Linalool modulates dermatophyte susceptibility to azole drugs.

This study investigated the monoterpene linalool and its resistance modulating activity involving ergosterol biosynthesis inhibitors (ketoconazole, fluconazole, and itraconazole) in strains of Microsporum spp. and Trichophyton spp. The minimum inhibitory concentration (MIC) of test-drugs were determined by microdilution. The modulating effect of linalool was evaluated by determining the MIC of the antifungals in the presence of subinhibitory concentrations of linalool. We also investigated the association effect (checkerboard) of linalool together with ketoconazole and itraconazole. The fungi became more sensitive to ketoconazole and itraconazole in the presence of linalool. The linalool and azole drug associations presented synergism.

Comparison of two inoculation methods for Microsporum canis culture using the toothbrush sampling technique.

BACKGROUND: The fungal culture toothbrush method is a common method for obtaining material for fungal cultures to diagnose dermatophytosis. The optimal technique for inoculation onto the agar surface has not been studied. HYPOTHESIS/OBJECTIVES: To compare two inoculation techniques; the first involved pressing the toothbrush onto the plate surface (Procedure A) and the second involved pressing the toothbrush onto the agar, as well as transferring hairs and scales entrapped in the bristles. (Procedure B). The hypothesis was that transferring hairs onto the plate would increase the likelihood of obtaining positive cultures. ANIMALS: Twenty-six cattery-housed cats were sampled using the toothbrush technique. Two toothbrush samples were obtained from each cat. METHODS AND MATERIALS: The two toothbrush samples from each cat were randomized to Procedure A or B, and the investigator was blinded to inoculation technique. Cultures were performed on a medium specific for dermatophytes. The number of positive plates, and the presence and abundance of colonies of dermatophytes and contaminant moulds were compared between the two techniques. RESULTS: Twenty-one cats were culture-positive for Microsporum canis. Procedure A resulted in a significantly higher number (P < 0.01) of positive plates (20 of 21; 95%) compared with Procedure B (seven of 21; 33%). These results were due mainly to higher plate invasion by contaminant moulds, using Procedure B. CONCLUSIONS AND CLINICAL IMPORTANCE: Based upon the findings of this study, the optimum inoculation technique is to press toothbrush bristles onto agar plates to maximize growth of M. canis and minimize introduction of contaminant inoculation.

Tinea corporis by Microsporum canis in mycological laboratory staff: Unexpected results of epidemiological investigation.

Microsporum canis is a zoophilic dermatophyte, which is very contagious, especially to cats and dogs. Asymptomatic animal carriers of M. canis are regarded a critical factor in the epidemiology of the disease. The aim of this study was to investigate the epidemiological origin of M. canis isolates using morphological traits in combination with molecular analysis. Identification of dermatophyte strains was carried out by correlating the clinical manifestation of the infection with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. A positive result of the culture examination was obtained from the samples with arthrospores in the direct analysis, that is, from a symptomatic cat and humans, and from a cat without any signs of infection. The microsatellite-primed PCR fingerprinting (MSP-PCR) electro-profiles were identical for all analysed strains. The melting profile-PCR (MP-PCR) electrophoregram indicated variability of the genomes of the strains. The search for the source of the infection indicated one cat that did not have any signs of dermatophytosis. PCR-fingerprinting techniques are useful tools for epidemiological investigation of the origin of dermatophyte infection. These methods can also be used in many cases for species identification of dermatophytes and clarification of the relationships among varieties of a species.

Resistance Mechanism in a Terbinafine-Resistant Strain of Microsporum canis.

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2-4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.

Activity of Salvia dolomitica and Salvia somalensis Essential Oils against Bacteria, Molds and Yeasts.

Essential oils (EOs) from Salvia dolomitica and Salvia somalensis, widely employed in the cosmetic and perfume industry, were analyzed for composition and tested against bacterial and fungal pathogens isolated from clinical and environmental specimens. The analyses were carried out against Staphylococcus aureus, Staphylococcus pseudointermedius, Pseudomonas aeruginosa, Escherichia coli, Streptococcus canis, Streptococcus pyogenes, Klebsiella pneumoniae, Proteus mirabilis, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Aspergillus niger, Aspergillus flavus, Candida albicans, Candida krusei, Mucor sp. and Trichothecium roseum. Both EOs showed similar percentages of total monoterpenes and sesquiterpene hydrocarbons. The main constituents were 1,8-cineole and ß-caryophyllene in S. dolomitica and bornyl acetate and camphor in S. somalensis. The selected EOs have no relevant antifungal or antibacterial activities if compared to conventional drugs.

