BACKGROUND: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. OBJECTIVES: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. METHODS: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. RESULTS: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. CONCLUSIONS: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

Biomedical Center Faculty of Medicine Charles University Pilsen Czech Republic

Department of Biology Saint George University of Beirut Beirut Lebanon

Department of Microbiology Faculty of Medicine University Hospital in Pilsen Charles University Pilsen Czech Republic

Department of Natural Sciences Lebanese American University Byblos Lebanon

Lebanese American University Medical Center Rizk Hospital Beirut Lebanon

School of Medicine Lebanese American University Beirut Lebanon

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Soulountsi V, Schizodimos T, Kotoulas SC. Deciphering the epidemiology of invasive candidiasis in the intensive care unit: is it possible? Infection. 2021;49(6):1107‐1131. doi:10.1007/s15010-021-01640-7

Rodrigues CF, Silva S, Henriques M. Candida glabrata: a review of its features and resistance. Eur J Clin Microbiol Infect Dis. 2014;33(5):673‐688. doi:10.1007/s10096-013-2009-3

Hassan Y, Chew SY, Than LTL. Candida glabrata: pathogenicity and resistance mechanisms for adaptation and survival. J Fungi. 2021;7(8):667. doi:10.3390/jof7080667

Fidel PL, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev. 1999;12(1):80‐96. doi:10.1128/cmr.12.1.80

El ZA, Istambouli R, Alkozah M, et al. Predominance of Candida glabrata among non‐albicans candida species in a 16‐year study of candidemia at a tertiary care center in Lebanon. Pathogens. 2021;10(1):1‐10. doi:10.3390/pathogens10010082

Husni R, Bou Zerdan M, Samaha N, et al. Characterization and susceptibility of non‐albicans Candida isolated from various clinical specimens in Lebanese hospitals. Front Public Health. 2023;11:1115055. doi:10.3389/fpubh.2023.1115055

Frías‐De‐león MG, Hernández‐Castro R, Conde‐Cuevas E, et al. Candida glabrata antifungal resistance and virulence factors, a perfect pathogenic combination. Pharmaceutics. 2021;13(10):1529. doi:10.3390/pharmaceutics13101529

Aldejohann AM, Herz M, Martin R, Walther G, Kurzai O. Emergence of resistant Candida glabrata in Germany. JAC‐Antimicrobial Resist. 2021;3(3):1‐10. doi:10.1093/jacamr/dlab122

Cowen LE, Sanglard D, Howard SJ, Rogers PD, Perlin DS. Mechanisms of antifungal drug resistance. Cold Spring Harb Perspect Med. 2015;5(7):a019752. doi:10.1101/cshperspect.a019752

Prasad R, Nair R, Banerjee A. Multidrug Transporters of Candida Species in Clinical Azole Resistance. Vol 132. Elsevier Inc.; 2019. doi:10.1016/j.fgb.2019.103252

Campoy S, Adrio JL. Antifungals. Biochem Pharmacol. 2017;133:86‐96. doi:10.1016/j.bcp.2016.11.019

Sanguinetti M, Posteraro B, Lass‐Flörl C. Antifungal drug resistance among Candida species: mechanisms and clinical impact. Mycoses. 2015;58(S2):2‐13. doi:10.1111/myc.12330

Healey KR, Zhao Y, Perez WB, et al. Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi‐drug resistance. Nat Commun. 2016;7:1‐10. doi:10.1038/ncomms11128

Pais P, Galocha M, Viana R, Cavalheiro M, Pereira D, Teixeira MC. Microevolution of the pathogenic yeasts Candida albicans and Candida glabrata during antifungal therapy and host infection. Microb Cell. 2019;6(3):142‐159. doi:10.15698/mic2019.03.670

D'Enfert C, Janbon G. Biofilm formation in Candida glabrata: what have we learnt from functional genomics approaches? FEMS Yeast Res. 2015;16(1):1‐13. doi:10.1093/femsyr/fov111

Araújo D, Henriques M, Silva S. Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 2017;25(1):62‐75. doi:10.1016/j.tim.2016.09.004

Valotteau C, Prystopiuk V, Cormack BP. Atomic force microscopy demonstrates that Candida glabrata. Am Soc Microbiol. 2019;4(3):1‐9.

