Impact of CYP51A mutations associated with azole-resistance on invitro growth rates and invivo virulence of clinical Aspergillus fumigatus isolates
Abstract number: O345
Mavridou E., Meletiadis J., Arendrup M., Melchers W.J., Mouton J.W., Verweij P.
Objectives: The emergence of multi-drug resistant Aspergillus fumigatus (AF) causing invasive aspergillosis has raised the question of whether the evolution of azole-resistance has an impact on virulence and therefore on clinical outcome. The aim of this study was to determine whether acquired azole resistance in fungi through mutations in CYP51A or other unknown genes comes with a cost in fitness.
Methods: The virulence study included a) 8 multi-azole resistant AF clinical isolates with different mutations in the CYP51A gene (3 strains with TR/L98H, 1 each with M220I, M220V, M220K, G54W, G138C) b) 3 wild-type azole susceptible (WT) strains as reference and c) 2 isogenic isolates with no CYP51A mutations (1 susceptible and 1 resistant strain) which were serially recovered from a single patient pre- and post-antifungal treatment. Microsatellite-typing method and CSP-method were performed to verify the genetic relationship of the 2 isolates. Susceptibility testing was performed based on the CLSI-M38A method. RT-PCR was performed to determine the CYP51A expression. In vitro growth rates were determined using a previously described kinetic system (Meletiadis et al JCM 2001) and defined as the incubation time needed to reach a hyphal growth of 70 microm. In vivo virulence was determined based on the 15-day mortality in each of 52 groups of 572 outbred i.v infected CD-1 mice (11 mice/group × 13 AF strains × 4 different CFU inocula).
Results: There was a marked reduction of virulence of the post-treatment azole-resistant isogenic isolate 2 (0% 15-day mortality) compared to a) the reference WT groups (57.6% average mortality, p < 0.05) b) the CYP51A groups (47.72% average mortality, p < 0.05) and c) the pre-treatment azole susceptible isolate 1 (90.91% mortality, p < 0.05). There was no difference (p < 0.05) in virulence for all CYP51A mutants compared to the WTs. The in vitro growth rates of the AF CYP51A mutants were not different compared to those of the AF WT control but it was significantly reduced when compared to the resistant isolate 2. Linear correlation was found between in vitro growth rates-and in vivo virulence (R2 0.99, slope 2.28, p < 0.001). Moreover, Cyp51A elevated RNA levels implicate a-demethylase in an important role in azole resistance but not in virulence.
Discussion: Acquired antifungal resistance can lead to dramatic decrease of virulence of AF. However, CYP51A mutations associated with acquired azole resistance did not affect AF virulence.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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