seconds before discarding the supernatant.Repeat the washing process and allow the beads to dry for 15 minutes. Remove the plate from the magnetic stand and add 52.5 microliters of resuspension buffer to the beads. Using a microplate shaker, agitate the plate for two minutes at 1, 800 RPM.Then after the shaker has stopped, incubate the plate at room temperature for two minutes. After the incubation period has ended, place the plate on a magnetic stand until the supernatant is clear. Finally, use a multi-channel pipette to transfer 50 microliters of the clear supernatant from the cleanup tray to a fresh hard-shell plate.First follow library normalization reagents and transfer 20 microliters of the clear supernatant from the hard-shell plate to a new deep-well plate. Add 45 microliters of a magnetic bead suspension to each well of the deep-well plate and use a plate sealer to seal the plate. Next, agitate the plate on a microplate shaker at 1, 800 RPM for 30 minutes.Place the plate on a magnetic stand for two minutes until the supernatant has cleared. Use a pipette to remove the supernatant and dispose of the supernatant into an appropriate hazardous waste container. Then remove the plate from the magnetic stand and use 45 microliters of wash buffer to wash the beads.Agitate the plate on a microplate shaker and place the plate on a magnetic stand for two minutes. After the supernatant clears, discard the supernatant and remove the plate from the magnetic stand. Repeat the washing process with wash buffer once more.Before removing the plate from the magnetic stand, add NaOH to each well. Then agitate the plate on a microplate shaker at 1, 800 RPM for five minutes before placing the plate on a magnetic stand until the liquid is clear. Add 30 microliters of elution buffer to each well of a new 96-well hard-shell thin-wall plate.Finally, transfer 30 microliters of supernatant from the normalization plate to each of the wells containing elution buffer. Now, the final normalized library plate is ready to be sequenced in further experiments. 13 C.glabrata isolates obtained before and after antifungal therapy comprised of C.glabrata ATCC 90030 and twelve isolates from a clinical laboratory were studied.The minimum inhibitory concentration, or MIC, was obtained for each isolate for nine antifungal agents. This table lists the MIC values for each isolate antifungal test condition. The presence of SNPs in genes conferring resistance in isolate correlated with elevated in-vitro MIC against antifungal drugs.This table summarizes the SNP positions in the clinical C.glabrata isolates and the drugs that the genes confer resistance to. After watching this video, viewers can replicate this technique in a diagnostic laboratory setting for accreditation in whole genome sequencing. It highlights the important steps in DNA library preparation and the essential quality control points for accurate and reproducible results.

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