A Candidozyma (Candida) auris-optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding FKS1 mutation

A Candidozyma (Candida) auris-optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding FKS1 mutation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A Candidozyma ( Candida ) auris -optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding FKS1 mutation Laura A. Doorley , Vanessa Meza-Perez , Sarah J. Jones , Jeffrey M. Rybak doi: https://doi.org/10.1101/2025.02.14.638356 Laura A. Doorley a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vanessa Meza-Perez a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sarah J. Jones a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeffrey M. Rybak a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: jrybak{at}stjude.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Objectives Mutations in the Candidozyma ( Candida ) auris β-glucan synthase gene ( FKS1 ) altering S639 are frequently associated with clinical echinocandin resistance. However, the direct impact of these mutations remains uncharacterized. We have developed a novel C. auris- optimized E pisomal P lasmid- I nduced C as9 (EPIC) gene-editing system capable of recyclable precision genome editing and demonstrate the contribution of FKS1 S639F mutation to echinocandin resistance. Methods The EPIC gene-editing system was generated for optimized use in C. auris , and ADE2 modification was evaluated in 5 C. auris clades. Mutations leading to Fks1 S639F and Fks1 WT were placed into echinocandin-susceptible and echinocandin-resistant isolates from Clade-III and -I, respectively, using the EPIC system. Echinocandin susceptibility was determined by CLSI broth microdilution, and cell wall abundance of chitin and β-glucan was assessed by staining with Calcofluor White (CFW) and Aniline Blue (AB). Results The EPIC system was capable of targeted ADE2 editing in C. auris isolates from 5 genetic clades and shown to be precise by confirmatory sequencing of 50 transformants. A single nucleotide change in FKS1 resulting in either the S639F substitution or a silent synonymous mutation was introduced in an echinocandin-susceptible Clade-III isolate. Precision FKS1 editing by the EPIC system was confirmed by whole genome sequencing. Subsequent susceptibility testing demonstrated introduction of the FKS1 S639F mutation to increase resistance to echinocandins. Moreover, introduction of a wildtype Fks1 sequence to an echinocandin-resistant Clade-I clinical isolate, correcting the FKS1 S639F mutation, resulted in a restoration of echinocandin sensitivity. Evaluation of cell wall composition showed isolates or strains harboring FKS1 S639F to contain significantly elevated β-glucan and chitin content relative to isogenic comparators. Conclusions These data demonstrate the potential of our EPIC system in its ability to introduce single nucleotide substitutions in multiple C. auris clade backgrounds while revealing the direct impact of the S639F encoding FKS1 mutation on echinocandin resistance. INTRODUCTION C. auris is an emerging fungal pathogen recognized by the Centers for Disease Control and Prevention (CDC) as an increasing threat to public health, calling for “urgent and aggressive action” [ 1 , 2 ]. Applying the current tentative CDC clinical breakpoints, approximately 90% of C. auris clinical isolates are found to be resistant to fluconazole, and up to 50% of isolates are resistant to amphotericin B [ 3 ]. Typically, echinocandin resistance is observed in only 1 to 5% of isolates, making echinocandins the front-line therapy of choice for many clinicians [ 3 ]. However, numerous cases of C. auris infections acquiring echinocandin resistance in host during treatment have been documented, leaving clinicians with no reliable therapeutic option when treating patients with C. auris infections [ 4 , 5 ]. Previous studies have shown that echinocandin resistance in species, such as Candida glabrata and Candida albicans is strongly associated with mutations in the genes encoding the echinocandin target β-glucan synthase ( GSC1 , also known as FKS1 , in C. albicans and the genes FKS1 and FKS2 in C. glabrata ) [ 6 - 8 ]. Furthermore, these mutations can be predictive of clinical failure for candidemia treatment [ 9 - 11 ]. The vast majority of FKS1 mutations cluster into 3 distinct “hot-spots”, all of