Pathogenic Dermatophytes Research Articles

Preliminary studies for ciclopirox olamine determination by thermal lens spectrophotometry

The spectrophotometric kinetic methods are broadly used in the determination of drugs in several types of matrices. Thermal Lens Spectrometry (TLS) has demonstrated to be a sensitive indirect spectrophotometric technique used in the field of applied sciences, such as environmental and biochemical sciences. Ciclopirox olamine (CXO) is a synthetic fungicide that inhibits the growth of pathogenic dermatophytes. This work presents the results of CXO determination by TLS, through a kinetic method, based on the CXO oxidation with KMnO4 in alkaline medium to form manganate (MnO42−). The Thermal Lens (TL) effect was generated by a He-Ne laser (TEM00, 20mW, 632nm). The TLS measurements were performed on a single beam assembly with a lens of f=7.5cm focal distance and 10Hz modulation frequency. The TL signal from the detector was acquired for each sample. The optimization was at 30min reaction time, KMnO4 8.0·10−4molL−1 and Na2SO4 0.6molL−1. Under these conditions a calibration curve was constructed. The analytical features were LOD=3.40μmolL−1 and LOQ=11.3μmolL−1. To examine accuracy and reliability, the determination method was successfully applied in tap water samples where known amounts of CXO were spiked.

Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

HapX Mediates Iron Homeostasis in the Pathogenic Dermatophyte Arthroderma benhamiae but Is Dispensable for Virulence.

For many pathogenic fungi, siderophore-mediated iron acquisition is essential for virulence. The process of siderophore production and further mechanisms to adapt to iron limitation are strictly controlled in fungi to maintain iron homeostasis. Here we demonstrate that the human pathogenic dermatophyte Arthroderma benhamiae produces the hydroxamate siderophores ferricrocin and ferrichrome C. Additionally, we show that the iron regulator HapX is crucial for the adaptation to iron starvation and iron excess, but is dispensable for virulence of A. benhamiae. Deletion of hapX caused downregulation of siderophore biosynthesis genes leading to a decreased production of siderophores during iron starvation. Furthermore, HapX was required for transcriptional repression of genes involved in iron-dependent pathways during iron-depleted conditions. Additionally, the ΔhapX mutant of A. benhamiae was sensitive to high-iron concentrations indicating that HapX also contributes to iron detoxification. In contrast to other pathogenic fungi, HapX of A. benhamiae was redundant for virulence and a ΔhapX mutant was still able to infect keratinized host tissues in vitro. Our findings underline the highly conserved role of the transcription factor HapX for maintaining iron homeostasis in ascomycetous fungi but, unlike in many other human and plant pathogenic fungi, HapX of A. benhamiae is not a virulence determinant.

Phytochemical property and assessment of antidermatophytic activity of some selected wild macrofungi against pathogenic dermatophytes

ABSTRACTThe phytochemical property and inhibitory potential of petroleum ether and ethanol extracts of Lenzites quercina, Ganoderma lucidum and Rigidoporus ulmarius were assessed. Standard method was adopted to quantify the phytochemicals in the mushroom extracts. Poisoned food technique was used to assess the inhibition of the extracts obtained from the macrofungi against some pathogenic dermatophytes. The phenolic content of the studied mushroom extracts ranged from 15.7 to 83.8 mg gallic acid equivalent (GAE)/g extract. Ethanolic extract of G. lucidum has the higher content of alkaloids (34.3 mg/g) and flavonoids (18.1 mg/g). Exactly 33.1 mg/g of terpenoids and 16.6 mg/g of saponins were also quantified in ethanolic extract of L. quercina. Extracts of L. quercina, G. lucidum and R. ulmarius exhibited wide range of mycelia inhibition at 50 mg/ml on the tested dermatophytes. The consistent inhibition displayed by the mushroom extracts against the dermatophytes affirms that these wild mushrooms contain bioactive compounds that are antifungal in nature and may possess the ability to cure dermatophyte infections.

