Molecular Mechanisms of Azole Resistance in Four Clinical Isolates.

Azole resistance constitutes a serious clinical problem in the management of infections caused by This study aimed to explore azole-resistant mechanisms in clinical isolates collected in Jinan, Shandong, China. In total, 22 samples were collected and analyzed. Among these, four isolates (28A, 28D, 28I, and 28J) exhibited high level of pan-azole-resistance that was Hsp90 dependent. Gene sequencing revealed that the four Hsp90-dependent strains contained different mutations that led to four novel amino acid substitutions (S265Y, N322D, N324S, and E355D) in Erg3. The role of these substitutions in azole resistance development was determined by constructing one copy of the mutated from the 28A, 28D, and 28I strains into CAI4, respectively. The minimum inhibitory concentration value of fluconazole (FLC) against CAI4- increased fourfold compared with the wild-type strain, suggesting that the novel combination of substitutions S265Y, N322D, and N324S played an important role in mediating azole resistance in 28I. Besides, we identified several different mechanisms in other three isolates. Strains 28A and 28D displayed increased efflux ability and overexpression of . Strain 28J showed high level of expression, but no mutation in its regulator Upc2 was observed. Our study revealed that multiple factors confer azole resistance in clinical isolates and combination therapy should be conducted clinically.