Abstract
Aspergillus fumigatus molecular typing has become increasingly more important for detecting outbreaks as well as for local and global epidemiological investigations and surveillance. Over the years, many different molecular methods have been described for genotyping this species. Some outstanding approaches are based on microsatellite markers (STRAf assay, which is the current gold standard), or based on sequencing data (TRESP typing improved in this work with a new marker and was renamed TRESPERG). Both methodologies were used to type a collection of 212 A. fumigatus isolates that included 70 azole resistant strains with diverse resistance mechanisms from different geographic locations. Our results showed that both methods are totally reliable for epidemiological investigations showing similar stratification of the A. fumigatus population. STRAf assay offered higher discriminatory power (D = 0.9993) than the TRESPERG typing method (D = 0.9972), but the latter does not require specific equipment or skilled personnel, allowing for a prompt integration into any clinical microbiology laboratory. Among azole resistant isolates, two groups were differentiated considering their resistance mechanisms: cyp51A single point mutations (G54, M220, or G448), and promoter tandem repeat integrations with or without cyp51A modifications (TR34/L98H, TR46/Y121F/A289T, or TR53). The genotypic differences were assessed to explore the population structure as well as the genetic relationship between strains and their azole resistance profile. Genetic cluster analyses suggested that our A. fumigatus population was formed by 6–7 clusters, depending on the methodology. Also, the azole susceptible and resistance population showed different structure and organization. The combination of both methodologies resolved the population structure in a similar way to what has been described in whole-genome sequencing works.
Highlights
Aspergillus fumigatus is a worldwide saprotrophic mold that produces very large numbers of airborne spores (Latgé, 1999)
The nucleotide and amino acid sequence of each repeat type are described in Supplementary Table S1 and the different ERG alleles identified among the global A. fumigatus population and the tandem repeat (TR) succession forming each allele are shown in Supplementary Table S2
Many different molecular methods have been described for A. fumigatus typing purposes, such as random amplified polymorphic DNA (RAPD) (Loudon et al, 1993), amplified fragment length polymorphism analysis (AFLP) (Warris et al, 2003), restriction fragment length polymorphism analysis (RFLP) (Neuveglise et al, 1996), microsatellite length polymorphism (MLP) (Bart-Delabesse et al, 1998), retrotransposon insertionsite context (RISC) typing, and multilocus sequence typing (MLST) (Bain et al, 2007)
Summary
Aspergillus fumigatus is a worldwide saprotrophic mold that produces very large numbers of airborne spores (Latgé, 1999). Most azole resistance mechanisms in A. fumigatus lay on overexpression and/or point mutations in cyp51A gene which encodes a 14-α sterol demethylase involved in the ergosterol biosynthesis pathway (Lepesheva and Waterman, 2007; Hargrove et al, 2015). In this context, two routes of azole resistance acquisition have been described. Typing of the isolates could give insight into the dynamics of azole resistance development within an A. fumigatus population that includes azole susceptible and resistant isolates with both types of azole resistance mechanisms
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