Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Abstract

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.

FIG. 1.

Alignment of the CSP repeat region. Representative CSP STs are shown as well as the N. fischeri ST. A dash indicates an insertion/deletion, and a dot indicates a nucleotide that is identical to the nucleotide in the top sequence.

FIG. 2.

Phylogeny of unique A. fumigatus STs based on the 564-bp CSP gene fragment. N. fischeri is the outgroup taxon used to root the tree. The tree is a strict consensus of 66,825 most parsimonious trees that were 386 steps in length generated using a heuristic search. Numbers along branches represent bootstrap values. Only unique STs were used to construct the tree. Strains listed below the tree are grouped with other strains that possess identical CSP STs; the numbers above each grouping correspond to the numerical superscripts for each representative ST included in the phylogeny. Isolates showed within the brace were recovered exclusively from clinical samples.

FIG. 3.

Phylogeny of A. fumigatus strains based on the 159-bp 12-mer repeat region. N. fischeri is the outgroup taxon used to root the tree. The tree is a strict consensus of 15 most parsimonious trees that were 172 steps in length generated using a branch-and-bound search. Numbers along branches represent bootstrap values. Only unique STs were used to construct the tree; strains having identical CSP STs are listed below the tree as groups. The numbers above each grouping correspond to the numerical superscripts for each representative ST included in the phylogeny. Isolates recovered from clinical samples are shown within the brace.