Antifungal properties of lecithin- and terbinafine-loaded electrospun poly(ε-caprolactone) nanofibres

We investigated the effect of terbinafine- and egg lecithin-loaded PCL mats on mechanical properties, swellability, biocompatibility andin vitroandex vivoantifungal efficacy against pathogenic moulds and dermatophytes.

Bio-analytical method development and validation for the estimation of Clotrimazole in human plasma by RP-HPLC method

The present study Estimation of drugs in biological media is increasingly important nowadays, which reveals information like bioavailability, bioequivalence, drug abuse, and pharmacokinetics and drug research. HPLC is the most suitable technique for the analysis of biological fluids owing to its well-developed characteristics and ruggedness. It is an extremely sensitive, precise, accurate, and rapid, separation technique. Clotrimazole is a broad spectrum antifungal agent that is used for the treatment of infections caused by various species of pathogenic dermatophytes, yeasts, and Malassezia furfur. The primary action of Clotrimazole is against dividing and growing organisms. Clotrimazole interacts with yeast 14-α demethylase, a cytochrome P-450 enzyme that converts lanosterol to ergosterol, an essential component of the membrane. In this way, Clotrimazole inhibits ergosterol synthesis, resulting in increased cellular permeability. Few analytical methods were reported for the quantitative determination of Clotrimazole and its combination with other drugs by HPLC. Only one method was reported for Clotrimazole in mice plasma by capillary electrophoresis. But there is no RP-HPLC method reported for the bio-analytical method development in human plasma using protein precipitation method under different chromatographic conditions. The present study is planned to develop newer analytical method for the determination of Clotrimazole by bio-analytical method development in human plasma using protein precipitation by RP-HPLC under different chromatographic conditions. CONCLUSION: A bioanalytical method was developed for the estimation of Clotrimazole by HPLC method. The method was validated for its transferability to other user or other laboratory. The HPLC method was developed by using 0.5%TEA in water (pH 3 adjusted with Orthophosphoric acid) and Acetonitrile in meticulous ratio. The peaks obtained for the drugs of interest by the present method are well resolved from each other without any interference and from the plasma endogenous proteins. The peaks are symmetrical with acceptable tailing factor. The retention time of Clotrimazole was within the limit. The results of linearity, intraday and interday precision study and capability of the extraction method were within the limits of bioanalytical method development. The method was linear with a correlation coefficient of acceptable agreement, which is suitable for the estimation of Clotrimazole in human plasma and other biological fluids. The method demonstrated relative recoveries with acceptable relative standard deviation. The limit of quantification (LOQ) and limit of detection (LOD) for Clotrimazole was found to be nanograms lesser than unity. Hence the developed method is sensitive for the estimation of Clotrimazole in trace amounts. Peak purity studies, with peak purity index values closer to unity reveals that the method developed was specific for the estimation of Clotrimazole in blood and other biological fluids. It can be concluded that the developed RP-HPLC method in human plasma was found to be very simple, reliable and selective for providing satisfactory accuracy and precision. The methods are suitable for routine quantitative analysis in pharmaceutical dosage forms. Hence this developed method can be used further in Bioequivalence and bioavailability studies of clotrimazole. Pharmacokinetic and bio-equivalence study centers

The physicochemical, safety and antimicrobial properties of Phyllanthus amarus herbal cream and ointment