Rodrigues CF, Rodrigues ME, Silva S, Henriques M. Candida glabrata biofilms: how far have we come? J Fungi. 2017;3(1):11. doi:10.3390/jof3010011

Malinovská Z, Čonková E, Váczi P. Biofilm formation in medically important Candida species. J Fungi. 2023;9(10):955. doi:10.3390/jof9100955

Castrejón‐Jiménez NS, Castillo‐Cruz J, Baltierra‐Uribe SL, Hernández‐González JC, García‐Pérez BE. Candida glabrata is a successful pathogen: an artist manipulating the immune response. Microbiol Res. 2022;260:127038. doi:10.1016/j.micres.2022.127038

Mitchell KF, Taff HT, Cuevas MA, Reinicke EL, Sanchez H, Andes DR. Role of matrix β‐1,3 glucan in antifungal resistance of non‐albicans Candida biofilms. Antimicrob Agents Chemother. 2013;57(4):1918‐1920. doi:10.1128/AAC.02378-12

Ueno K, Namiki Y, Mitani H, Yamaguchi M, Chibana H. Differential cell wall remodeling of two chitin synthase deletants Δchs3A and Δchs3B in the pathogenic yeast Candida glabrata. FEMS Yeast Res. 2011;11(5):398‐407. doi:10.1111/j.1567-1364.2011.00728.x

Fattouh N, Khalaf RA, Husni R. Candida glabrata hospital isolate from Lebanon reveals micafungin resistance associated with increased chitin and resistance to a cell‐surface‐disrupting agent. J Glob Antimicrob Resist. 2024;37:62‐68. doi:10.1016/j.jgar.2024.02.012

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114‐2120. doi:10.1093/bioinformatics/btu170

Bankevich A, Nurk S, Antipov D, et al. SPAdes: a new genome assembly algorithm and its applications to single‐cell sequencing. J Comput Biol. 2012;19(5):455‐477. doi:10.1089/cmb.2012.0021

Reslan L, Araj GF, Finianos M, et al. Molecular characterization of Candida auris isolates at a major tertiary Care Center in Lebanon. Front Microbiol. 2022;12:1‐8. doi:10.3389/fmicb.2021.770635

Wang Y, Liu JY, Shi C, et al. Mutations in transcription factor Mrr2p contribute to fluconazole resistance in clinical isolates of Candida albicans. Int J Antimicrob Agents. 2015;46(5):552‐559. doi:10.1016/j.ijantimicag.2015.08.001

Fattouh N, Hdayed D, Geukgeuzian G, Tokajian S, Khalaf RA. Molecular mechanism of fluconazole resistance and pathogenicity attributes of Lebanese Candida albicans hospital isolates. Fungal Genet Biol. 2020;2021(153):103575. doi:10.1016/j.fgb.2021.103575

Kapteyn JC, Hoyer LL, Hecht JE, et al. The cell wall architecture of Candida albicans wild‐type cells and cell wall‐defective mutants. Mol Microbiol. 2000;35(3):601‐611. doi:10.1046/j.1365-2958.2000.01729.x

Toutounji M, Tokajian S, Khalaf RA. Genotypic and phenotypic characterization of Candida albicans Lebanese hospital isolates resistant and sensitive to caspofungin. Fungal Genet Biol. 2018;2019(127):12‐22. doi:10.1016/j.fgb.2019.02.008

Silva S, Henriques M, Martins A, Oliveira R, Williams D, Azeredo J. Biofilms of non‐Candida albicans Candida species: quantification, structure and matrix composition. Med Mycol. 2009;47(7):681‐689. doi:10.3109/13693780802549594

Khalaf RA, Fattouh N, Medvecky M, Hrabak J. Whole genome sequencing of a clinical drug resistant Candida albicans isolate reveals known and novel mutations in genes involved in resistance acquisition mechanisms. J Med Microbiol. 2021;70(4):1351. doi:10.1099/JMM.0.001351

Garnaud C, Botterel F, Sertour N, et al. Next‐generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles. J Antimicrob Chemother. 2015;70(9):2556‐2565. doi:10.1093/jac/dkv139

Castanheira M, Woosley LN, Messer SA, Diekema DJ, Jones RN, Pfaller MA. Frequency of fks mutations among Candida glabrata isolates from a 10‐year global collection of bloodstream infection isolates. Antimicrob Agents Chemother. 2014;58(1):577‐580. doi:10.1128/AAC.01674-13

Bienvenu AL, Leboucher G, Picot S. Comparison of fks gene mutations and minimum inhibitory concentrations for the detection of Candida glabrata resistance to micafungin: a systematic review and meta‐analysis. Mycoses. 2019;62(9):835‐846. doi:10.1111/myc.12929

Ceballos‐Garzon A, Monteoliva L, Gil C, et al. Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin‐resistant Candida glabrata. J Antimicrob Chemother. 2022;77(3):585‐597. doi:10.1093/jac/dkab454

Lim HJ, Choi MJ, Byun SA, et al. Whole‐genome sequence analysis of Candida glabrata isolates from a patient with persistent Fungemia and determination of the molecular mechanisms of multidrug resistance. J Fungi. 2023;9(5):515. doi:10.3390/jof9050515