which encode residues spatially near a single hydrophobic convex surface predicted to interact with both plasma membrane lipids and echinocandins [ 7 , 12 - 14 ] While the association between these mutations and clinical echinocandin resistance is undeniable, confirmation of the direct impact of these β-glucan synthase mutations by genetic interrogation has been hindered both by limited genetic tools and the practicality of manipulating the gene of interest (essentiality, large size: approximately 5.7kb, and proximity to flanking features). Previous characterization of β-glucan synthase mutations has relied on methods requiring exposure of cells to echinocandins (possibly introducing confounding adaptations) either directly through in vitro evolution studies or indirectly as a component of selection media to identify potential transformants [ 15 , 16 ] Alternatively, clinically derived Candida FKS1 mutations have been recreated in situ with the addition of large selection cassettes or through heterologous expression studies by site-directed mutagenesis in the model organism Saccharomyces cerevisiae [ 7 , 17 ]. Consequently, studies demonstrating the direct impact of mutations in Candida β-glucan synthase on clinical echinocandin resistance without these potential confounding variables are limited. In this work, we describe the creation of the C. auris- optimized E pisomal P lasmid I nduced C as9 (EPIC) genetic manipulation system, a transient and recyclable tool capable of single nucleotide editing without the need to integrate a dominant marker into the C. auris genome. We demonstrate system functionality in clinical isolates representing different clades of C. auris , confirm system precision using whole genome sequencing (WGS), and utilize the system to interrogate direct impacts of the FKS1 mutation leading to S639F substitution in two distinct C. auris clinical isolate backgrounds. MATERIALS AND METHODS Isolate, strains, and growth media All clinical isolates and derived strains used in this study are listed in Supplementary Table 1 . Long-term C. auris isolates and strains were maintained in 40% glycerol at -80°C and cultured in YPD media (1% yeast extract, 2% peptone, 2% dextrose) at 35°C, 220 RPM, unless noted otherwise. Construction of EPIC system components The C. auris- optimized EPIC vector, pJMR19 (accession: pending ) was constructed from pJMR17v3 utilizing restriction digest and ligation to remove the C. albicans ORI410 sequence and to replace the C. albicans ACT1 promoter, with the 1kb sequence upstream of the C. auris ACT1 gene (B9J08_000486) [ 18 ]. Vector verification via whole plasmid sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation. Duplexed guide sequence primers targeting the gene of interest were ligated into pJMR19 digested by Lgu1 (Thermo Scientific™), as previously described [ 18 , 19 ]. Transformation repair templates were created by PCR amplification of gBlock sequence (Integrated DNA technologies) followed by QIAquick PCR Purification Kit (Qiagen) or by duplexing approximately 80bp complementary primers (Integrated DNA Technologies) as previously described [ 18 , 19 ]. All vectors, primers, and templates are listed in Supplementary Table 1 . EPIC-based C. auris transformation C. auris was grown overnight in 25mL YPD to an OD 600 of 1.8-2.2. Cultures were pelleted at 4000 RPM for three minutes, washed with 1mL water, and pellets were resuspended in 1mL TE-LiAc (1M Tris-EDTA and 1M lithium acetate pH7.5) and centrifuged once more at 8000 RPM for 30 seconds. Pellets were resuspended in a minimally required TE-LiAc volume. Transformation reactions were assembled with 10µL boiled then cooled salmon sperm DNA, 8µg pJMR19, 8µg repair template DNA, 50µL resuspended pellet, and 300µL TE-LiAC + 55% polyethylene glycol (PEG). Transformations were incubated for 90 minutes with intermittent manual agitation, heat-shocked at 45°C for 15 minutes and pelleted at 8000 RPM for 30 seconds. Pellets were resuspended in 2mL YPD and incubated for 4 hours. Transformants were cultured on nourseothricin (200µg/mL)-supplemented YPD agar plates for selection. Single colonies were patched, screened and replica plated for plasmid ejection as previously described [ 18 , 19 ]. Validation of EPIC manipulation and specificity Genomic DNA (gDNA) was extracted from overnight C. auris transformant