Phenotypic Variants of Azole-Resistant Aspergillus Fumigatus that Co-exist in Human Respiratory Samples are Genetically Highly Related

Abstract

Respiratory specimens obtained from patients with chronic forms of aspergillosis contain phenotypic variants of azole-resistant Aspergillus fumigatus (ARAF) that co-exist in the airway. Here we aimed to study whether phenotypic variants of ARAF that co-exist in clinical specimens were genetically distinct. A panel of six phenotypic variants of ARAF cultured from two sputum samples collected from two patients with chronic aspergillosis were included. Preliminary identification of all isolates was obtained using MALDI–ToF mass spectrometry and confirmed by AsperGenius® real-time PCR assay. Antifungal susceptibility testing was determined using EUCAST E.Def 9.3 microbroth dilution. Genomic DNA libraries were constructed with the Illumina TruSeq Nano kit. Prepared whole-genome libraries were sequenced on an Illumina HiSeq 2500. Whole genome data were converted into presence/absence of a SNP with respect to the Af293 reference genome. Colonies of ARAF that co-existed in one respiratory sample demonstrated marked phenotypic diversity. Two cyp51A polymorphisms were found among azole-resistant isolates: TR34/L98H/T289A/I364V/G448S was consistently present in four variants with a pan-azole resistant phenotype and TR34/L98H was detected in two variants (itraconazole MIC > 16 mg/L). WGS typing showed that despite marked phenotypic variation, each sample contained a population of highly genetically related azole-resistant A. fumigatus variants. Our SNP analysis suggest that mechanisms additional to genetic-based variation are responsible for phenotypic diversity. Our data demonstrate that the phenotypic variants of ARAF that co-exist in clinical specimens are highly clonal and strongly suggest their origination from a single common ancestor.

Emerging Antifungal Resistance in Fungal Pathogens

“…The TR 34 /L98H resistance allele, along with TR 46 /Y121F/T289A (Table 1 ), is associated with itraconazole and voriconazole resistance respectively and has been isolated within and outside the clinic [ 27 ]. A contributing factor for the generation of novel drug resistance alleles could be attributed to the high recombination rate observed in A. fumigatus [ 28 ]; a recent example of a pan-azole resistance phenotype, attributed to TR 34 /L98H/T289A/I364V/G448S, could be the result of such recombination [ 29 ]. Thus, the pandemic potential of drug-resistant A. fumigatus has already been demonstrated, primarily due to human activity by using clinical antifungals in horticultural products [ 30 ], and global spread is likely due to ease of dispersal via conidia on air currents [ 2 ].…”

Risk factors and the value of microbiological examinations of COVID-19 associated pulmonary aspergillosis in critically ill patients in intensive care unit: the appropriate microbiological examinations are crucial for the timely diagnosis of CAPA

“…Fungal species were identified by the analysis of the mass spectrometer (MALDI-ToF, Bruker Daltonics, Germany). We consider a score of 2 or higher to be species-level reliable (Abdolrasouli and Rhodes, 2022).…”