Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences

In the present study, the nucleotide sequences of the CHS1 gene from dermatophytes and related fungi in the genera Chrysosporium, Epidermophyton, Microsporum and Trichophyton were investigated using molecular methods. About 440-bp genomic DNA fragments of the CHS1 gene from 21 species were amplified by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these fungi showed more than 83% similarity. The molecular taxonomy of the CHS1 gene sequences revealed that Microsporum was genetically distinct from Chrysosporium and Trichophyton, as classified by morphological characteristics. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phytogeny of Epidermophyton floccosum and other dermatophytes

Eleven strains of Epidermophyton floccosum were compared with 5 Microsporum and 5 Trichophyton species with respect to the restriction fragment length polymorphism (RFLP) of the mitochondrial DNA to reveal their phylogenetic relationships. The phylogeny of 11 species showed that the three dermatophyte genera could not be separated from each other and could be considered to be congeneric. This result is not inconsistent with the results from ribosomal RNA sequences.     

Updating the Taxonomy of Dermatophytes of the BCCM/IHEM Collection According to the New Standard: A Phylogenetic Approach

Recent taxonomical revisions based on multilocus gene sequencing have provided some clarifications to dermatophyte (Arthrodermataceae) family tree. These changes promoted us to investigate the impact of the changed nomenclature of the dermatophyte strains in the BCCM/IHEM fungal collection, which contains strains of all dermatophyte genera except for Ctenomyces. For 688 strains from this collection, both internal transcribed spacer region (ITS) and partial β-tubulin (BT) sequences were aligned and a multilocus phylogenetic tree was constructed. The ITS?+?BT phylogentic tree was able to distinguish the genera Arthroderma, Lophophyton, Microsporum, Paraphyton, Nannizzia and Trichophyton with high certainty. Epidermophyton, which is widely considered as a well-defined genus with E. floccosum as the only representative, fell within the Nannizzia clade, whereas the phylogenetic analysis, based on the ITS region alone, differentiates Epidermophyton from Nannizzia as a separate genus. Re-identification and reclassification of many strains in the collection have had a profound impact on the composition of the BCCM/IHEM dermatophyte collection. The biggest change is the decline of prevalence of Arthroderma strains; starting with 103 strains, only 22 strains remain in the genus after reassessment. Most Arthroderma strains were reclassified into Trichophyton, with A. benhamiae and A. vanbreuseghemii leaving the genus. The amount of Microsporum strains also dropped significantly with most of these strains being reclassified into the genera Paraphyton and Nannizzia.

     

The growth of Epidermophyton floccosum and E. stockdaleae at different temperatures

The ability of 17 strains of genus Epidermophyton (15 strains belonging to Epidermophyton floccosum, one to E. floccosum var. nigricans and one to E. stockdaleae) to grow at different temperatures (4 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C) was stated.The strains were inoculated on Sabouraud Dextrose Agar and regularly controled over a period of 14 days when the plates were incubated at 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C, and over a period of 70 days when the temperature was 4 °C. The optimal growth of E. floccosum was observed at 28 °C and 31 °C, and no signs of growth were recorded neither at 4 °C nor at 40 °C. The optimal development of E. stockdaleae was observed at 25 °C and 28 °C. This species grew from 4 °C to 31 °C.     

Carbohydrate composition of the isolated cell walls of dermatophytes

In an attempt to evaluate taxonomic character of sugar composition of dermatophytes, the purified cell walls from 13 species are analyzed on neutral sugar composition by gas liquid chromatography. The results were principally compatible with those obtained by conventional morphological examination. Neutral sugar components of dermatophytes cell walls were mannose and glucose in the ratio of 1∶2.7 for Epidermophyton and 1∶1.4 for Microsporum. There were two types in Trichophyton, in which the ratios of mannose to glucose were 1∶1.6 and 1∶3.8. The cases of Trichophyton ferrugineum and Trichophyton mentagrophytes were exceptional. The ratio of the former was 1∶1.4, which implied the relation to Microsporum group, and the ratio of the latter was 1∶2.3, which was supposed to be the intermediate of two types of Trichophyton group. Albino type cell wall of Epidermophyton floccosum was more rich in glucose than pigmented type one.     