The ethanol extract of Phyllanthus amarus (Schum and Thonn), a plant of ethnomedicinal importance, was formulated into herbal cream and ointment and evaluated using physicochemical, safety and antimicrobial properties. The extract was obtained by maceration and the antimicrobial properties tested on clinically isolated pathogenic bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and dermatophytes (Trichophyton rubrum and Candidaalbicans) using established methods. Cream and ointment formulations containing 1–10 % w/w extract was prepared and 4 % w/w humectant (glycerin) was incorporated into the cream while 5 % w/w surfactant (cetomacrogol 1000) was incorporated to the ointment. The results showed that the pH of the formulations was acidic and the viscosity ranged from 1250 to 4950 cP for ointments and 570–1233 cP for creams. The presence of humectant and surfactant significantly (p < 0.05) reduced the viscosities of the formulations. The results of dermal irritation showed negligible irritation index while sub-chronic toxicity tests showed that the formulations did not cause any visible lesions in the skin of the animals after application for twenty-one days. The in vitro antimicrobial properties of formulations were concentration-dependent with the creams showing higher activity. Furthermore, the in vivo activity of the cream on S. aureus showed increased antibacterial activity with increase in extract concentration and humectant presence. The herbal cream and ointment of Phyllanthus amarus extract had acceptable physicochemical and safety profiles with significant (p < 0.05) in vitro and in vivo antimicrobial activity. Thus, the formulations could be useful in the treatment of skin infections instead of using the extract for bathing and rubbing.

Comparison of Antifungal Activity of Extracts of Ten Plant Species and Griseofulvin Against Human Pathogenic Dermatophytes

Background: This study was conducted to measure the antifungal activity of the extracts of 10 plant species used in traditional Iranian medicine against human pathogenic dermatophytes. Materials and Methods: In this experimental study, the leaves of these plants (Calendula officinalis , Acacia arabica, Altheae officinalis , Ginkgo biloba, Juglans regia, Osimum basilicum, Solanum nigrum, Hypericum perforatum, Urtica dioica, and Anagalis arvensis) were taken and extractions were made in methanol and were tested against Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, and Epidermophyton floccosum . The minimum inhibitory concentration (MIC) was determined using broth macrodilution method. The effects of plants extracts were compared with those of griseofulvin. Results: Plants under review showed antifungal activity against all the dermatophytes tested with MIC values ranging from 0.001 to 0.016 mg/mL using inhibitory zone estimation, 0.3 to 12.8 mg/mL using agar dilution method and 0.2 to 12.5 mg/mL using broth dilution method. The minimum fungicidal concentration (MFC) of the extracts ranged from 0.8 to 15.62 mg/mL. Conclusions: The results obtained suggested that H. perforatum, A. arvensis, and A. arabica have anti-dermatophyte activity.

Inactivation of human pathogenic dermatophytes by non-thermal plasma

Non-thermal plasma (NTP) was tested as an in vitro deactivation method on four human pathogenic dermatophytes belonging to all ecological groups including anthropophilic Trichophyton rubrum and Trichophyton interdigitale, zoophilic Arthroderma benhamiae, and geophilic Microsporum gypseum. The identification of all strains was confirmed by sequencing of ITS rDNA region (internal transcribed spacer region of ribosomal DNA). Dermatophyte spores were suspended in water or inoculated on agar plates and exposed to NTP generated by a positive or negative corona discharge, or cometary discharge. After 15min of exposure to NTP a significant decrease in the number of surviving spores in water suspensions was observed in all species. Complete spore inactivation and thus decontamination was observed in anthropophilic species after 25min of exposure. Similarly, a significant decrease in the number of surviving spores was observed after 10–15min of exposure to NTP on the surface of agar plates with full inhibition after 25min in all tested species except of M. gypseum. Although the sensitivity of dermatophytes to the action of NTP appears to be lower than that of bacteria and yeast, our results suggest that NTP has the potential to be used as an alternative treatment strategy for dermatophytosis and could be useful for surface decontamination in clinical practice.

In vitro antifungal activities of Actinomyces species isolated from soil samples against Trichophyton mentagrophytes.

