Mechanisms of azole resistance in 52 clinical isolates of Candida tropicalis in China

Objectives: To explore the mechanisms underlying azole resistance in clinical isolates of Candida tropicalis collected in China by focusing on their efflux pumps, respiratory status and azole antifungal target enzyme. Methods: Fifty-two clinical isolates of C. tropicalis were collected from five hospitals in four provinces of China and antifungal susceptibility tests were performed. Rhodamine 6G and rhodamine 123 were used to investigate the efflux pumps and respiratory status, respectively. Transporter-related genes CDR1 and MDR1, mitochondrial gene CYTb, as well as ERG11, were quantified by real-time RT-PCR. Meanwhile, ergosterol content was analysed using liquid chromatography-mass spectrometry/mass spectrometry. An ERG11-deficient (erg11 Delta) Saccharomyces cerevisiae strain was generated to study the function of mutations in ERG11. Results: MICs showed that 31 isolates were resistant to at least one type of azole antifungal. Flow cytometry using rhodamine 123 revealed increased respiration for the azole-resistant isolates, but CYTb was not overexpressed. No significant difference in the efflux of rhodamine 6G was found, which was consistent with the comparable expression levels of CDR1 and MDR1. In contrast, the azole-resistant isolates overexpressed ERG11 and showed increased ergosterol content. Moreover, the isolates resistant to three azole antifungals expressed higher levels of ERG11 mRNA than those resistant to only fluconazole or itraconazole. Two ERG11 mutations, Y132F and S154F, were found in azole-resistant isolates and could be shown to mediate azole resistance by expression in S. cerevisiae. Conclusions: The up-regulation and mutations of ERG11 mediate azole resistance of C. tropicalis.