Discovery of novel septacidin congeners from a high yield heterologous expression strain Streptomyces albus 1597.

Septacidin is an adenine nucleoside antibiotic with antifungal and antitumor activities. During the efforts to construct a better septacidin producer, we obtained a high yield strain S. albus 1597 by putting the biosynthetic gene cluster (BGC) of septacidin under the control of the constitutive strong promoter ermE*. S. albus 1597 could produce new septacidin congeners SEP-538 and SEP-552 with shorter fatty acyl chains. Moreover, SEP-624 with an unprecedented hydroxylated fatty acyl chain was also isolated from this titre improved strain, enriching the diversity of septacidins. SEP-552 showed moderate inhibitory effects against Epidermophyton floccosum 57312 with MIC value 62.5 µM, while SEP-538 and SEP-624 only exhibited weak antifungal activities. The structure-activity relationship investigation revealed that the antifungal activity of septacidins is significantly influenced by the length of and the decoration on their fatty acyl chains.

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 ß-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.

Molecular and Phenotypic Characterization of Nannizzia (Arthrodermataceae).

Phylogenetic studies of the family Arthrodermataceae have revealed seven monophyletic dermatophyte clades representing the genera Trichophyton, Epidermophyton, Nannizzia, Lophophyton, Paraphyton, Microsporum, and Arthroderma. Members of the genus Nannizzia are geo- or zoophiles that occasionally infect humans. With the newly proposed taxonomy, the genus Nannizzia comprises thirteen species, i.e., Nannizzia aenigmatica, N. corniculata, N. duboisii, N. fulva, N. graeserae, N. gypsea, N. nana, N. incurvata, N. perplicata, N. persicolor, N. praecox, and two novel species. Nannizzia polymorpha sp. nov. was isolated from a skin lesion of a patient from French Guiana. For the strain originally described as Microsporum racemosum by Borelli in 1965, we proposed Nannizzia lorica nom. nov. The species are fully characterized with five sequenced loci (ITS, LSU, TUB2, RP 60S L1 and TEF3), combined with morphology of the asexual form and physiological features. A key to the species based on phenotypic and physiological characters is provided.

Epidermophyton floccosum: nucleotide sequence analysis and antifungal susceptibility testing of 40 clinical isolates.

Purpose. Epidermophyton floccosum is an anthropophilic dermatophyte species, which is one of the common causative agents of dermatophytosis in different parts of the world. The aim of the present investigation was to evaluate the genetic diversity of E. floccosum strains isolated from different parts of Iran and to define the in vitro susceptibility profiles of seven antifungal drugs against these clinical isolates.Methodology. Forty clinical strains of E. floccosum isolated from 40 patients with dermatophytosis were subjected to DNA extraction and PCR amplification of the ITS rDNA region using universal primers ITS1 and ITS4. The in vitro activities of griseofulvin, itraconazole, voriconazole, posaconazole, caspofungin, ketoconazole and terbinafine were determined using a broth microdilution method according to the CLSI-M-38A2 protocol.Results. A mean genetic similarity of 99.5 % was found between E. floccosum strains, with intraspecies differences ranging from 0 to 3 nt. The geometric mean (GM) MICs and minimum effective concentrations (MECs) across all isolates were, in increasing order, as follows: terbinafine (GM=0.018 mg l-1), posaconazole (GM=0.022 mg l-1), itraconazole (GM=0.034 mg l-1) and voriconazole (GM=0.045 mg l-1), which had low MICs against all tested strains, whereas caspofungin (GM=0.22 mg l-1), ketoconazole (GM=0.41 mg l-1) and griseofulvin (GM=0.62 mg l-1) demonstrated higher MICs.Conclusion. Our study showed low intraspecies variation within strains of E. floccosum. Furthermore, terbinafine, posaconazole, itraconazole and voriconazole were shown to be the most potent antifungal drugs against E. floccosum strains.

Characterizing the clinical isolates of dermatophytes in Hamadan city, Central west of Iran, using PCR-RLFP method.