Background and Purpose:Cutaneous infections arise from a homogeneous group of keratinophilic fungi, known as dermatophytes. Since these pathogenic dermatophytes are eukaryotes in nature, use of chemical antifungal agents for treatment may affect the host tissue cells. In this study, we aimed to evaluate the antifungal activity of Actinomyces species against Trichophyton mentagrophytes (abbreviated as T. mentagrophytes). The isolates were obtained from soil samples and identified by polymerase chain reaction (PCR) technique.Material and Methods:In total, 100 strains of Actinomyces species were isolated from soil samples in order to determine their antagonistic activities against T. mentagrophytes in Kerman, Iran. The electron microscopic study of these isolates was performed, based on the physiological properties of these antagonists (e.g., lipase, amylase, protease and chitinase), using relevant protocols. The isolates were identified using gene 16S rDNA via PCR technique. Results: Streptomyces flavogriseus, Streptomyces zaomyceticus strain xsd08149 and Streptomyces rochei were isolated and exhibited the most significant antagonistic activities against T. mentagrophytes. Images were obtained by an electron microscope and some spores, mycelia and morphology of spore chains were identified. Molecular, morphological and biochemical characteristics of these isolates were studied, using the internal 16S rDNA gene. Active isolates of Streptomyces sequence were compared to GenBank sequences. According to nucleotide analysis, isolate D5 had maximum similarity to Streptomyces flavogriseus (99%).Conclusion:The findings of this study showed that Streptomyces isolates from soil samples could exert antifungal effects on T. mentagrophytes.

In Vitro Evaluation of Enzymatic and Antifungal Activities of Soil-Actinomycetes Isolates and Their Molecular Identification by PCR.

Background:Human cutaneous infection caused by a homogeneous group of keratinophilic fungi called dermatophytes. These fungi are the most common infectious agents in humans that are free of any population and geographic area. Microsporum canis is a cause of dermatophytosis (Tinea) in recent years in Iran and atypical strain has been isolated in Iran. Its cases occur sporadically due to M. canis transmission from puppies and cats to humans. Since this pathogenic dermatophyte is eukaryotes, chemical treatment with antifungal drugs may also affect host tissue cells.Objectives:The aim of the current study was to find a new antifungal agent of soil-Actinomycetes from Kerman province against M. canis and Actinomycete isolates were identified by PCR.Materials and Methods:A number of hundred Actinomycete isolated strains were evaluated from soil of Kerman province, for their antagonistic activity against the M. canis. M. canis of the Persian Type Culture Collection (PTCC) was obtained from the Iranian Research Organization for Science and Technology (IROST). Electron microscope studies of these isolates were performed based on the physiological properties of these antagonists including lipase, amylase, protease and chitinase activities according to the relevant protocols and were identified using gene 16SrDNA.Results:In this study the most antagonist of Actinomycete isolates with antifungal activity against M. canis isolates of L1, D5, Ks1m, Km2, Kn1, Ks8 and Ks1 were shown in vitro. Electron microscopic studies showed that some fungal strains form spores, mycelia and spore chain. Nucleotide analysis showed that Ks8 had maximum homology (98%) to Streptomyces zaomyceticus strain xsd08149 and L1 displayed 100% homology to Streptomyces sp. HVG6 using 16SrDNA studies.Conclusions:Our findings showed that Streptomyces has antifungal effects against M. canis.

Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

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.

Cutaneous bacterial species from Lithobates catesbeianus can inhibit pathogenic dermatophytes.

Antibiotics are being successfully used to fight many infectious diseases caused by pathogenic microorganisms. However, new infectious diseases are continuously being identified, and some known pathogens are becoming resistant against known antibiotics. Furthermore, many antifungals are causing serious side effects in long-term treatments of patients, and many skin infections caused by dermatophytes are difficult to cure. The beneficial roles of resident cutaneous microbiota to inhibit pathogenic microorganisms have been shown for many vertebrate species. Microbial symbionts on the amphibian skin for example can be a source of powerful antimicrobial metabolites that can protect amphibians against diseases, such as chytridiomycosis, caused by a fungal pathogen. In this research, we investigated whether cutaneous bacterial species isolated from Lithobates catesbeianus (North American bullfrog), an invasive amphibian species that is resistant to chytridiomycosis, produce secondary metabolites that can be used to inhibit the growth of three species of dermatophytes (Microsporum gypseum, Epidermophyton floccosum, and Trichophyton mentagrophytes) which are known to cause topical or subdermal skin infections in humans. Strongly anti-dermatophyte bacterial species that belonged to the Bacillaceae, Streptomycetaceae, Pseudomonadaceae, Xanthomonadaceae, Aeromonadaceae, and Enterobacteriaceae were identified. This research has provided evidence of the presence of cutaneous anti-dermatophyte bacteria from L. catesbeianus which might provide a basis for health care providers to experiment with new antifungals in the future.

