Background: The mechanisms of resistance of moulds isolated from patients enrolled in two Isavuconazole Phase III clinical trials were characterized. Specifically, we determined whether the underlying resistance mechanism/s are due to alterations of sterol synthesis or up-regulation of drug efflux pumps.
Methods: Strains with elevated MICs to Isavuconazole, posaconazole and voriconazole were Rhizopus (n=7) Fusarium (n=2) and Aspergillus (A. niger, n=3; and A. fumigatus, n= 2). Isavuconazole susceptible strains (1 for each species) were used as comparators. Extracted sterols were derivatized using a mixture of hexamethyldisilazane and trimethylchlorosilane and analyzed by Gas Liquid Chromatography. Functional activities of efflux pumps were assayed fluorometrically using Rhodamine 123 (Rh123), while transcriptional analysis of efflux pump expression was performed using gene-specific primer/probes for MDR1, MDR2, MDR3 following RNA extraction (Qiagen kit), reverse transcribed to get cDNA, and analyzed using efflux pumps genes of A. fumigatus isolates.
Results: Sterol analysis showed a decrease in the ergosterol content of the resistant Rhizopus strains (2.6%) compared to the ergosterol content of the susceptible isolate (76.93%). Moreover, squalene, the first intermediate in the ergosterol biosynthesis, was elevated in the resistant Rhizopus isolates compared to susceptible control one (65% vs 9% content), while resistant Fusarium strains showed an elevated level of squalene and a decrease in obtusifolial. Our data showed that retention of Rh123 was significantly higher in ISA-sensitive Fusarium isolates compared to the ISA-resistant isolates (P <= 0.01). Similarly, both A. fumigatus and A. niger strains had a significantly higher retention of Rh123 in susceptible isolates compared to resistant ones (P ≤ 0.04) indicating up-regulation of efflux pump activity in resistant strains. In contrast, no significant difference in Rh123 levels between ISA-sensitive and ISA-resistant Rhizopus isolates was noted. Finally, expression of MDR2 was elevated in ISA-resistant A. fumigatus compared to control.
Conclusions: Our results indicate that changes in sterol content are the main contributor to azole resistance in Rhizopus, while resistance in Fusarium and Aspergillus is due to elevated activity of efflux pumps.
- ICAAC 54th (2014)