Morpho-Molecular Characterization of Soil Inhabitant Dermatophytes from Ahvaz, Southwest of Iran, a High Occurrence of Microsporum fulvum.

Occurrence and diversity of dermatophyte mycoflora in 298 soil samples from Ahvaz, Southwest of Iran was investigated by using the hair-baiting technique. The samples were collected during spring (n = 210) and autumn (n = 88) of 2015, and the fungal isolates were identified based on the macro- and micro-morphology of colonies and with further ITS-rDNA RFLP and sequencing. Totally, 60 soil samples (20.1%) were positive for dermatophyte growth whose pH varied from 7.0 to 7.9. The highest (26.6%) and the lowest (14.3%) recovery rates were from the animal resorts and the streets soils samples, respectively. Seasonally, 16.7% of the spring samples and 28.4% of the autumn samples were positive. Based on molecular identification, three species of two genera were identified viz. M. fulvum (n = 57), M. canis (n = 2) and zoophilic Trichophyton interdigitale (n = 1). As a specific goal in the study, differentiation of the species in Microsporum gypseum complex was established by measuring the mean length and width of macroconidia in some strains of M. gypseum, M. fulvum and M. incurvatum. Mean size for macroconidia length and width in three species showed that M. gypseum and M. incurvatum can morphologically be differentiated from M. fulvum but not from each other. M. fulvum was the most abundant species isolated from the soils of Ahvaz; however, to comprehensively specify the distribution pattern of geophilic dermatophytes in the soils of this city further investigations are needed. Identification based on micro-morphometric is not effective for species distinction in M. gypseum complex, while molecular procedures based on sequencing of certain DNA regions are the most reliable and applicable strategies for this purpose.

MODELLING AND BIOPHARMACEUTICAL EVALUATION OF CICLOPIROX OLAMINE GELS.

The ciclopirox olamine (CPO) has a broad antimicrobial profile including dermatophytes, yeasts and is used in various pharmaceutical forms. The aim of this study is to evaluate the quality of the CPO gels according to biopharmaceutial tests in vitro and antifungal activity assay. Hydroxypropyl cellulose, chitosan and poloxamer 407 were selected as agents gelificants. The effects of gelling agent properties and concentration on the consistency and flow characteristics have been studied by rheometer. CPO release rates from gel were measured with Franz type diffusion cells. The antifungal activity of gels was tested using agar well diffusion technique. The results of the experimental study have shown that the rheological properties of the medications depend on the selected gelling agent and the amount of it. The higher amounts of CPO were released from the poloxamer 407 gels. Though all tested CPO gels showed great inhibition of Microsporn canis.

Antidermatophytic Action of Resorcinol Derivatives: Ultrastructural Evidence of the Activity of Phenylethyl Resorcinol against Microsporum gypseum.

In this work, we evaluated the antidermatophytic activities of three resorcinol derivatives that have a history of use in dermo-cosmetic applications to discover molecules with multiple dermatological activities (i.e., multi-target drugs), thereby reducing the cost and time necessary for new drug development. The antidermatophytic activities of the three skin lighteners were evaluated relative to the known antifungal drug fluconazole on nine dermatophytes responsible for the most common dermatomycoses: Microsporum gypseum, Microsporum canis, Trichophyton violaceum, Arthroderma cajetani, Trichophyton mentagrophytes, Epidermophyton floccosum, Nannizzia gypsea, Trichophyton rubrum and Trichophyton tonsurans. Among the three tested resorcinols, only two showed promising properties, with the ability to inhibit the growth of all tested dermatophytes; additionally, the IC50 values of these two resorcinols against the nine dermatophytes confirmed their good antifungal activity, particularly for phenylethyl resorcinol against M. gypseum. Ultrastructural alterations exhibited by the fungus were observed using scanning electron microscopy and transmission electron microscopy and reflected a dose-dependent response to treatment with the activation of defence and self-preservation strategies.

Antifungal activity of berberine hydrochloride and palmatine hydrochloride against Microsporum canis -induced dermatitis in rabbits and underlying mechanism.