Isolation of keratinophilic fungi from floors in Roman kindergarten and secondary schools

We examined the dust collected from the floors of forty classrooms, twenty in kindergarten schools (children aged 2–5) and twenty in secondary schools (students aged 11–14) in order to determine the diffusion of keratinophilic fungi in respect to such different factors as human presence and children's age. In the kindergarten schools 268 colonies of keratinophilic fungi were isolated: 50 were Microsporum, 6 Trichophyton and 212 Chrysosporium species. Members of the Chrysosporium genus were found the widely diffused. It is interesting to note the isolation of M. gypseum in two schools. In the secondary schools 847 colonies of keratinophilic fungi were isolated: 727 were Chrysosporium, 81 Microsporum, 38 Trichophyton and 1 Epidermophyton species. Again the Chrysosporium species were the most widely diffused. It is remarkable to point out the isolation of pathogenic species such as Epidermophyton floccosum, Microsporum canis, Microsporum gypseum and the rather rare Microsporum vanbreuseghemii.     

Lipolytic activity of dermatophytes

Lipolytic activity of dermatophytes was tested by the method generally used forCandida lipolytica. Most of the freshly isolated strains ofMicrosporum canis, M. gypseum, Epidermophyton floccosum andTrichophyton mentagrophytes gave positive reactions, whereas, only few strains ofT. schoenleini, T. violaceum, T. megnini, T. rubrum andT. tonsurans yielded such reactions. Among subcultured strains, it was found a decreased lipolytic activity inMicrosporum species and an increased lipolytic activity inEpidermophyton and someTrichophyton species.Part of this work was performed at Serviço de Doenças Infecto-Contagiosas (Secção de Dermatologia), Hospital de Curry Cabral - Lisboa.     

Sodium chloride tolerance in strains ofEpidermophyton floccosum andEpidermophyton stockdaleae

The ability of strains of genusEpidermophyton to grow at different concentrations of NaCl (ranged from 0 to 10%) was studied. A reduction in the diameter of colonies was observed as the concentration of NaCl increased. A nearly complete absence of macroconidia were shown even at the lowest concentration assayed. The geophilic speciesE. stockdaleae tolerated higher concentrations of NaCl (> 7%) thanE. floccosum (< 3%).     

Characterization of siderophores produced by different species of the dermatophytic fungiMicrosporum andTrichophyton

The dermatophytic fungiTrichophyton spp andMicrosporum spp secrete ferrichrome type siderophores under low iron conditions. Three different species ofMicrosporum, i.e.M. qypseum, M. canis andM. audouinii, as well asT. rubrum produce ferrichrome C and ferricrocin, whereasT. mentagrophytes andT. tonsurans produce only ferrichrome. The identification of the siderophores was established by means of thin layer chromatography, high performance liquid chromatography and mass spectroscopy.     

Incidence of dermatophytes and cyclohexamide resistantfungi on healthy children hairs and nails in nurseries

In order to estimate the prevalence of dermatophytes and other fungi on healthy children hairs and nails, 92 hair samples and 85 nail samples (groups of 10 finger nails from each child) were collected from 5 nurseries (children aged 9 months up to 4 years) in Assiut city. From hair samples 22 species were collected, Trichophyton (2 species) and Microsporum (2 species) were the only recovered dermatophytes in addition to well known keratinophilic genus Chrysosporium (4 species). From nail samples, 18 species were identified, Trichophyton was represented by 4 species, Microsporum, 2 species and Chrysosporium, 4 species. Also, several other saprophytes and cycloheximide resistant fungi were isolated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Toward a Novel Multilocus Phylogenetic Taxonomy for the Dermatophytes

