P006 Resistance of Aspergillus flavus clinical isolates and associated fitness-cost

Abstract Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Aspergillus flavus and closely related species could be pathogenic for humans, animals, and plants and could also produce mycotoxins. The members of the Flavi section are morphologically quite similar making precise identification to the species level difficult. In this study, we present the antifungal susceptibility profiles of French clinical isolates belonging to the Flavi section. Isolates have been characterized by molecular methods and the potential fitness-cost associated with azole-resistance has been determined. Methods A total of 120 isolates phenotypically identified as A. flavus were included in the study. These clinical isolates were recovered over a 15-year period (2001-2015). For all isolates, specific identification was confirmed by sequencing a part of the β-tubulin and calmodulin genes. The isolates were first screened for their susceptibility to azoles antifungal agents by using 3-sectors agar plates containing itraconazole, voriconazole, and a drug-free control. Susceptibility to six antifungal drugs was further determined by using the EUCAST reference microdilution broth technique. Fitness cost was evaluated by growth curve kinetics in RPMI and by evaluation of virulence in a Galleria mellonella invertebrate animal model. Results Out of 120 isolates, molecular analysis of the partial β-tubulin and calmodulin sequences showed that 117 isolates were A. flavus sensu stricto and the three remaining corresponded to A. parasiticus, A. nomius, and A. tamarii. Two isolates were azole-resistant by the screening test. For the A. flavus sensu stricto isolates, the geometric mean MIC values (range) of amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole, and caspofungin were 1.84 (0.25-16), 0.29 (0.125-2), 0.82 (0.5-8), 0.27 (0.06-2), 1.15 (0.25-8), and 0.061 (0.03-0.125) μg/ml, respectively. For A. parasiticus, A. nomius, and A. tamarii, MICs were in the same range. Two A. flavus sensu stricto isolates (AfR1 and AfR2) had voriconazole and isavuconazole MICs at 8 μg/ml. Compared to susceptible isolates, these two azole-resistant isolates had a delayed growth in RPMI liquid medium. In the G. mellonella model, the mortality was 100% for susceptible isolates. In contrast, the Galleria infected by AfR1 and AfR2 showed a significantly lower mortality rate. Conclusion Antifungal susceptibility to six drugs was determined on a large collection of clinical isolates belonging to Aspergillus Flavi section. Most of the isolates were identified as A. flavus sensu stricto and most of them were susceptible to antifungal drugs. Nevertheless, the occurrence of two resistant isolates highlights the need for susceptibility testing for A. flavus. It seems that azole-resistance is associated with a fitness-cost including a lower growth rate and a lower virulence.