Martínez‐Jiménez V, Ramírez‐Zavaleta CY, Orta‐Zavalza E, et al. Sir3 polymorphisms in Candida glabrata clinical isolates. Mycopathologia. 2013;175(3–4):207‐219. doi:10.1007/s11046-013-9627-2

Ferrari S, Ischer F, Calabrese D, et al. Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence. PLoS Pathog. 2009;5(1):e1000268. doi:10.1371/journal.ppat.1000268

Merdan O, Şişman AS, Aksoy SA, et al. Investigation of the defective growth pattern and multidrug resistance in a clinical isolate of Candida glabrata using whole‐genome sequencing and computational biology applications. Microbiol Spectr. 2022;10(4):e0077622. doi:10.1128/spectrum.00776-22

Hou X, Xiao M, Wang H, et al. Profiling of PDR1 and MSH2 in Candida glabrata bloodstream Isolates from a multicenter study in China. Antimicrob Agents Chemother. 2018;62(6):1‐5. doi:10.1128/AAC.00153-18

Singh A, Healey KR, Yadav P, et al. Absence of azole or echinocandin resistance in Candida glabrata isolates in India despite background prevalence of strains with defects in the dna mismatch repair pathway. Antimicrob Agents Chemother. 2018;62(6):2‐10. doi:10.1128/AAC.00195-18

Byun SA, Won EJ, Kim MN, et al. Multilocus sequence typing (MLST) genotypes of Candida glabrata bloodstream isolates in Korea: association with antifungal resistance, mutations in mismatch repair gene (Msh2), and clinical outcomes. Front Microbiol. 2018;9:1‐10. doi:10.3389/fmicb.2018.01523

Bhattacharya S, Sae‐Tia S, Fries BC. Candidiasis and mechanisms of antifungal resistance. Antibiotics. 2020;9(6):1‐19. doi:10.3390/antibiotics9060312

Vazquez JA, Sobel JD. Candidiasis. In: Kauffman CA, Pappas PG, Sobel JD, Dismukes WE, eds. Essentials of Clinical Mycology. 2nd ed. Springer; 2011:167‐206. doi:10.1007/978-1-4419-6640-7

Yazbek S, Barada G, Basma R, Mahfouz J, Khalaf RA. Significant discrepancy between real‐time PCR identification and hospital identification of C. albicans from Lebanese patients. Med Sci Monit. 2007;13(5):MT7‐MT12.

Barada G, Basma R, Khalaf RA. Microsatellite DNA identification and genotyping of Candida albicans from Lebanese clinical isolates. Mycopathologia. 2008;165(3):115‐125. doi:10.1007/s11046-008-9089-0

Araj GF, Asmar RG, Avedissian AZ. Candida profiles and antifungal resistance evolution over a decade in Lebanon. J Infect Dev Ctries. 2015;9(9):997‐1003. doi:10.3855/jidc.6550

Awad L, Tamim H, Ibrahim A, et al. Correlation between antifungal consumption and distribution of Candida spp. in different departments of a Lebanese hospital. J Infect Dev Ctries. 2018;12(21):1‐11. doi:10.3855/jidc.10105

Ghaddar N, El Roz A, Ghssein G, Ibrahim JN. Emergence of vulvovaginal candidiasis among Lebanese pregnant women: prevalence, risk factors, and species distribution. Infect Dis Obstet Gynecol. 2019;2019:1‐8. doi:10.1155/2019/5016810

Ghaddar N, Anastasiadis E, Halimeh R, et al. Prevalence and antifungal susceptibility of Candida albicans causing vaginal discharge among pregnant women in Lebanon. BMC Infect Dis. 2020;20:1‐9. doi:10.1186/s12879-019-4736-2

Brown H, Esher SK, Alspaugh JA. Chitin: a"hidden Figure" In the Fungal Cell Wall. Vol 425. Springer Nature; 2020:83‐111. doi:10.1007/82_2019_184

Castaño I, Pan SJ, Zupancic M, Hennequin C, Dujon B, Cormack BP. Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol. 2005;55(4):1246‐1258. doi:10.1111/j.1365-2958.2004.04465.x

Erwig LP, Gow NAR. Interactions of fungal pathogens with phagocytes. Nat Rev Microbiol. 2016;14(3):163‐176. doi:10.1038/nrmicro.2015.21

Perlin DS. Echinocandin resistance in Candida. Clin Infect Dis. 2015;61(Suppl 6):S612‐S617. doi:10.1093/cid/civ791

Tan L, Chen L, Yang H, Jin B, Kim G, Im YJ. Structural basis for activation of fungal sterol receptor Upc2 and azole resistance. Nat Chem Biol. 2022;18(11):1253‐1262. doi:10.1038/s41589-022-01117-0

Phenotypic and genotypic characterization of Candida parapsilosis complex isolates from a Lebanese hospital