The opportunistic pathogen Candida parapsilosis is a major causative agent of candidiasis leading to death in immunocompromised individuals. Azoles are the first line of defense in their treatment. The purpose of this study was to characterize eight fluconazole-resistant and sensitive C. parapsilosis hospital isolates through a battery of phenotypic tests that target pathogenicity attributes such as virulence, biofilm formation, stress resistance, and ergosterol content. Whole genome sequencing was carried out to identify mutations in key pathogenicity and resistance genes. Phylogenetic comparison was performed to determine strain relatedness and clonality. Genomic data and phylogenetic analysis revealed that two isolates were C. orthopsilosis and C. metapsilosis misidentified as C. parapsilosis. Whole genome sequencing analysis revealed known and novel mutations in key drug resistance and pathogenicity genes such as ALS6, ALS7, SAPP3, SAP7, SAP9, CDR1, ERG6, ERG11 and UPC2. Phylogenetic analysis revealed a high degree of relatedness and clonality within our C. parapsilosis isolates. Our results showed that resistant isolates exhibited an increase in biofilm content compared to the sensitive isolates. In conclusion, our study is the first of its kind in Lebanon to describe phenotypic and genotypic characteristics of nosocomial C. parapsilosis complex isolates having a remarkable ability to form biofilms.

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 PO Box 36 Byblos Lebanon

Lebanese American University Medical Center Rizk Hospital Beirut Lebanon

School of Medicine Lebanese American University Beirut Lebanon

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Res Sq. 2024 May 30:rs.3.rs-4169036. doi: 10.21203/rs.3.rs-4169036/v1. PubMed

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Brown, G. D. et al. Hidden killers: human fungal infections. Sci. Transl Med.4, 165rv13. 10.1126/scitranslmed.3004404 (2012). PubMed

Branco, J., Miranda, I. M. & Rodrigues, A. G. Candida parapsilosis virulence and Antifungal Resistance mechanisms: a Comprehensive Review of Key determinants. J. Fungi9, 80. 10.3390/jof9010080 (2023). PubMed PMC

Husni, R. et al. Characterization and susceptibility of non-albicans Candida isolated from various clinical specimens in Lebanese hospitals. Front. Public. Heal 11. 10.3389/fpubh.2023.1115055 (2023). PubMed PMC

Tóth, R. et al. Candida parapsilosis: from genes to the bedside. Clin. Microbiol. Rev.32, 10–1128. 10.1128/CMR.00111-18 (2019). PubMed PMC

Silva, S. et al. Candida Glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol. Rev.36, 288–305. 10.1111/j.1574-6976.2011.00278.x (2012). PubMed

Tavanti, A., Davidson, A. D., Gow, N. A. R., Maiden, M. C. J. & Odds, F. C. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J. Clin. Microbiol.43, 284–292. 10.1128/JCM.43.1.284-292.2005 (2005). PubMed PMC

Daneshnia, F. et al. Worldwide emergence of fluconazole-resistant Candida parapsilosis: current framework and future research roadmap. Lancet Microbe4, e470–e480. 10.1016/S2666-5247(23)00067-8 (2023). PubMed PMC

O’Brien, C. E. et al. Identification of a novel Candida metapsilosis isolate reveals multiple hybridization events. G3 genes. Genomes Genet.12, jkab367. 10.1093/G3JOURNAL/JKAB367 (2022). PubMed PMC

Gabaldón, T. Threats from the Candida parapsilosis complex: the surge of multidrug resistance and a hotbed for new emerging pathogens. Microbiol. Moleular Biol. Rev. n.d.:e-00029-23. 10.1128/mmbr.00029-23 PubMed PMC

Schröder, M. S. et al. Multiple origins of the pathogenic yeast Candida orthopsilosis by separate hybridizations between two parental species. PLoS Genet.12, 1006404. 10.1371/journal.pgen.1006404 (2016). PubMed PMC

Pryszcz, L. P. et al. The genomic Aftermath of hybridization in the opportunistic Pathogen Candida Metapsilosis. PLoS Genet.11, e1005626. 10.1371/journal.pgen.1005626 (2015). PubMed PMC

Gómez-Gaviria, M., García-Carnero, L. C., Baruch-Martínez, D. A. & Mora-Montes, H. M. The Emerging Pathogen Candida metapsilosis: Biological aspects, virulence factors, diagnosis, and treatment. Infect. Drug Resist.17, 171–185. 10.2147/IDR.S448213 (2024). PubMed PMC

Cavalheiro, M. & Teixeira, M. C. Candida Biofilms: threats, challenges, and promising strategies. Front. Med.5, 28. 10.3389/fmed.2018.00028 (2018). PubMed PMC

Gácser, A., Schäfer, W., Nosanchuk, J. S., Salomon, S. & Nosanchuk, J. D. Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models. Fungal Genet. Biol.44, 1336–1341. 10.1016/j.fgb.2007.02.002 (2007). PubMed