OBJECTIVE: Dermatophytosis is one of the most common mycotic infections, which considered as a public health problem in the major of countries. This study evaluated the molecular epidemiology of dermatophytosis in patients referred to Farshchian hospital in Hamadan city with PCR-RFLP method. MATERIALS AND METHODS: Four hundred and five specimens from clinically suspected patients of dermatophytosis were collected and analyzed by direct microscopic and culture. The isolates were identified by PCR-RFLP method using the MvaI restriction enzyme. RESULTS: Of the 405 specimens, 88 specimens were positive in direct examination and culture. Among the patients, 64.8% were males and35.2% females. Tinea pedis (31.8%) was the most common type of dermatophytosis followed by tinea corporis (22.7%), tinea cruris (20.5%), tinea capitis (10.2%), tinea manuum (5.7%), tinea faciei (4.6%) and tinea unguium (4.6%). Trichophyton interdigitale (36.4%) was the most common isolate followed by Trichophyton rubrum (27.3%), Epidermophyton floccosum (17%), Trichophyton tonsurans (11.4%), Microsporum canis (4.5%), Microsporum gypseum (2.3%) and Trichophyton benhamiae (1.1%). CONCLUSION: Our finding showed that the anthropophilic dermatophyte species causing dermatophytosis are increasing, and molecular methods are reliable assays for accurse identification of dermatophyte species in epidemiological studies.

PRP8 intein in dermatophytes: Evolution and species identification.

Dermatophytes are keratinophilic fungi belonging to the family Arthrodermataceae. Despite having a monophyletic origin, its systematics has always been complex and controversial. Sequencing of nuclear ribosomal ITS and D1/D2 rDNA has been proposed as an efficient tool for identifying species in this group of fungi, while multilocus analyses have been used for phylogenetic species recognition. However, the search for new markers, with sequence and size variation, which enable species identification in only one polymerase chain reaction (PCR) step, is very attractive. Inteins seems to fulfill these characteristics. They are self-splicing genetic elements present within housekeeping coding genes, such as PRP8, that codify the most important protein of the spliceosome. The PRP8 intein has been described for Microsporum canis in databases but has not been studied in dermatophytes in any other published work. Thus, our aim was to determine the potential of this intervening element for establishing phylogenetic relationships among dermatophytes and for identifying species. It was found that all studied species have a full-length PRP8 intein with a Homing Endonuclease belonging to the family LAGLIDADG. Phylogenetic analyses were consistent with other previous phylogenies, confirming Epidermophyton floccosum in the same clade of the Arthroderma gypseum complex, Microsporum audouinii close to M. canis, differentiating A. gypseum from Arthroderma incurvatum, and in addition, better defining the Trichophyton interdigitale and Trichophyton rubrum species grouping. Length polymorphism in the HE region enables identification of the most relevant Microsporum species by a simple PCR-electrophoresis assay. Intein PRP8 within dermatophytes is a powerful additional tool for identifying and systematizing dermatophytes.

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 ß-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.

Molecular epidemiology, phylogeny and evolution of dermatophytes.

Dermatophytes are fungi that invade and propagate in the keratinized skin of mammals, including humans, often causing contagious infections. The species of medical concern belong to the genera Microsporum, Trichophyton, Epidermophyton (in their anamorphic state) and Arthroderma (in their telomorphic state), which were traditionally identified based on their morphology and biochemical characters. Nonetheless, limitations linked to the differentiation of closely related agents at species and strains level have been recently overcome by molecular studies. Indeed, an accurate identification of dermatophytes is pivotal for the establishment of effective control and prevention programs as well as for determining the most appropriate and effective antifungal therapies to be applied. This article reviews the DNA techniques and the molecular markers used to identify and to characterize dermatophyte species, as well as aspects of their phylogeny and evolution. The applications of typing molecular strain to both basic and applied research (e.g., taxonomy, ecology, typing of infection, antifungal susceptibility) have also been discussed.

Molecular analysis of dermatophytes suggests spread of infection among household members.

Dermatophyte infection from the same strains may be an important route for transmission of dermatophytoses within a household. In this study, we used molecular methods to identify dermatophytes in members of dermatophyte-infected households and evaluated variables associated with the spread of infection. Fungal species were identified by polymerase chain reaction (PCR) using primers targeting the internal transcribed spacer (ITS) regions (ITS1 and ITS4). For strain differentiation, fungal DNA was probed with a ribosomal DNA-specific probe (containing ITS1, 5.8S ribosomal DNA, and ITS2) to detect restriction fragment length polymorphisms (RFLPs). Associations between the spread of a dermatophyte infection and fungal/host variables were determined using χ² and logistic regression analyses. Among the 50 households enrolled in this study, 18 included multiple infected members (MIMs). Trichophyton rubrum was the most commonly isolated dermatophyte species, followed by Trichophyton mentagrophyts and Epidermophyton floccosum. Sixteen T rubrum strains (TR-A to TR-P) were identified, with spread of infection detected in 8 MIM households. Factors that were significantly (P<.05) associated with the spread of infection included the presence of strains TR-B or TR-D, a history of concomitant tinea pedis and onychomycosis, and plantar scaling and/or nail discoloration. This study is unique in that it used molecular evidence to demonstrate the association of certain strains with the spread of dermatophyte infection among members of the same household.

