Rapid induction of multiple resistance mechanisms in Aspergillus fumigatus during azole therapy: a case study and review of the literature

Abstract

Nine consecutive isogenic Aspergillus fumigatus isolates cultured from a patient with aspergilloma were investigated for azole resistance. The first cultured isolate showed a wild-type phenotype, but four azole-resistant phenotypes were observed in the subsequent eight isolates. Four mutations were found in the cyp51A gene of these isolates, leading to the substitutions A9T, G54E, P216L, and F219I. Only G54 substitutions were previously proved to be associated with azole resistance. Using a Cyp51A homology model and recombination experiments in which the mutations were introduced into a susceptible isolate, we show that the substitutions at codons P216 and F219 were both associated with resistance to itraconazole and posaconazole. A9T was also present in the wild-type isolate and thus considered a Cyp51A polymorphism. Isolates harboring F219I evolved further into a pan-azole-resistant phenotype, indicating an additional acquisition of a non-Cyp51A-mediated resistance mechanism. Review of the literature showed that in patients who develop azole resistance during therapy, multiple resistance mechanisms commonly emerge. Furthermore, the median time between the last cultured wild-type isolate and the first azole-resistant isolate was 4 months (range, 3 weeks to 23 months), indicating a rapid induction of resistance.

Fig 1

Alignment of fungal Cyp51 proteins. In case Cyp51 was specified as either Cyp51A or Cyp51B, this is indicated after the name of the fungus. The positions of A. fumigatus A9, G54, P216, and F219 are indicated in black; aligned positions in the other fungal species are indicated in gray. For each of the amino acids we are interested in, only 10 amino acids of surrounding sequence is added to the alignment. Other surrounding sequence is indicated by ***. The A. flavus Cyp51B sequence was relatively short compared to the other sequences. The alignment of A. flavus Cyp51B started at the position of residue 55 in A. fumigatus Cyp51A, so there is no comparable amino acid for A. fumigatus A9 and G54 in A. flavus Cyp51B.

Fig 2

Locations of residues P216 and F219 indicated in the Cyp51A homology model. The green tubes represent the two ligand access channels leading to the heme center of the enzyme.

Fig 3

Reported cases of acquired azole resistance in A. fumigatus (2, 3, 5, 6, 14). The treatment schedules of all eight patients are indicated with bars, and the A. fumigatus isolates obtained from the patients are indicated with triangles. The corresponding resistance mechanisms are also indicated. wt, azole-susceptible wild-type isolate; ?, resistant isolate without any cyp51A mutations; ND, cyp51A sequence not determined; ITC, itraconazole; VRC, voriconazole; CAS, caspofungin; POS, posaconazole; AMB, amphotericin B (in various formulations). In the isolates marked with an asterisk, the F219I resistance mechanism was found in cyp51A. However, this isolate continued to evolve further azole resistance by an additional and yet unknown non-cyp51A-related resistance mechanism. Information regarding the treatment of patients 1, 2, and 3 was kindly provided by the author (S. Howard, personal communication).