cultures using the MasterPure™ Yeast DNA Purification kit (Biosearch Technologies). C. auris FKS1 (B9J08_000964) and ‘landing-pad’ sequences were amplified by PCR and purified with QIAquick PCR Purification Kits (Qiagen). FKS1 sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology. C. auris ADE2 (B9J08_03951) deep sequencing was performed by the Center for Advanced Genome Engineering at St. Jude Children’s Research Hospital (SJCRH) utilizing a two-step PCR system and the Illumina Miseq sequencing platform. WGS was performed for the clinical isolate RVA001 and select RVA001-derived strains. Sample libraries were prepared from gDNA by the Hartwell Center at SJCRH using KAPA HyperPrep (Roche) and sequencing targeting 100x coverage/ 150bp reads was performed via Illumina HiSeq. Code4DNA ( Code4DNA.com ) provided bioinformatics analysis. Two sequence library runs per sample were merged for each paired end R1 and R2. Overlapping regions were error corrected using bbmerge 38.94. Reads were aligned to GCA_002775015.1 using bwa (0.7.17-r1188). Alignments were sorted and indexed by Samtools v1.13 and duplicates were marked with picard (2.26.0). Variants called by running chromosomal based Freebayes (1.3.4) on GNU parallel (20180722) were annotated using SnpEff (4.5covid19). Variants found in each affected sample but not the control were extracted with Vcftools (0.1.15) and manually evaluated using Jbrowse (jbrowse-desktop-v1.5.3). (SRA: pending ). Antifungal susceptibility testing Minimum inhibitory concentrations (MIC) for anidulafungin (Sigma-Aldrich), caspofungin (Fisher Scientific), micafungin (Sigma-Aldrich), and rezafungin (MedChemExpress) were determined by broth microdilution in accordance with the M27-A4 from the Clinical Laboratory Standards Institute [ 20 ]. All susceptibility testing was performed in biological triplicate. Relative cell wall constituent abundance C. auris strains grown on Sabouraud-dextrose (SD) agar were subsequently cultured in RPMI (RPMI1640, MOPS, 2% glucose, pH7) to mid-log phase. Suspensions were washed to remove media and fixed with 3.7% (v/v) formaldehyde. Pellets were washed, normalized for cell density, aliquoted, and stained for chitin or β-glucans with 25mg/L CFW (Fluorescent Brightener 28: Fisher Scientific) or 50mg/L AB (Fisher Scientific), respectively. BioTek Cytation7 with Gen5 software (Agilent) used to measure absorbance (OD 600 ) and fluorescence intensity (CFW: 380nm/475nm; AB: 395nm/495nm). RESULTS The EPIC genetic manipulation system is functional in multiple C. auris clinical isolate backgrounds The C. auris gene B9J08_03951 ( ADE2 ), predicted to encode phosphoribosylaminoimidazole carboxylase, was selected to demonstrate the functionality of the EPIC genetic manipulation system. Successful ADE2 disruption ( ADE2 dis ) produces red-pink colony coloration on standard culture media (SD or YPD) due to accumulation of pigmented metabolites in the adenine biosynthetic pathway ( Figure 1 ) . ADE2 dis transformations were carried out with the EPIC vector containing a guide sequence targeting the 5’ region of ADE2 (pJMR19- dADE2 ) and a DNA repair template encoding approximately 1000 bases of the 5’ region of ADE2 in five clinical isolates representing five C. auris clades. Transformants from each background including RVA001 (Clade-III, FKS1 WT ) and SKU067 (Clade-I, FKS1 S639F ), the C. auris isolates to be studied in subsequent FKS1- related experiments, yielded red-pink colonies. ( Supplementary Figure 1 ). Download figure Open in new tab Figure 1. Genetic manipulation of ADE2 via EPIC system. Schematic pJMR19 representing nourseothricin resistance transformation selection marker, Cas9, guide sequence, and autonomous replicating sequence-7 integration into pJMR19. Cas9 mediated disruption of ADE2 leading to formation of red-pink colonies in a Clade-I C. auris isolate, SKU067, on SD agar supplemented with nourseothricin 200mg/L. To assess incorporation of the ADE2 dis repair template, 50 red-pink colonies generated in RVA001 and SKU067 were selected for sequencing of the ADE2 locus (25 per isolate background and representing 3 independent transformations) ( Figure 2A ) . Of the 50 red-pink transformants sequenced, 45 were found to have the targeted manipulation (21 in RVA001 and 24 in SKU067) ( Figure 2B ) . The remaining 5 transformants harbored one of 3 