Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions

Abstract

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.

FIG. 1

EcoRI restriction map of the rDNA repeat unit of T. rubrum. The fragment between restriction sites Ec 1 and Ec 2 may encompass the whole of the 25S gene and is of constant length (∼3 kb) in all strains. The fragment between sites Ec 2 and Ec 3 represents the NTS region and the 18S gene and shows fragment length polymorphisms in several T. rubrum strains. One hypothesis to account for these length variations is the presence of a repetitive element located in the NTS region.

FIG. 2

Southern blot of EcoRI-digested genomic DNA from 14 strains of T. rubrum, probed as described in Materials and Methods. Two distinct types of restriction fragment length polymorphism are present in the rDNA repeat of these strains. The six strains (types A to F) illustrated in panel A have a single variable fragment in the size range 4.7 to 5.8 kb, while the eight pattern types (G to N) shown in panel B have multiple variable fragments (either two or four) in the same region. An additional high-molecular-weight variable fragment(s) is present in the 9.0-kb region of all strains. The single, invariant band at 3.0 kb representing fragment Ec 1 to Ec 2 (Fig. 1) is not shown. Molecular weights of standards (in thousands) are given to the right of each panel. λ = molecular weight marker (HindIII-digested lambda DNA).

FIG. 3

Agarose gel electrophoresis of PCR-amplified ITS regions (A) and MvaI restriction digests of amplified ITS products (B) from 17 dermatophyte species. All products were electrophoresed in 2% agarose gels and stained with ethidium bromide. Lanes: 1, E. floccosum DS.EF; 2, M. audouinii SJ.EM 4875; 3, M. canis 97/12400; 4, M. gypseum SJ.EM 3928; 5, M. persicolor NCPF 356; 6, T. concentricum NCPF 600; 7, T. erinacei LM 47; 8, T. quinkeanum LM 54; 9, T. schoenleinii SJ.EV 327; 10, T. verrucosum 98/4545; 11, T. rubrum NCPF 295; 12, T. soudanense 98/7676; 13, T. violaceum SJ.EM 7115; 14, T. mentagrophytes DS.TMvM; 15, T. equinum SJ.A1; 16, T. tonsurans SJ.EM 6717; 17, T. terrestre LM 39.