新居 鉄平

BACKGROUND: Azoles target Cyp51A and Cyp51B in Aspergillus fumigatus. Mutations in cyp51A are known as the primary mechanisms of azole resistance. However, not all of them cause azole resistance. Among them, mutations related to improved susceptibility have not been reported so far. We found that two isolates that carry frameshift or nonsense mutations in cyp51A are more susceptible to azoles, even to fluconazole (FLCZ) (IC50: frameshift, 32 μg/mL; nonsense, 32 μg/mL) compared to other azole-susceptible strains (IC50: > 256 μg/mL). OBJECTIVES: We investigated the contribution of these two mutations to azole sensitivity and their effect on Cyp51A functions. METHODS: We transformed an experimental strain, AfS35, by replacing cyp51AWT with each of the mutated cyp51A and measured its MICs to azoles. We also evaluated the functions of mutated Cyp51A after suppression of Cyp51B, based on the notion that Cyp51A and Cyp51B complement each other. RESULTS: Induction of mutated cyp51A in AfS35 led to higher susceptibility to FLCZ (IC50: frameshift, 32-64 μg/mL; nonsense, 32 μg/mL). Transformants carrying either of the mutated cyp51A could not survive when cyp51B was suppressed, indicating that these cyp51A mutations result in Cyp51A dysfunction. Furthermore, a cyp51A-deleted mutant strain also showed increased susceptibility to FLCZ (IC50: 32 μg/mL), similar to cyp51A dysfunctional strains, while a cyp51B-deleted mutant strain showed unchanged susceptibility (IC50: > 256 μg/mL) from AfS35. CONCLUSIONS: It was suggested that FLCZ can inhibit Cyp51B rather than Cyp51A and that this unequal inhibition leads to higher azole susceptibility of the two isolates harbouring Cyp51A dysfunction.

BACKGROUND: Approximately 60% of individuals with cystic fibrosis (CF) are affected by Aspergillus fumigatus infection. This condition is correlated with a decline in lung function and is identified as an independent risk factor contributing to hospital admissions among CF patients. This study investigates the dynamic interplay of A. fumigatus within the context of CF patients, tracing its evolution over time, with a specific emphasis on colonization dynamics. METHODS: An analysis was conducted on 83 sequential A. fumigatus isolates derived from sputum samples of six patients receiving care at a renowned CF hospital in Brazil. Employing microsatellite genotyping techniques, alongside an investigation into cyp51A gene mutations, this research sheds light on the genetic variations, colonization, and resistance of A. fumigatus within the CF respiratory environment. RESULTS: Our research findings indicate that CF patients can harbor A. fumigatus strains from the same clonal complexes for prolonged periods. Additionally, we identified that clinical isolates have the potential to spread among patients in the same healthcare facility, evidencing hospital contamination. Two patients who underwent long-term Itraconazole treatment did not show phenotypic resistance. However, one of these patients exhibited mutations in the cyp51A gene, indicating the need to monitor resistance to azoles in these patients colonized for long periods by A. fumigatus. We also observed co-colonization or co-infection involving multiple genotypes in all patients over time. CONCLUSION: This comprehensive examination offers valuable insights into the pathogenesis of A. fumigatus infections in CF patients, potentially shaping future therapeutic strategies and management approaches. This enhanced understanding contributes to our knowledge of A. fumigatus impact on disease progression in individuals with cystic fibrosis. Additionally, the study provides evidence of cross-contamination among patients undergoing treatment at the same hospital.

Abstract Aspergillus fumigatus is a pathogenic fungus with a global distribution. The emergence of azole-resistant A. fumigatus (ARAf) other than the TR-mutants is a problem in Japan. Additionally, the genetic diversity of A. fumigatus strains in Japan remains relatively unknown. Here we show the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. First, we analyzed the genome sequences of 171 strains from Japan as well as the antifungal susceptibility of these strains. Next, we conducted a population analysis of 876 strains by combining the available genomic data for strains isolated worldwide, which were grouped in six clusters. Finally, a genome-wide association study identified the genomic loci associated with ARAf strains, but not the TR-mutants. These results highlight the complexity of the genomic mechanism underlying the emergence of ARAf strains other than the TR-mutants.