Evaluation of antifungal activities of the essential oil and various extracts of Nigella sativa and its main component, thymoquinone against pathogenic dermatophyte strains

Plant extracts and plant-derived compounds are valuable sources as folk medicine for the treatment and prevention of a wide range of diseases including infectious diseases. In the present study, the antifungal activities of the essential oil and various extracts Nigella sativa and its active principle, thymoquinone against Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum as pathogenic dermatophyte strains have been evaluated. In addition, the cytotoxic effects of N. sativa against murine macrophage cells were determined. In this study, the antifungal activity was studied by disk diffusion method and assessment of minimum inhibitory concentration (MIC) of extracts using broth macrodilution method. In addition, the cytotoxic activity of N. sativa was evaluated by colorimetric assay (MTT). The components of the N. sativa essential oil were also identified by gas chromatography/mass spectroscopy (GC/MS) analysis. The results showed that the essential oil and various extracts of N. sativa particularly thymoquinone have potent antifungal effects on T. mentagrophytes, M. canis and M. gypseum as pathogenic dermatophyte strains. In the assessment of the cytotoxicity activity, it could be observed that N. sativa had no significant cytotoxicity in the murine macrophages at low concentrations. While, thymoquinone in comparison with essential oil and various extracts of N. sativa showed higher cytotoxicity on murine macrophage cells. In the GC/MS analysis, thymoquinone (42.4%), p-cymene (14.1%), carvacrol (10.3%) and longifolene (6.1%) were found to be the major components of N. sativa essential oil. The findings of this study suggest a first step in the search of new antidermatophytic drugs and aid the use of N. sativa seeds in the traditional medicine for dermatophytic infections.

Antifungal activity of a soil isolate of Pseudomonas chlororaphis against medically important dermatophytes and identification of a phenazine-like compound as its bioactive metabolite

The increasing importance of dermatophytoses and emerging resistance of dermatophytes to current synthetic antifungals have stimulated the search for safer and more effective alternative drugs from natural sources. The present study was carried out to identify antagonistic bacteria of soil origin with strong inhibitory activities on the growth of major human pathogenic dermatophytes. Antifungal activity of isolated soil bacteria was screened against the dermatophytes from three genera Microsporum (M.canis, M.gypseum), Epidermophyton (E.floccosum) and Trichophyton (T.mentagrophytes, T.rubrum, T.violaceum, T.tonsurans) by using visual plate agar assay method. A Pseudomonas chlororaphis isolate S105, identified at the species level by 16S ribosomal RNA sequence analysis, was reported as the strongest antagonistic bacterium. P.chlororaphis S105 culture supernatant (PCCS) was examined against tested dermatophytes by GY (glucose-yeast extract) broth bioassay in 6-well microplates. Antifungal compound of the bacterium was partially purified from the culture supernatant through a purification scheme of methanol extraction, Diaion HP20 ion-exchange chromatography and preparative thin layer chromatography. P.chlororaphis S105 was the most potent inhibitor of fungal growth for all tested dermatophytes with a percent inhibition ranged from 57.1% to 99.8%. The PCCS suppressed the growth of all fungi tested in the range of 18.5% to 84.8%. Partially purified antifungal compound of the bacterium was identified as a phenazine-like compound with an Rf value of 0.51. The compound inhibited fungal growth by 73.6% to 97.9% on GY broth. Fungal growth inhibition was significant for all dermatophytes tested in comparison with the controls (Anova, P<0.05). With respect to the strong inhibitory activity of P.chlororaphis against pathogenic dermatophytes reported here, it may be considered as a rich source of useful metabolites with potential application in antifungal drug discovery.

