Clinical isolation, biofilm formation, and pathogenicity analysis of different species of the Stephanoascus ciferrii complex

The <i>Stephanoascus ciferrii</i> complex, comprising <i>Stephanoascus ciferrii, Candida allociferrii</i>, and <i>Candida mucifera</i>, is an emerging fungal pathogen with increasing isolation rates and antifungal resistance. However, detailed information about clinical isolation rates and pathogenicity comparisons among the three species are lacking. In order to fill in this information gap, this study aimed to investigate and compare the clinical isolation rates and pathogenicity of the three species. Twenty-seven <i>S. ciferrii</i> complex strains isolated from the secretion specimens of patients admitted to Nanjing Drum Tower Hospital between 2012 and 2023 were included. According to the results of ITS sequencing, there were 15 strains of <i>S. ciferrii</i>, 7 strains of <i>C. allociferrii</i>, and 5 strains of <i>C. mucifera</i>. Antifungal susceptibility testing demonstrated that the <i>S. ciferrii</i> complex exhibited high MICs against azole antifungal agents, particularly fluconazole, while it showed lower MICs against echinocandins. <i>S. ciferrii</i> displayed higher MICs against caspofungin than <i>C. allociferrii</i> (<i>P</i> < 0.05). The results of biofilm quantification using crystal violet staining indicated <i>C. allociferrii</i> exhibited stronger biofilm-forming ability than <i>S. ciferrii</i> in RPMI-1640 medium (<i>P</i> < 0.05), but there was no significant difference between <i>C. allociferrii</i> and <i>C. mucifera</i> or between <i>S. ciferrii</i> and <i>C. mucifera</i>. The results were similar with the metabolic activity by using XTT assay. The <i>G. mellonella</i> larvae infection experiments revealed that the survival rates of larvae infected by strains of the <i>S. ciferrii</i> complex were 60%, 50%, and 48% at 24 h, 48 h, and 72 h, respectively. Furthermore, the <i>G. mellonella</i> larvae lethality caused by <i>C. allociferrii</i> and <i>C. mucifera</i> were significantly higher than that caused by <i>S. ciferrii</i> (<i>P</i> < 0.001). This study is the first to describe and compare the pathogenicity and biofilm formation ability of the three species of <i>S. ciferrii</i> complex in the clinical context. Our research reveals the high prevalence of <i>S. ciferrii</i> in the complex and elucidates the correlation between fungal drug resistance, biofilm formation, and virulence, thus providing essential empirical evidence for further study of the clinical pathogenic characteristics of each species in the complex and treatment strategies.