BACKGROUND: Phellodendron amurense, exhibits antifungal activity mainly by bioactive components including berberine hydrochloride and palmatine hydrochloride. This study was conducted to evaluate the antifungal effects of berberine hydrochloride, palmatine hydrochloride, and a mixture of both substances against Microsporum canis in vivo and in vitro. METHODS: The minimal inhibitory concentrations (MICs) of monomers and clotrimazole were determined using 1.5 % tryptic soy agar. The effects of these drugs on Microsporum canis growth was detected by determining dry weight. Transmission electron microscopy (TEM) was performed to observe the effect of chemicals on cell ultrastructure. Differential mRNA expressions of eight genes of M. canis treated with berberine or palmatine or their combination at different time points were determined by real-time PCR. NADH enzyme concentration was also detected. Clinical evaluation via in-vivo antifungal assay was also performed. Skin histology PAS staining was also carried out. RESULTS: Results showed that MICs of berberine, palmatine and clotrimazole were 1, 1, and 0.015 mg/mL, respectively. No significant difference was observed among the growth curves of the three groups before 18 h was reached. TEM showed that these drugs could destroy the cell membrane and organelles of M. canis at different time points. After 30 h of incubation, relative mRNA expressions of the genes in the combined group were significantly higher than those in the other groups including the clotrimazole group (P < 0.05); Palmatine initially induced the mRNA up-regulation of PGAL4, FSH1, PQ-LRP, NADH1 and NDR in M. canis; by contrast, berberine maintained a high expression level of these genes to shorten fungal life cycle and eradicate M. canis. Clinical results showed that combined treatment was more effective than single administration of each monomer or clotrimazole. Hence, berberine mixed with palmatine significantly elicited antifungal activities and could be used to treat M. canis in rabbits. CONCLUSION: These results provide a comprehensive view of the mechanism of berberine and palmatine in anti-M. canis activity.

Development of topical hydrogels of terbinafine hydrochloride and evaluation of their antifungal activity.

The purpose of this study was to prepare hydrogels and microemulsion (ME)-based gel formulations containing 1% terbinafine hydrochloride (TER-HCL) and to evaluate the use of these formulations for the antifungal treatment of fungal infections. Three different hydrogel formulations were prepared using chitosan, Carbopol® 974 and Natrosol® 250 polymers. A pseudo-ternary phase diagram was constructed, and starting from ME formulation, a ME gel form containing 1% Carbopol 974 was prepared. We also examined the characteristic properties of the prepared hyrogels. The physical stability of hydrogels and the ME -based gels were evaluated after storage at different temperatures for a period of 3 months. The release of TER-HCL from the gels and the commercial product (Lamisil®) was carried out by using a standard dialysis membrane in phosphate buffer (pH 5.2) at 32 °C. The results of the in vitro release study showed that the Natrosol gel released the highest amount of drug, followed by Carbopol gel, chitosan gel, commercial product, and the microoemulsion-based gel in that order. In vitro examination of antifungal activity revealed that all the prepared and commercial products were effective against Candida parapsilosis, Penicillium, Aspergillus niger and Microsporum. These results indicate that the Natrosol®-based hydrogel is a good candidate for the topical delivery of TER-HCL.

A Multi-Target Approach toward the Development of Novel Candidates for Antidermatophytic Activity: Ultrastructural Evidence on α-Bisabolol-Treated Microsporum gypseum.

Multi-target strategies are directed toward targets that are unrelated (or distantly related) and can create opportunities to address different pathologies. The antidermatophytic activities of nine natural skin lighteners: α-bisabolol, kojic acid, ß-arbutin, azelaic acid, hydroquinone, nicotinamide, glycine, glutathione and ascorbyl tetraisopalmitate, were evaluated, in comparison with the known antifungal drug fluconazole, on nine dermatophytes responsible for the most common dermatomycoses: Microsporum gypseum, Microsporum canis, Trichophyton violaceum, Nannizzia cajetani, Trichophyton mentagrophytes, Epidermophyton floccosum, Arthroderma gypseum, Trichophyton rubrum and Trichophyton tonsurans. α-Bisabolol showed the best antifungal activity against all fungi and in particular; against M. gypseum. Further investigations were conducted on this fungus to evaluate the inhibition of spore germination and morphological changes induced by α-bisabolol by TEM.

In vitro antifungal activity of naftifine hydrochloride against dermatophytes.