alternative disruptions ( Figure 2B ). Download figure Open in new tab Figure 2. Sequence confirmation of ADE2 editing. A) Wildtype ADE2 sequence along with repair template showing intended manipulation highlighted in red to create ADE2 dis strains. B) Identification and distribution of four variant sequences of ADE2 dis strains sequenced from SKU067 and RVA001 ADE2 dis transformations. Variants identified via sequencing by the Center for Advanced Genome Engineering (SJCRH). Plotted values represent distribution for each variant among 50 screened red-pink colonies (25 per isolate background) from 3 independent transformations. The C. auris EPIC genetic manipulation system is capable of single nucleotide editing The C. auris EPIC system was further characterized by evaluating the direct impact of a single nucleotide FKS1 mutation frequently associated with clinical echinocandin resistance in the echinocandin-susceptible clinical isolate RVA001 [ 21 ]. Transformations were carried out using an FKS1 hot spot 1 (HS1)-guided EPIC vector (pJMR19- FKS1 :HS1a) alongside a PCR-generated repair template containing either the FKS1 sequence encoding an S639F substitution ( FKS1 S639F ) or the manipulation control sequence ( FKS1 WTs ; encoding a synonymous nucleotide substitution at codon S639). Following initial FKS1 sequencing confirmation, two independent transformants for both RVA001- FKS1 WTs and RVA001- FKS1 S639F were passaged in YPD media without nourseothricin to promote loss of the transiently maintained EPIC system vector. gDNA from the 4 transformants and the parental clinical isolate (RVA001) was then extracted for WGS confirmation. Incorporation of the intended single nucleotide substitutions in FKS1 was found in all 4 transformants. Three of the four transformants were found to have no other nucleotide variations in the genome relative to the parental isolate RVA001, while one strain, RVA001- FKS1 WTs -A, was found to have one additional single nucleotide variation in a non-coding region (Supplemental Table 2) . The S639F substitution in C. auris FKS1 confers resistance to clinically available echinocandins The MIC for all four clinically available echinocandins (anidulafungin, caspofungin, micafungin, and rezafungin) were then assessed for RVA001 and each of the derivative FKS1 strains. Strains harboring the S639F mutation in FKS1 were observed to exhibit elevations in MIC for all clinically available echinocandins relative to the parental RVA001 ( Figure 3 ) . Anidulafungin MIC increased 64-fold, caspofungin MIC increased 8-fold, micafungin MIC increased 256-fold, and rezafungin MIC increased 16-fold. Conversely, no change in echinocandin MIC was found between RVA001 and the FKS1 WTs control strains ( Figure 3 ). Download figure Open in new tab Figure 3. Effects of single FKS1 mutation on echinocandin susceptibility. Micafungin, anidulafungin, caspofungin, and rezafungin MICs were determined as outlined by the Clinical Laboratory Standards Institute (CLSI) guidelines using broth microdilution. Vertical green lines denote the threshold for 50% growth inhibition compared the growth control well. To further confirm the impact of the S639F mutation in C. auris FKS1 , we then used the EPIC system to introduce a sequence encoding wild-type (matching B8441 reference) Fks1 into the echinocandin-resistant clinical isolate SKU067, correcting the S639F substitution. Two independent strains were generated and MIC for all 4 clinically available echinocandins were found to have decreased below current clinical breakpoints ( Figure 3 ), definitively demonstrating that the FKS1 S639F mutation alone is sufficient to confer clinical echinocandin resistance in two independent clinical isolate backgrounds representing both Clade-I and Clade-III. The S639F substitution in C. auris FKS1 confers an increase in the relative abundance of beta-glucans and chitin Mutations in FKS1 have previously been associated with altered relative abundance of β-glucan and chitin in Candida albicans [ 22 ]. We next sought to evaluate the relative abundance of these fungal cell wall constituents in our isogenic sets of C. auris FKS1 mutation strains by staining with AB and CFW as previously described. Relative abundance of both β-glucan and chitin were observed to increase upon the introduction of the S639F-encoding mutation into both