Identification of Zoophilic Dermatophytes Using MALDI-TOF Mass Spectrometry

Abstract

Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the in-house database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents.

Keywords: Microsporum; Trichophyton; Trichophyton erinacei; dermatophytoses; geophilic; hedgehog; zoonoses; zoophilic.

Figure 1

Photographs of the colony morphology of selected Arthroderma (A.) spp. (A–F) and Trichophyton (T.) erinacei cultures (G–L) isolated from symptomatic and asymptomatic hedgehogs. Cultures were grown on Sabouraud-Dextrose agar (2%) at 28°C for 3d, species identity was confirmed by sequencing of the ITS region of fungal rDNA. The first column shows the typical morphology of A. chiloniense with a white, fluffy obverse (A) and a beige to yellow reverse (B). T. erinacei 218315 (G, H) showed a very similar appearance rendering those two isolates hardly distinguishable solely by the assessment of phenotypic traits. The typical colony morphology of T. erinacei is depicted in (K) (obverse: white to yellow, granular) and (L) (reverse: yellow to bright orange). Intermediate, rather untypical morphologies (fluffy, folded, elevated in the center) with varying color shades from white to cream to beige are seen with other Arthroderma spp. and T. erinacei isolates.

Figure 2

Phylogenetic tree based on fungal rDNA ITS sequences for the investigated dermatophyte isolates (Tamura-Nei-model, Neighbor-Join and BioNJ algorithms, MCL approach, 1000 replicates). The tree is drawn to scale with branch lengths measured in the number of substitutions per site and indicated support values; the analysis involved 37 nucleotide sequences including the dermatophytes isolated during this study and from veterinary routine diagnostics (vet; vac refers to a vaccine strain used in bovine practice), human-derived isolates from the Laboratory of Medical Microbiology (hum) and sequences derived from the NCBI database. Generally, each dermatophyte species forms an own subclade with mostly very robust support; the different origins of isolation (vet vs. hum) are not reflected. The tree comprises two main clades: the upper one contains all geophiles, i.e. all Arthroderma spp.; the lower one all isolates of the genera Trichophyton and Microsporum. The latter form individual subclades in this lower clade; the very closely related species T. benhamiae and T. erinacei as well as T. mentagrophytes and T. quinckeanum are separated in distinct subclades.

Figure 3

Sabouraud-Dextrose agar (2%) covered with a sterilized filter paper promoted fungal growth successfully and facilitated sampling of merely fungal material for MALDI-TOF MS analysis. Different Trichophyton (T.), Arthroderma (A.) and Microsporum (M.) isolates are exemplarily shown after an incubation time of 5d at 28°C (T. verrucosum: 37°C). Species identity was confirmed by sequencing of the ITS region of fungal rDNA.

Figure 4

Examples of mass spectrometric profiles (raw spectra) of different Trichophyton (T.) erinacei-isolates cultured in liquid Sabouraud-2% Dextrose-broth over-night with gentle rotation at room temperature (liquid, over-night) and on solid agar plates for 3d at 28°C (solid, 3d) are shown. Species identity was confirmed by sequencing of the ITS region of fungal rDNA. Overall, the mass spectrometric profiles were very similar for both cultivation methods. For some isolates grown in liquid media, more and/or more intense peaks were observed in the lower mass range (indicated by the black parenthesis). On the other hand, a peak at around 7300 m/z (black box) was predominantly found in solid cultures. However, a cluster of peaks around 4000 m/z (blue box) seems somewhat species specific for T. erinacei since it was found in most examined isolates regardless of the cultivation method.

Figure 5

A score-oriented distance dendrogram based on the mass spectrometric data (MSP) was generated to identify similarities and clusters of closely related zoophilic and geophilic dermatophytes (Distance Measure Correlation mode, MBT Compass Explorer, Bruker Daltonik GmbH). Isolates of Trichophyton (T.) erinacei, Arthroderma (A.) spp. as well as others from the genera Trichophyton and Microsporum, respectively, from animal (vet, vac) and human patients (hum) were included (ref - reference, derived from NCBI database). The different species cluster in two main clades: the lower one comprises geophilic isolates only, i.e. A. insingulare, A. quadrifidum, A. chiloniense and A. crocatum. The isolates of A. tuberculatum group into the upper clade but therein in the lowest position, i.e. closest to the other Arthroderma spp. The upper clade further comprises the Trichophyton spp. and the M. canis-isolates (and S. brevicaulis). All T. erinacei, T. verrucosum and T. benhamiae-isolates form one subclade indicating their close relation. Species identity was previously confirmed by sequencing of the ITS region of fungal rDNA.