A novel combination of cyp51A mutations in Aspergillus fumigatus induces a significant reduction in the in-vitro susceptibility to voriconazole and posaconazole

BRITO DEVOTO T; MACEDO D; POLA SJ; FINQUELIEVICH JL; DAN FRANCO O; CUESTAS ML; GARCIA-EFFRON G

Congreso; 16TH INFOCUS; 2018

IntroductionSince the late 2000s, a steady increase in worldwide reports of azole resistance in Aspergillus fumigatus has been a matter of serious clinical concern. Some resistant strains can develop during therapy in an individual patient, while others may arise in the environment through exposure to certain triazole fungicides used in agriculture. The resistance mechanism most frequently identified in environmental and clinical A. fumigatus strains is the combination of a 34-base pair (bp) tandem repeat (TR34) in the promoter region of cyp51A along with a substitution of leucine 98 to histidine (TR34/L98H). The aim of the present study was to analyze the resistance profile of three A. fumigatus isolates obtained from a patient with cystic fibrosis (CF) which exhibited a pattern of reduced susceptibility to triazoles. Material and methodsThree A. fumigatus isolates recovered from different sputum samples from an Argentinean CF patient, collected in October 2015, November 2016 and June 2017 (identified as A, B and C respectively). The patient had received treatment with ITC (400 mg/day) during 2015-2016 and with VCZ (200 mg/day) during 2017. MICs of itraconazole (ITC), voriconazole (VCZ) and posaconazole (PSC) were determined according to CLSI methodology described in the M38-3.ed document. Species were identified by sequencing of the β-tubulin and calmodulin genes. In addition, cyp51A gene was sequenced. ResultsSusceptibility testing showed that isolates A, B and C had MIC values above the reported epidemiological cut-off values (ECVs) to ITC (MIC > 2 µg/ml) and PSC (MIC > 1 µg/ml), while only isolate B showed MIC above the ECV for VCZ (MIC > 1 µ/ml) and a higher MIC value for PSC (MIC > 2 µg/ml). Sequence analysis of the promoter region revealed the presence of a tandem repeat of 34 bp (Tr34) in the alignment of isolates A, B and C accompanied by mutations L98H and S297T in the cyp51A coding region.Only isolate B had an additional point mutation at position 65 (R65K) which has never been reported in literature. To test whether this new mutation was responsible for the increased PSC and VCZ MIC values, gene replacement experiments were carried out using a WT strain of A. fumigatus (KU80 deletant). Transformants that incorporated the Tr34-R65K-L98H mimicked the MIC increase to VCZ and PSC observed in the clinical strain B. These data confirm that the association of these three modifications in the cyp51A?s promoter and coding region are responsible for the described phenotype.ConclusionsTo the best of our knowledge, this is the first report of a novel combination of Cyp51A mutations in A. fumigatus that lead to a significant reduction in the in vitro susceptibility to VRC and PSC.