Genetic diversity of human pathogenic members of the Fusarium oxysporum complex inferred from multilocus DNA sequence data and amplified fragment length polymorphism analyses: evidence for the recent dispersion of a geographically widespread clonal lineage and nosocomial origin

Abstract

Fusarium oxysporum is a phylogenetically diverse monophyletic complex of filamentous ascomycetous fungi that are responsible for localized and disseminated life-threatening opportunistic infections in immunocompetent and severely neutropenic patients, respectively. Although members of this complex were isolated from patients during a pseudoepidemic in San Antonio, Tex., and from patients and the water system in a Houston, Tex., hospital during the 1990s, little is known about their genetic relatedness and population structure. This study was conducted to investigate the global genetic diversity and population biology of a comprehensive set of clinically important members of the F. oxysporum complex, focusing on the 33 isolates from patients at the San Antonio hospital and on strains isolated in the United States from the water systems of geographically distant hospitals in Texas, Maryland, and Washington, which were suspected as reservoirs of nosocomial fusariosis. In all, 18 environmental isolates and 88 isolates from patients spanning four continents were genotyped. The major finding of this study, based on concordant results from phylogenetic analyses of multilocus DNA sequence data and amplified fragment length polymorphisms, is that a recently dispersed, geographically widespread clonal lineage is responsible for over 70% of all clinical isolates investigated, including all of those associated with the pseudoepidemic in San Antonio. Moreover, strains of the clonal lineage recovered from patients were conclusively shown to genetically match those isolated from the hospital water systems of three U.S. hospitals, providing support for the hypothesis that hospitals may serve as a reservoir for nosocomial fusarial infections.

FIG. 1.

(A) Distribution of the 88 human isolates among the 21 EF-1α-IGS rDNA sequence haplotypes. (B) One of 16 most-parsimonious phylograms inferred from the combined EF-1α-IGS rDNA sequence data rooted with sequences of F. oxysporum f. sp. cubense NRRL 25603 and 26024 from clade 1 of the FOC (21). All ingroup strains are from humans, except for strain NRRL 26395 from a whale and the 18 environmentalisolates indicated by shading. Note that 82 of the ingroup strains are members of a widespread clonal lineage. The number 1 or 2 following the five-digit NRRL culture collection number indicates that the strain was typed by the mating-type (MAT) idiomorph PCR assay as MAT1-1 or MAT1-2, respectively. All strains of the clonal lineage and the five most closely related strains are MAT1-1 (identified by boldface internodes). Internodes supported by bootstrap values of ≥70% are indicated.

FIG. 2.

(A) One of four most-parsimonious midpoint rooted phylograms inferred from the combined EF-1α-IGS rDNA-mtSSU rDNA sequence data for the 80-taxon matrix. (B) One of >64,000 most-parsimonious phylograms inferred from the AFLP data, indicating the seven AFLP genotypes (AG1 to -7) for 74 strains of the widespread clonal lineage. Geographic origin and year isolated are indicated. A, San Antonio, Tex., hospital A, reporting the pseudoepidemic; B, Houston, Tex., hospital B, reporting the water system as a potential reservoir of nosocomial fusariosis (1, 14). Internodes supported by bootstrap values of ≥70% are indicated. (C) Distribution of 74 clinical and environmental strains of the widespread clonal lineage among the seven AFLP genotypes.

FIG. 3.

(A) MAT1-1 idiomorph showing coding and noncoding regions and directions of transcription of the three MAT genes. PCR and sequencing primers are indicated by half-arrows (Table 2). The two intergenic regions are arbitrarily designated A and B. (B) Single most-parsimonious midpoint rooted phylogram inferred from the MAT1-1 nucleotide sequence data. Note that strains representing six AFLP genotypes (AG1 to -6) of the clonal lineage are identical to one another and to outgroup strain NRRL 26370 (100% bootstrap support). The AB011379 MAT1-1 sequence was obtained from GenBank.

FIG. 4.

Phylogenetic diversity of human isolates within the F. oxysporum complex inferred from parsimony analysis of the combined EF-1α-mtSSU rDNA sequence data. The human isolates exhibit a polyphyletic distribution among three of the four clades. The widespread clonal lineage is nested within clade 3. Internodes supported by bootstrap values of ≥70% are indicated. Sequences of Fusarium commune NRRL 22903 and Fusarium sp. strain NRRL 25184 were used as outgroups to root the phylogram.