Abstract
<title>Abstract</title> <p> <bold>Objectives:</bold> This study aimed to investigate the species distribution, molecular characteristics, antifungal susceptibility profiles, resistance-associated genes, and virulence properties of azole-resistant <italic>C. parapsilosis</italic> complex isolates in Kayseri, Türkiye. <bold>Methods:</bold> Thirty <italic>C. parapsilosis</italic> complex isolates, initially identified from clinical specimens by conventional methods and MALDI-TOF MS over a two-year period, were analyzed. Whole-genome sequencing (WGS) was performed for species-level re-identification, phylogenetic analysis, and resistance gene mapping. Antifungal susceptibility testing was conducted using broth microdilution and gradient strip methods in accordance with CLSI and EUCAST guidelines. Virulence-related phenotypes, including phospholipase, proteinase, and esterase activity, and biofilm formation, were evaluated. <bold>Results:</bold> Of the 30 isolates, 28 were re-identified as <italic>C. parapsilosis</italic> sensu stricto and two as <italic>C. orthopsilosis</italic> using WGS. Most isolates (84%) were obtained from intensive care unit (ICU) patients, predominantly in the pediatric age group (67%). Blood was the most common source of isolates (90%). Isolates were resistant to (100%) fluconazole and itraconazole, and (70%) voriconazole, and susceptible to amphotericin B and caspofungin. High rates of proteinase (92.8%), phospholipase (71.4%), esterase activity (64.3%), and biofilm formation (78.6%) were observed among isolates. The most frequently detected resistance-associated genes were <italic>ERG3</italic> (60%), <italic>HOG1</italic> (56.6%), <italic>PDR16</italic> (53.3%), <italic>ERG5</italic> (53.3%), <italic>ERG27</italic> (50%), and <italic>FKS1</italic> (46.6%). Phylogenetic analysis revealed genetic diversity, with evidence of close evolutionary relationships among some isolates. <bold>Conclusion:</bold> Universal fluconazole resistance and the high virulence potential of these isolates, especially in pediatric ICUs, highlight a critical clinical challenge. The presence of diverse resistance-associated genes, even in the absence of canonical <italic>ERG11</italic> mutations in some strains, suggests complex, multi-pathway resistance mechanisms. WGS provided higher discriminatory resolution than conventional methods for distinguishing cryptic species within the <italic>C. parapsilosis</italic> complex. WGS data were analyzed to determine phylogenetic relationships and identify resistance-associated genes using the ResFungi database. </p>