Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus

Abstract

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.

FIG. 1.

ML tree of partial nucleotide sequences of genes for β-tubulin (a) and rodlet A (b) from the variant isolates (in bold), A. fumigatus, and isolates assigned to Aspergillus section Fumigati. Asterisks indicate sequences of strains derived from the GenBank database. ML trees were generated with the PAUP* software; bootstrap values generated from 1,000 pseudoreplicates are shown.

FIG. 2.

ML tree of partial nucleotide sequences of the salt-responsive gene (a), ITS (b), and mtcytb (c) regions of the variant isolates (in bold), A. fumigatus, and other isolates assigned to Aspergillus section Fumigati whose sequences were available in the GenBank database (denoted by asterisks). Bootstrap values generated from 1,000 pseudoreplicates are shown. ML trees and bootstrap values were generated as outlined in Materials and Methods.

FIG. 3.

ML (a) and maximum-parsimony (b) trees generated from sequences from all five loci. Bootstrap values generated from 1,000 pseudoreplicates are shown.

FIG. 4.

DIC microscopy images of conidial heads of variant FH5 (a) and A. fumigatus Af293 (b) and SEM images of variant FH5 (c) and A. fumigatus Af293 (d). Both images were produced with a magnification of ×2,000. SEM images at a magnification of ×20,000 of conidia of variant FH5 (e) and A. fumigatus Af293 (f) are also shown.