Type and reference strains of members of the onygenalean family Arthrodermataceae have been sequenced for rDNA ITS and partial LSU, the ribosomal 60S protein, and fragments of β-tubulin and translation elongation factor 3. The resulting phylogenetic trees showed a large degree of correspondence, and topologies matched those of earlier published phylogenies demonstrating that the phylogenetic representation of dermatophytes and dermatophyte-like fungi has reached an acceptable level of stability. All trees showed Trichophyton to be polyphyletic. In the present paper, Trichophyton is restricted to mainly the derived clade, resulting in classification of nearly all anthropophilic dermatophytes in Trichophyton and Epidermophyton, along with some zoophilic species that regularly infect humans. Microsporum is restricted to some species around M. canis, while the geophilic species and zoophilic species that are more remote from the human sphere are divided over Arthroderma, Lophophyton and Nannizzia. A new genus Guarromyces is proposed for Keratinomyces ceretanicus. Thirteen new combinations are proposed; in an overview of all described species it is noted that the largest number of novelties was introduced during the decades 1920–1940, when morphological characters were used in addition to clinical features. Species are neo- or epi-typified where necessary, which was the case in Arthroderma curreyi, Epidermophyton floccosum, Lophophyton gallinae, Trichophyton equinum, T. mentagrophytes, T. quinckeanum, T. schoenleinii, T. soudanense, and T. verrucosum. In the newly proposed taxonomy, Trichophyton contains 16 species, Epidermophyton one species, Nannizzia 9 species, Microsporum 3 species, Lophophyton 1 species, Arthroderma 21 species and Ctenomyces 1 species, but more detailed studies remain needed to establish species borderlines. Each species now has a single valid name. Two new genera are introduced: Guarromyces and Paraphyton. The number of genera has increased, but species that are relevant to routine diagnostics now belong to smaller groups, which enhances their identification.     

The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera, Apidae, Meliponini): characterization and phylogenetic analysis

Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flanking regions from three Melipona species were PCR-amplified, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approximately 80 bp. The low variation level between M. quadrifasciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological characters.Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004.     

Construction of a fosmid library of cucumber (Cucumis sativus) and comparative analyses of the eIF4E and eIF(iso)4E regions from cucumber and melon (Cucumis melo)

A fosmid library of cucumber was synthesized as an unrestricted resource for researchers and used for comparative sequence analyses to assess synteny between the cucumber and melon genomes, both members of the genus Cucumis and the two most economically important plants in the family Cucurbitaceae. End sequencing of random fosmids produced over 680 kilobases of cucumber genomic sequence, of which 25% was similar to ribosomal DNAs, 25% to satellite sequences, 20% to coding regions in other plants, 4% to transposable elements, 13% to mitochondrial and chloroplast sequences, and 13% showed no hits to the databases. The relatively high frequencies of ribosomal and satellite DNAs are consistent with previous analyses of cucumber DNA. Cucumber fosmids were selected and sequenced that carried eukaryotic initiation factors (eIF) 4E and iso(4E), genes associated with recessively inherited resistances to potyviruses in a number of plants. Indels near eIF4E and eIF(iso)4E mapped independently of the zym, a recessive locus conditioning resistance to Zucchini yellow mosaic virus, establishing that these candidate genes are not zym. Cucumber sequences were compared with melon BACs carrying eIF4E and eIF(iso)4E and revealed extensive sequence conservation and synteny between cucumber and melon across these two independent genomic regions. This high degree of microsynteny will aid in the cloning of orthologous genes from both species, as well as allow for genomic resources developed for one Cucumis species to be used for analyses in other species. Names are necessary to report factually on available data; however, the US Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Experimental dermatophytoses produced by E. floccosum in guinea pigs

Experimental dermatophytoses were tested in guinea pigs by 17 strains of genus Epidermophyton. Only three strains of E. floccosum produced lesions. These lesions were detected 5–7 d after inoculation, and no crust formation or alopecia was observed. These lesions were also studied using microbiological and histopathological techniques. No infection occurred after cutaneous application of E. floccosum var. nigricans and E. stockdaleae.     