RVA001. Conversely, the abundance of these cell wall constituents also decreased upon correction of the S639F-encoding mutation in SKU067 ( Figure 4 ). Download figure Open in new tab Figure 4. Relative quantification of cell wall components following FKS1 manipulation via EPIC system. A) Assessment of β-glucan abundance measured by fluorescence intensity of Aniline Blue staining for excitation 395nm and emission 495nm. B) Assessment of chitin abundance measured by CFW fluorescence intensity with excitation 380nm and emission 475nm. P-values derived from ordinary one-way ANOVA statistical analysis. DISCUSSION C. auris is a healthcare-associated pathogen of increasing clinical concern and represents a substantial threat to global public health. While the growing body of research has revealed much about C. auris , a lack of available molecular tools impedes progress and many important questions pertaining to this organism remain unanswered. The C. auris- optimized EPIC system described here represents an important new genetic tool for the study of C. auris . In addition to the precise single-nucleotide editing of ADE2 and FKS1 , the EPIC system can also be used to introduce larger gene cassettes, such as those expressing fluorescent proteins, as demonstrated by the generation of a dTOM-fluorescent strain in the derived RVA001- FKS1 S639F strain (Supplemental Figure 2) . Further demonstrating the utility and recyclability of the EPIC system. Using this novel tool, we show the impact of one of the most commonly observed C. auris FKS1 mutations associated with clinical echinocandin resistance, encoding S639F. Demonstrated by manipulations both introducing and correcting this mutation in two independent clinical isolate backgrounds representing two different genetic clades of C. auris , we have delineated the direct contribution of the mutation encoding S639F to clinical echinocandin resistance. The S639F mutation alone is sufficient to confer increased resistance to all 4 clinically available echinocandins, increasing echinocandin MIC by as much as 256-fold and elevating rezafungin and micafungin MIC above available clinical breakpoints in both clinical isolate backgrounds tested [ 3 , 23 ]. Further, using our isogenic sets of FKS1 mutant strains, we have shown this mutation also alters the relative abundance of both chitin and β-glucans, suggesting the impact of these mutations may extend beyond impeding echinocandin interaction with Fks1. Taken together, the findings of this study definitively demonstrate the S639F-encoding FKS1 mutation confers dramatically increased resistance to clinically available echinocandins, including the recently approved agent rezafungin. Further, we show the potential utility of a novel C. auris -optimized EPIC gene-editing system, a recyclable tool capable of single nucleotide editing which we hope will enable future research seeking to characterize and overcome C. auris and the infections caused by this emerging pathogen of global concern. NOTES Transparency declaration No reported conflicts. Authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. This work was supported through the St. Jude Children’s Research Hospital Children’s Infection Defense Center grant and the Society of Infectious Diseases Pharmacists Young Investigator Research Award granted to J.M.R.. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Acknowledgments This research was supported in part by ALSAC and the National Cancer Institute grant P30 CA021765. Preliminary results of these studies were presented on the 23 rd of April, 2022 at the “New mechanisms of antifungal resistance in emerging yeast pathogens” oral session as part of the 32nd European Congress of Clinical Microbiology & Infectious Diseases with the support of the 32nd ECCMID 2022 Travel Grant. The authors would like to thank Lisa Lombardi and Geraldine Butler for kindly providing the pCP-tRNA vector, and Lisa Lombardi, Geraldine Butler, and Judith Berman for helpful discussions relating to this manuscript. REFERENCES 1. ↵ Centers for Disease Control and Prevention . Antibiotic Resistance Threats in the United States. Atlanta, GA: U.S. Department of Health and Human Services, CDC ; 2019 . 2. ↵ Lockhart SR , Etienne KA , Vallabhaneni S , Farooqi J , Chowdhary A , Govender NP , et al. 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