Phenotypic and genomic characterization of azole resistance in Portuguese Candida parapsilosis isolates

Alexandru-Marian Papuc¹Cristina Veríssimo¹Helena Simões¹Cristina Toscano²João Paulo Gomes^3,4Verónica Mixão^3*Raquel Sabino^5,6,7*

• ¹Reference Unit for Parasitic and Fungal Infections, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Av. Padre Cruz, 1649-016, Lisbon, Portugal
• ²Microbiology Laboratory, Unidade Local de Saúde Lisboa Ocidental, Hospital Egas Moniz, 1349-019, Lisbon, Portugal
• ³Genomics and Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Av. Padre Cruz, 1649-016, Lisbon, Portugal
• ⁴Animal and Veterinary Research Center (CECAV), Faculty of Veterinary Medicine, Lusófona University - Lisbon University Centre, Lisbon, Portugal
• ⁵Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisbon, Portugal
• ⁶Instituto de Saúde Ambiental, Faculdade de Medicina, Universidade de Lisboa, 1649-028, Lisbon, Portugal
• ⁷Laboratório Associado TERRA, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017, Lisbon, Portugal

Candida parapsilosis is a clinically important etiological agent of systemic infections associated with hospital outbreaks, which prevalence has been increasing in the last decade. Moreover, in recent years, fluconazole resistance in this species has been emerging in different countries, being a subject of significant interest and concern. In this context, the present study aims to determine the frequency of fluconazole resistance in C. parapsilosis sensu stricto isolates collected in Portugal (2003-2007 and 2017-2024), understand its associated molecular mechanisms, and relate it all with worldwide genomic data. To this end, we performed phenotypic assays of 145 isolates of C. parapsilosis collected from different biological and environmental products in Portugal (majority from the Lisbon Metropolitan area), and explored the genomic features of the fluconazole-resistant ones. We found eight C. parapsilosis fluconazole-resistant isolates between 2017 and 2024, corresponding to a frequency of 8.5% in this period, and contrasting with the absence of fluconazole-resistant isolates collected before 2007. Sequencing of the ERG11 gene showed that all fluconazole-resistant isolates had the Y132F and R398I mutations. A phylogenomic analysis including publicly available isolates from other countries revealed that our Portuguese isolates are more closely related to those from the USA and Germany than to the isolates sequenced thus far from the neighbor country, Spain. Furthermore, although three distinct C. parapsilosis genetic clades were found in our dataset, all the fluconazole-resistant isolates detected in this study cluster together, raising the question of whether the increased fluconazole-resistance in the country could possibly be associated with the emergence or introduction of this particular lineage. Altogether, these results provide valuable insights on fluconazole resistance in a set of Portuguese C. parapsilosis isolates and their associated mechanisms, representing an important step towards a better understanding of the increasing C. parapsilosis fluconazole resistance in Southern Europe.