Dermatophyte and non dermatophyte fungi in Riyadh City,Saudi Arabia

BackgroundDermatophytes are a scientific label for a group of three genera (Microsporum, Epidermophyton and Trichophyton) of fungus that causes skin disease in animals and humans. Conventional methods for identification of these fungi are rapid and simple but are not accurate comparing to molecular methods.ObjectiveThis study aimed to isolate human pathogenic dermatophytes which cause dermatophytosis in Riyadh City, Saudi Arabia and to identify these fungi by using conventional and molecular methods.MethodsThe study was conducted in Medical Complex, Riyadh and King Saud University. Samples of infected skin, hairs and nails were collected from 112 patients. Diagnosis of skin infections, direct microscopic test, isolation and identification of dermatophytes by conventional and molecular methods were carried out.ResultsThe results indicated that the tinea capitis infection had the highest prevalence among the patients (22.3%) while Tinea barbae had the lowest. In this study the identified dermatophyte isolates belong to nine species as Trichophyton violaceum, Trichophyton verrucosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton schoenleinii, Trichophyton concentricum, Microsporum canis, Microsporum audouinii and Epidermophyton floccosum which cause skin infections were isolated during this study. Non dermatophyte isolates included 5 isolates from Aspergillus spp. 4 isolates from Acremonium potronii and 15 isolates from Candida spp. M. canis were the most common species (25% of isolated dermatophytes). Out of the 52 dermatophyte isolates identified by conventional methods, there were 45 isolates identified by the molecular method.ConclusionsThe results concluded that approximately M. canis caused a quarter of dermatophyte cases, tinea capitis infection was prevalent and the molecular method was more accurate than conventional methods.     

Molecular Analysis of Chitin Synthase 1 (CHS1) Gene Sequences of Trichophyton mentagrophytes Complex and T. rubrum

Nucleotide sequences of chitin synthase 1 (CHS1) gene of dermatophytes, Arthroderma benhamiae, A. simii, A. vanbreuseghemii, Trichophyton mentagrophytes var. interdigitale (T. interdigitale), and T. rubrum were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these five dermatophytes showed more than 90% similarity between the species. The phylogenetic analysis of their sequences revealed that A. benhamiae, A. simii, A. vanbreuseghemii, and T. rubrum were genetically distinct from one another, but T. interdigitale was genetically very close to A. vanbreuseghemii. On the other hand, a specific restriction endonuclease site of HinfI was present in the CHS1 gene fragment of T. rubrum but not in those of A. benhamiae, A. simii, A. vanbreuseghemii and T. interdigitale. The molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution. Received: 6 March 1998 / Accepted: 4 May 1998     

Sensitivity of some strains of the genus Epidermophyton to different antifungal agents

The inhibitory activity of some antifungal agents against 17 strains of genus Epidermophyton have been studied. The behaviour of Epidermophyton stockdaleae against antifungal agents tested is clearly different from that observed in the strains belonging to Epidermophyton floccosum, since all of the latter were sensitive to the antifungal agents used and the geophilic species showed resistance to griseofulvin, isoconazole, natamycin and nystatin, and intermediate sensitivity to ketoconazole and miconazole. The low sensitivity of E. stockdaleae (and other geophilic dermatophytes) to antifungal agents could be a problem in the current therapy if further studies or case reports demonstrate their pathogenic role.     

Using progesterone 5β-reductase, a gene encoding a key enzyme in the cardenolide biosynthesis, to infer the phylogeny of the genus Digitalis

Summary The progesterone 5β-reductase (5β-POR), a key enzyme in the cardenolide biosynthesis, was sequenced for 21 species of Digitalis and Isoplexis to infer phylogenetic and biogeographic relationships. This new secondary metabolism molecular marker was compared to the previously applied nuclear ITS and plastid trnL-F sequences. The results from separate analyses show high congruence within the genus Digitalis and support the conclusion that all species of Isoplexis have a common origin and are embedded in Digitalis. The genus Isoplexis therefore should be reduced to sectional rank within the genus Digitalis. The sequence analyses give further evidence that additional sequence data increase support for relationships. It demonstrates that poorly supported relationships in smaller data sets may lead to erroneous conclusions about the evolution of the investigated taxa.     

A significant geographical area for the study of the epidemiological and ecological aspect of mediterranean sporothricosis

The ability of 17 strains of Epidermophyton to perforate hair in vitro using the Ajello & Georg's test procedure and a modification of Lu's method has been studied.Following the Ajello & Georg's test procedure only E. stockdaleae perforated hair. Sporadically some strains of E. floccosum perforated horse hair. We noted as well unusual perforations originated from inside to outside of the hair.By the other technique, all strains, excepting E. floccosum var. nigricans in child hair, perforated hair. E. floccosum showed these perforations later than E. stockdaleae.