Pfaller, M. A., Moet, G. J., Messer, S. A., Jones, R. N. & Castanheira, M. Candida bloodstream infections: comparison of species distributions and antifungal resistance patterns in community-onset and nosocomial isolates in the SENTRY Antimicrobial Surveillance Program, 2008–2009. Antimicrob. Agents Chemother.55, 561–566. 10.1128/AAC.01079-10 (2011). PubMed PMC

Bhattacharya, S., Sae-Tia, S. & Fries, B. C. Candidiasis and mechanisms of antifungal resistance. Antibiotics9, 312. 10.3390/antibiotics9060312 (2020). PubMed PMC

Clinical and Laboratory Standards Institute. Performance Standards for Antifungal Susceptibility Testing of Yeasts. (1st ed, CLSI supplement M60). Wayne, PA: Clinical and Laboratory Standards Institute. (2017).

Asadzadeh, M., Dashti, M., Ahmad, S., Alfouzan, W. & Alameer, A. Whole-genome and targeted-amplicon sequencing of Fluconazole-Susceptible and -resistant Candida parapsilosis isolates from Kuwait reveals a previously undescribed N1132D polymorphism in CDR1. Antimicrob. Agents Chemother.65, e01633–e01620. 10.1128/aac.01633-20 (2021). PubMed PMC

Štefánek, M., Garaiová, M., Valček, A., Jordao, L. & Bujdáková, H. Comparative Analysis of Two Candida parapsilosis isolates originating from the same patient harbouring the Y132F and R398I mutations in the ERG11 gene. Cells12, 1579. 10.3390/cells12121579 (2023). PubMed PMC

Arastehfar, A. et al. First Report of Candidemia Clonal Outbreak caused by emerging fluconazole-resistant Candida parapsilosis isolates harboring Y132F and/or Y132F_K143R in Turkey. Antimicrob. Agents Chemptherapy64, e01001–e01020. 10.1128/aac.01001-20 (2020). PubMed PMC

Arastehfar, A. et al. Clonal candidemia outbreak by Candida parapsilosis carrying Y132F in Turkey: evolution of a persisting challenge. Front. Cell. Infect. Microbiol.11, 676177. 10.3389/fcimb.2021.676177 (2021). PubMed PMC

Ceballos-Garzon, A. et al. Emergence and circulation of azole-resistant C. Albicans, C. Auris and C. parapsilosis bloodstream isolates carrying Y132F, K143R or T220L Erg11p substitutions in Colombia. Front. Cell. Infect. Microbiol.13, 1136217. 10.3389/fcimb.2023.1136217 (2023). PubMed PMC

Alcoceba, E. et al. Fluconazole-resistant Candida parapsilosis clonally related genotypes: first report proving the presence of endemic isolates harbouring the Y132F ERG11 gene substitution in Spain. Clin. Microbiol. Infect.28, 1113–1119. 10.1016/j.cmi.2022.02.025 (2022). PubMed

Castanheira, M., Deshpande, L. M., Messer, S. A., Rhomberg, P. R. & Pfaller, M. A. Analysis of global antifungal surveillance results reveals predominance of Erg11 Y132F alteration among azole-resistant Candida parapsilosis and Candida tropicalis and country-specific isolate dissemination. Int. J. Antimicrob. Agents55, 105799. 10.1016/j.ijantimicag.2019.09.003 (2020). PubMed

Corzo-Leon, D. E., Peacock, M., Rodriguez-Zulueta, P., Salazar-Tamayo, G. J. & MacCallum, D. M. General hospital outbreak of invasive candidiasis due to azole-resistant Candida parapsilosis associated with an Erg11 Y132F mutation. Med. Mycol.59, 664–671. 10.1093/mmy/myaa098 (2021). PubMed PMC

Magobo, R. E., Lockhart, S. R. & Govender, N. P. Fluconazole-resistant Candida parapsilosis strains with a Y132F substitution in the ERG11 gene causing invasive infections in a neonatal unit, South Africa. Mycoses63, 471–477. 10.1111/myc.13070 (2020). PubMed PMC

Thomaz, D. Y. et al. Environmental clonal spread of azole-resistant candida parapsilosis with erg11-y132f mutation causing a large candidemia outbreak in a Brazilian cancer referral center. J. Fungi7, 259. 10.3390/jof7040259 (2021). PubMed PMC

Vumbaca, M. et al. Genomic characterization of a persistent, azole-resistant C. parapsilosis strain responsible for a hospital outbreak during the first COVID-19 wave. BioRxiv2024, 2024–2011. 10.1101/2024.11.11.622614.