Prevalence of dermatophytes and other superficial fungal organisms in asymptomatic guinea pigs in Southern Italy

Guinea pigs have been indicated as a potential source of zoophilic dermatophytes that cause human dermatomycosis. The purpose of this study was to evaluate the prevalence of dermatophytes as well as saprophytic fungi in asymptomatic pet guinea pigs in Southern Italy. Two-hundred pet guinea pigs were enrolled from both private veterinary clinics and pet shops in the Campania region, Italy, from August 2012 to September 2013. Samples were collected using the MacKenzie's toothbrush technique. The plates were incubated for four weeks at 25°C and identification of the fungal colonies was based on both macroscopic and microscopic characteristics. Two pathogenic dermatophytes were isolated in 9 (4·5%) of 200 guinea pigs; Epidermophyton species in 2 (1%) and Scopulariopsis species in 7 (3·5%). Saprophytic dermatophytes were isolated from 151 (75·5%) animals enrolled. No fungal growth was observed in 40 (20%) guinea pigs. The results of this study indicate a low prevalence of pathogenic dermatophytes in pet guinea pigs in Southern Italy but the presence of Epidermophyton and Scopulariopsis species in asymptomatic pet guinea pigs.

Antifungal, Antileishmanial, and Cytotoxicity Activities of Various Extracts of Berberis vulgaris (Berberidaceae) and Its Active Principle Berberine.

In this study, in vitro antidermatophytic activity against Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis, and Microsporum gypseum was studied by disk diffusion test and assessment of minimum inhibitory concentration (MIC) using CLSI broth macrodilution method (M38-A2). Moreover, antileishmanial and cytotoxicity activity of B. vulgaris and berberine against promastigotes of Leishmania major and Leishmania tropica were evaluated by colorimetric MTT assay. The findings indicated that the various extracts of B. vulgaris particularly berberine showed high potential antidermatophytic against pathogenic dermatophytes tested with MIC values varying from 0.125 to >4 mg/mL. The results revealed that B. vulgaris extracts as well as berberine were effective in inhibiting L. major and L. tropica promastigotes growth in a dose-dependent manner with IC50 (50% inhibitory concentration) values varying from 2.1 to 26.6 μg/mL. Moreover, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages with CC50 (cytotoxicity concentration for 50% of cells) values varying from 27.3 to 362.6 μg/mL. Results of this investigation were the first step in the search for new antidermatophytic and antileishmanial drugs. However, further works are required to evaluate exact effect of these extracts in animal models as well as volunteer human subjects.

English

A Streptomyces sp. strain named 5K10, isolated from soil samples of Kaziranga National Park, Assam, India was found to secrete an antimicrobial proteinaceous compound, showing in vitro antagonistic effect against some pathogenic bacteria, yeasts, dermatophytes and filamentous fungi. Capacity to produce extracellular antimicrobial substances by the strain 5K10 was assessed by conventional spot inoculation method in agar media against test microorganisms as well as fungal spore germination inhibition assay by microscopic observations. The fermented culture broth with ammonium sulfate precipitation revealed a biologically active proteinaceous compound and molecular weight estimated to be about 14.3 kDa on SDS-PAGE gel. Using Candida albicans MTCC 227 as the indicator strain, antagonistic activity of the proteinaceous compound in the region of the SDS-PAGE gel was confirmed. Bioactivity of the extracellular protein was sensitive to proteinases and partially susceptible to prolonged heat treatment. The results suggest that Streptomyces sp. 5K10 might be an important bioresource of lead molecules for developing antimicrobial agents to control bacterial and fungal infections. &nbsp; Key words: Antifungal activity, extracellular protein, dermatophytes, Streptomyces.

Restriction Analysis of β-Tubulin Gene for Differentiation of the Common Pathogenic Dermatophytes

Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, β-tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile. The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR-amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis. After digestion of partially amplified β-tubulin gene with the restriction enzyme FatI, three dermatophyte species, that is, Microsporum gypseum, M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii, and T. violaceum, were identified by further restriction digestion by Alw21I, MwoI, and HpyCH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis. The two-step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes.