Determination of azole antifungal drug resistance mechanisms involving Cyp51A gene in clinical isolates of Aspergillus fumigatus and Aspergillus niger

Aims: The main aim of this research is to investigate azole resistance mechanisms in A. fumigatus and A. niger which involve Cyp51A gene that encodes 14-α sterol demethylase enzyme. Methodology and Results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1%) A. fumigatus and 7/23 (30.4%) A. niger isolates were resistant to Itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/ml to 3.0 μg/ml. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8%) and M220 mutation in 3/13 (23%) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13%) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r (13)=0.041038, p<0.05) with Itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r (15)=0.038434, p<0.05) with Itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50%) A. fumigatus isolates and 7/12 (58.3%) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates; however the difference in level of expression between resistant isolates and susceptible isolates is not significant. This may be due to similar MIC values in resistant and susceptible isolates. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards Itraconazole antifungal resistance, however other factors may also be involved.