Kim, T. Y. et al. Evolution of Fluconazole Resistance mechanisms and Clonal types of Candida parapsilosis isolates from a Tertiary Care Hospital in South Korea. Antimicrob. Agents Chemother.66, e00889–e00822. 10.1128/aac.00889-22 (2022). PubMed PMC

Mctaggart, L. R. et al. First Canadian report of transmission of fluconazole-resistant Candida parapsilosis within two hospital networks confirmed by genomic analysis. J. Clin. Microbiol.10.1128/jcm.01161-23 PubMed PMC

Won, E. J. et al. Nationwide Surveillance of Antifungal Resistance of Candida Bloodstream isolates in South Korean hospitals: two Year Report from Kor-GLASS. J. Fungi8, 996. 10.3390/jof8100996 (2022). PubMed PMC

Daneshnia, F. et al. Candida parapsilosis isolates carrying mutations outside FKS1 hotspot regions confer high echinocandin tolerance and facilitate the development of echinocandin resistance. Int. J. Antimicrob. Agents62, 106831. 10.1016/j.ijantimicag.2023.106831 (2023). PubMed

Franconi, I., Rizzato, C., Poma, N., Tavanti, A. & Lupetti, A. Candida parapsilosis sensu stricto Antifungal Resistance mechanisms and Associated Epidemiology. J. Fungi9, 798. 10.3390/jof9080798 (2023). PubMed PMC

Martí-Carrizosa, M., Sánchez-Reus, F., March, F., Cantón, E. & Coll, P. Implication of Candida parapsilosis FKS1 and FKS2 mutations in reduced echinocandin susceptibility. Antimicrob. Agents Chemother.59, 3570–3573. 10.1128/AAC.04922-14 (2015). PubMed PMC

Khalifa, H. O., Watanabe, A. & Kamei, K. Antifungal resistance and genotyping of clinical Candida parapsilosis Complex in Japan. J. Fungi10, 4. 10.3390/jof10010004 (2024). PubMed PMC

Morio, F. et al. Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis. J. Antimicrob. Chemother.74, 2230–2238. 10.1093/jac/dkz204 (2019). PubMed

Rizzato, C. et al. CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates. J. Antimicrob. Chemother.73, 1815–1822. 10.1093/jac/dky122 (2018). PubMed

Arthington-Skaggs, B. A., Jradi, H., Desai, T. & Morrison, C. J. Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J. Clin. Microbiol.37, 3332–3337. 10.1128/jcm.37.10.3332-3337.1999 (1999). PubMed PMC

El Hachem, S. et al. Sequential induction of Drug Resistance and characterization of an initial Candida albicans Drug-Sensitive isolate. J. Fungi10, 347. 10.3390/jof10050347 (2024). PubMed PMC

Plaine, A. et al. Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity. Fungal Genet. Biol.45, 1404–1414. 10.1016/j.fgb.2008.08.003 (2008). PubMed PMC

Lockhart, S. R., Messer, S. A., Pfaller, M. A. & Diekema, D. J. Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis. J. Clin. Microbiol.46, 2659–2664. 10.1128/JCM.00803-08 (2008). PubMed PMC

Neji, S. et al. Virulence factors, antifungal susceptibility and molecular mechanisms of azole resistance among Candida parapsilosis complex isolates recovered from clinical specimens. J. Biomed. Sci.24, 1–16. 10.1186/s12929-017-0376-2 (2017). PubMed PMC

Silva, A. P. et al. Transcriptional profiling of azole-resistant Candida parapsilosis strains. Antimicrob. Agents Chemother.55, 3546–3556. 10.1128/AAC.01127-10 (2011). PubMed PMC

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

Khalaf, R. A., 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.70, 001351. 10.1099/JMM.0.001351 (2021). PubMed PMC

Grossman, N. T., Pham, C. D., Cleveland, A. A. & Lockhart, S. R. Molecular mechanisms of fluconazole resistance in Candida parapsilosis isolates from a U.S. surveillance system. Antimicrob. Agents Chemother.59, 1030–1037. 10.1128/AAC.04613-14 (2015). PubMed PMC

Singh, A. et al. Emergence of clonal fluconazole-resistant Candida parapsilosis clinical isolates in a multicentre laboratory-based surveillance study in India. J. Antimicrob. Chemother.74, 1260–1268. 10.1093/jac/dkz029 (2019). PubMed

Seemann, T. & Snippy Fast Bacterial Variant Calling From NGS Reads, San Francisco, CA:github. (2015).

Reslan, L. et al. Molecular characterization of Candida Auris isolates at a major Tertiary Care Center in Lebanon. Front. Microbiol.12, 770635. 10.3389/fmicb.2021.770635 (2022). PubMed PMC

Silva, S. et al. Biofilms of non-candida albicans Candida species: quantification, structure and matrix composition. Med. Mycol.47, 681–689. 10.3109/13693780802549594 (2009). PubMed

Fattouh, N., Husni, R., Finianos, M., Bitar, I. & Khalaf, R. A. Adhesive and biofilm-forming Candida Glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins. Mycoses67, e13750. 10.1111/myc.13750 (2024). PubMed

Wang, N., Strugnell, R., Wijburg, O. & Brodnicki, T. Measuring bacterial load and immune responses in mice infected with Listeria monocytogenes. J. Vis. Exp.10.3791/3076 (2011). PubMed PMC

Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the arrive guidelines for reporting animal research. PLoS Biol.8, e1000412. 10.1371/journal.pbio.1000412 (2010). PubMed PMC