Molecular epidemiology of dermatophytosis in Tehran, Iran, a clinical and microbial survey.

In the framework of a survey on dermatophytoses, 14,619 clinical specimens taken from outpatients with symptoms suggestive of tinea and referred to a Medical Mycology laboratory in Tehran, Iran, were analyzed by direct microscopy and culture. In total, 777 dermatophyte strains recovered in culture were randomly identified by a formerly established RFLP analysis method based on the rDNA ITS regions. For confirmation of species identification, 160 isolates representing the likely entire species spectrum were subjected to ITS-sequencing. Infection was confirmed in 5,175 collected samples (35.4%) by direct microscopy and/or culture. Tinea pedis was the most prevalent type of infection (43.4%), followed by tinea unguium (21.3%), tinea cruris (20.7%), tinea corporis (9.4%), tinea manuum (4.2%), tinea capitis (0.8%) and tinea faciei (0.2%). Trichophyton interdigitale was the most common isolate (40.5%) followed by T. rubrum (34.75%), Epidermophyton floccosum (15.6%), Microsporum canis (3.9%), T. tonsurans (3.5 %) and M. gypseum (0.5%). Other species included M. ferrugineum, T. erinacei, T. violaceum, T. schoenleinii, and a very rare species T. eriotrephon (each one 0.25%). The two strains of T. eriotrephon isolated from tinea manuum and tinea faciei are the second and third reported cases worldwide. Application of DNA-based methods is an important aid in monitoring trends in dermatophytosis in the community.

Molecular typing of Epidermophyton floccosum isolated from patients with dermatophytosis by RAPD-PCR.

We evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type Epidermophyton floccosum isolates recovered from patients with dermatophytosis originating from different regions of Iran. A total of 13 clinical isolates of E. floccosum obtained from Iranian patients were analyzed by RAPD with 7 arbitrary primers (OPN16, OPD18' OPU15, OPX19, R28, OPA04 and OPAA17). Among the applied primers, OPN16 produced banding patterns from all the isolates. In addition, some of the isolates had very close relation. The phenon line which represented the mean similarities was at the value of 73%. At this level, 4 groups were characterized. Two isolates of a patient had different molecular patterns, suggesting infection transmission from different sources in the case of a single patient. RAPD-PCR provided a rapid and practical tool for identification of E. floccosum isolates, which was independent of morphological characteristics, and enhanced laboratory diagnosis of dermatophytosis.

Epidermophyton fungal keratitis following laser-assisted subepithelial keratectomy.

A 40-year-old man developed pain, decreased vision, and a corneal infiltrate 10 days after laser-assisted subepithelial keratectomy. Treatment with conventional topical and systemic antibiotic agents did not improve the symptoms. Approximately 2 weeks after surgery, the patient was referred to Kim's Eye Hospital, presenting with counting fingers visual acuity, moderate anterior chamber reaction, and multifocal stromal infiltrates in the left eye. The corneal infiltrate findings were suggestive of fungal keratitis, and corneal smears were positive for septate fungal hyphae. Treatment with topical amphotericin B was initiated, but there was little response. After mycology culture and molecular analysis identified Epidermophyton floccosum as the infectious organism, topical natamycin was added and the infiltrate gradually resolved. Three weeks after treatment with natamycin, the corrected visual acuity was 20/25. Treatment with topical natamycin was effective against Epidermophyton keratitis.

Recent dermatophyte divergence revealed by comparative and phylogenetic analysis of mitochondrial genomes.

