Molecular phylogenetic diversity of dermatologic and other human pathogenic fusarial isolates from hospitals in northern and central Italy

Abstract

Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among the Fusarium solani species complex (FSSC; n = 18), the F. oxysporum species complex (FOSC; n = 20), and the Gibberella (Fusarium) fujikuroi species complex (GFSC; n = 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates as F. verticillioides, 2 as F. sacchari, and 1 as F. guttiforme. This is the first report that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate of F. guttiforme was able to induce fusariosis of pineapple, heretofore its only known host.

FIG. 1.

One of six most-parsimonious trees inferred from MP analysis of the combined three-locus data set for 18 NRRL isolates within the FSSC. Sequences of two FSSC clade 2 species, F. virguliforme and F. tucumaniae, were used to root the phylogram. The in-group species and their multilocus STs are identified by Arabic numbers and lowercase roman letters, respectively. All in-group isolates except for NRRL 53120 and NRRL 53128 were from Sesto San Giovanni Hospital in Milan; NRRL 53120 was from Ancona (A), and NRRL 53128 was from Torino (T). Gray shading is used to identify two isolates (46437 and 46438) representing different STs recovered from the toe of a patient with onychomycosis in April and May 2007. The numbers above the nodes represent MP bootstrap support of ≥70% from 1,000 pseudoreplicates of the data. PIC, parsimony informative characters; CI, consistency index; RI, retention index.

FIG. 2.

One of 300 most-parsimonious phylograms inferred from MP analysis of the combined two-locus data set for 20 isolates within the FOSC. Sequences of the sister taxon of the FOSC, F. foetens, were used to root the phylogeny by the outgroup method. With the exception of NRRL 53121 from Milan hospital 2 (designated hospital M-2), all of the remaining 19 in-group isolates were obtained from Sesto San Giovanni Hospital in Milan. The two-locus ST is indicated to the right of the five-digit NRRL number. Two isolates of ST 33 (46436 and 46595) and three isolates of ST 93 (52681, 52682, and 52683), shaded in gray, were isolated, from separate patients with onychomycosis, respectively, on separate occasions during 2007. MP bootstrap support of ≥70% from 1,000 pseudoreplicates of the data is indicated above the nodes.

FIG. 3.

One of 251 phylograms inferred from MP analysis of the combined two-locus data set for 20 isolates within the GFSC by using sequences of F. sacchari to root the tree. MP bootstrap clade support of ≥70% from 1,000 pseudoreplicates of the data is indicated above nodes. The 11 dermatological isolates from onychomycotic and paronychial infections were from Sesto San Giovanni Hospital in Milan. The remaining nine isolates were from hospitals 3 and 4 in Milan (designated hospitals M-3 and M-4, respectively), Novara (N), Ancona (A), and Varese (V). The two shaded isolates of F. sacchari (44901 and 44905) and F. verticillioides (44891 and 44894) were isolated successively during 2005 and 2006, respectively, from separate patients with onychomycosis.

FIG. 4.

Fusarium guttiforme NRRL 53131 isolated from paronychia of the hand. (A) Prostrate conidiophore formed in culture on SNA, forming aerial conidia in false heads on the tips of phialides. Bar, 50 μm. (B) Polyphilalides formed on aerial conidiophores. Bar, 20 μm. (C) Short clavate to elliptical, 0-septate aerial conidia. Bar, 20 μm. (D) Conidiogenesis of falcate multiseptate conidia on agar. Bar, 50 μm. (E) Enlargement of a multiseptate conidium. Bar, 20 μm.