BACKGROUND: Dermatophytes are fungi that cause superficial infections of the skin, hair, and nails. They are the most common agents of fungal infections worldwide. Dermatophytic fungi constitute three genera, Trichophyton, Epidermophyton, and Microsporum, and the evolutionary relationships between these genera are epidemiologically important. Mitochondria are considered to be of monophyletic origin and mitochondrial sequences offer many advantages for phylogenetic studies. However, only one complete dermatophyte mitochondrial genome (E. floccosum) has previously been determined. RESULTS: The complete mitochondrial DNA sequences of five dermatophyte species, T. rubrum (26,985 bp), T. mentagrophytes (24,297 bp), T. ajelloi (28,530 bp), M. canis (23,943 bp) and M. nanum (24,105 bp) were determined. These were compared to the E. floccosum sequence. Mitochondrial genomes of all 6 species were found to harbor the same set of genes arranged identical order indicating that these dermatophytes are closely related. Genome size differences were largely due to variable lengths of non-coding intergenic regions and the presence/absence of introns. Phylogenetic analyses based on complete mitochondrial genomes reveals that the divergence of the dermatophyte clade was later than of other groups of pathogenic fungi. CONCLUSION: This is the first systematic comparative genomic study on dermatophytes, a highly conserved and recently-diverged lineage of ascomycota fungi. The data reported here provide a basis for further exploration of interrelationships between dermatophytes and will contribute to the study of mitochondrial evolution in higher fungi.

Single-step PCR using (GACA)4 primer: utility for rapid identification of dermatophyte species and strains.

Dermatophytes are fungi that belong to three genera: Epidermophyton, Microsporum, and Trichophyton. Identification of dermatophyte species is essential for appropriate diagnosis and treatment of dermatophytosis. Routine identification depends on macroscopic and microscopic morphology, which is time-consuming and does not identify dermatophyte strains. In this study, two PCR-based methods were compared for their abilities to identify 21 dermatophyte isolates obtained from Egyptian patients to the species and strain levels. The first method employed a two-step method: PCR amplification, using ITS1 and ITS4 as primers, followed by restriction enzyme digestion using the endonuclease MvaI. The second method employed a one-step approach employing the repetitive oligonucleotide (GACA)(4) as a primer. Dermatophyte strains were also identified using a conventional culture method. Our results showed that the conventional culture method identified four species: Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton violaceum. Moreover, both PCR methods agreed with the diagnosis made using the conventional approach. Furthermore, ITS1/ITS4-based PCR provided no strain differentiation, while (GACA)(4)-based PCR identified different varieties among the T. mentagrophytes isolates. Taken together, our results suggest that (GACA)(4)-based PCR has utility as a simple and rapid method for identification of dermatophyte species as well as utility for differentiation of T. mentagrophytes variants.

The effect of griseofulvin on the gene regulation of beta-tubulin in the dermatophyte pathogen Trichophyton rubrum.

Griseofulvin - a mold metabolite produced by Penicillium griseofulvum - is an antifungal drug whose interaction with tubulin is poorly understood. In this study we cloned the beta-tubulin gene in Trichophyton rubrum, which encodes a polypeptide with 447 amino acids (accession number AAV33733). Nucleotide sequence comparison in data banks for both the partial DNA and its deduced amino acid sequence revealed significant homology with members of the eukaryotic beta-tubulin gene family. Investigation based on real-time polymerase chain reaction (PCR) indicated that griseofulvin treatment of T. rubrum fungal cells decreased the expression of the beta-tubulin gene in a dose-dependent manner. However, fluconazole had no effect on the beta-tubulin transcript level under the same circumstances. The identification of this vital gene, which is potentially involved in many cellular functions in pathogenic dermatophyte fungi, is the first step towards understanding the functional characteristics of beta-tubulin in such fungi and may help in the development of new antifungal drugs against dermatophytes.

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum.

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.

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.

Application of PCR to distinguish common species of dermatophytes.

This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)(4). The primer was able to amplify all the strains, producing species-specific profiles for Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton ajelloi, and Epidermophyton floccosum. Intraspecific variability was not observed for these species. Instead, three different profiles were observed in the Trichophyton mentagrophytes group.

Identification of several clinical isolates of dermatophytes based on the nucleotide sequence of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA.

Nucleotide sequences of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA from seven morphologically unidentified dermatophyte isolates were determined. The sequences were compared with those of typical isolates of Trichophyton (T.) mentagrophytes var. interdigitale, T. rubrum, and Epidermophyton floccosum. Two of the isolates were classified as T. rubrum and the other five as T. mentagrophytes var. interdigitale. The results did not conflict with identifications using other molecular techniques, including random amplification of polymorphic DNA (RAPD) analysis and restriction enzyme analysis of mitochondrial DNAs. Thus, the nuclotide sequence of ITS 1 is possibly a good molecular marker for identification of these major anthropophilic dermatophyte species.