The incidence of superficial dermatophytoses is high in developed countries, and there remains a need for effective topical antifungals. In this study, we evaluated the in vitro antifungal activity of naftifine hydrochloride, the active ingredient in naftifine hydrochloride cream and gel 1% and 2%, against dermatophytes. The MICs and minimum fungicidal concentrations (MFCs) of naftifine hydrochloride against 350 clinical strains, including Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Epidermophyton floccosum, and Microsporum canis, were determined using the CLSI methodology. Subsets from this test panel were subsequently tested in a time-kill assay at 0.125×, 0.25×, 0.5×, and 1× the MFC for each isolate. CFU counts were performed over a period of 48 h of incubation. Additionally, in order to determine the potential for resistance development, six strains were subjected to 15 serial passages in concentrations higher than the MIC for each strain. MICs were determined following each passage. The MIC range against the dermatophyte isolates tested was 0.015 to 1.0 µg/ml, with naftifine hydrochloride being fungicidal against 85% of the Trichophyton species. The time-kill assay showed dose-dependent activity, with the greatest reduction in the numbers of CFU corresponding to the highest drug concentration. There was no increase in MIC for any strains following repeated exposure to naftifine hydrochloride. Naftifine hydrochloride demonstrated potent activity against all dermatophytes tested, and none of the isolates within this test panel demonstrated the potential for the development of resistance. Thus, future clinical studies of naftifine hydrochloride against dermatophytes may be warranted for the treatment of superficial dermatophytoses.

Inflammatory Tinea capitis: a 12-year study and a review of the literature.

Inflammatory Tinea capitis (TC) is a rare form of TC. The aim of this study was to review epidemiological, clinical and mycological profile of inflammatory TC. We present a retrospective study (1999-2010), enrolled all the cases of inflammatory TC observed at a referral hospital in the northern Tunisia. One hundred and twenty-one patients with inflammatory TC, 83 male patients (68.6%) and 38 female patients (31.4%) were enrolled. The mean age was about 8 years. A majority of TC (71.9%) were in patients lesser than 10 years of age. Positive family history and contact with animals were noted in seven and 35 cases respectively. Direct examination was positive in 110 cases (59 ectothrix, 51 endothrix) and positive cultures were obtained in 105 patients (49 Trichophyton violaceum, 31 Microsporum canis, 13 Trichophyton interdigitale complex, 12 Trichophyton verrucosum). Systemic treatment was carried out in 115 patients with griseofulvin, in one with terbinafine. A complete recovery was noted in 88 cases; and persistent alopecia in 28 cases. The inflammatory TC is rare, but more common in rural families. The disease mostly affected male genders (68.6%) and T. violaceum remains the common pathogen of inflammatory TC in northern Tunisia.

Characterization and biological studies of bis- and tera-acetyl derivatives of hydrocarbon-bridged diamines-I.

A systematic study of the pharmaceutically important, double ended, chelating agents of the types CH(3)CONH(CH(2))nNHCOCH(3) and (CH(3)CO)(2)N(CH(2))n N(COCH(3))(2), where n= 2, 3, 4, 5 and 6, prepared by the bis- and tetra-acetylation of the corresponding diamino-polymethylenes, have been carried out. Bis- and tertra-acetyl derivatives have been characterized by their elemental analysis and the FTIR spectra, Mass spectra and H-NMR spectra of these compounds have been reported to establish their structures. In the present work, FTIR spectra have been found an excellent means for distinguishing the bis-acetyl derivatives from their tetra-acetyl counterparts. The structures of these bis- and tetra-acetyl compounds have further been established by their H-NMR and Mass Spectra. The selective pharmacological screening of the derivatives was carried out according to the standard procedures. The compounds were screened for their antibacterial and antifungal activities and it was found that majority of these compounds did not possess any remarkable activity. Only the compound BA1,2-DAE, showed significant antifungal activity against Microsporum canis (80%).

Inhibition of dermatophytes by the antimicrobial peptides human ß-defensin-2, ribonuclease 7 and psoriasin.

Previous studies have described some antibacterial effects of antimicrobial peptides (AMPs) expressed in human skin, but little is known about their possible activity against dermatophytes. Therefore we have tested the effects of human ß-defensin 2 (hBD-2), ribonuclease 7 (RNase 7) and psoriasin on the in vitro growth of four dermatophyte species. Germinating conidia of Trichophyton rubrum, T. mentagrophytes, Microsporum canis and Epidermophyton floccosum were exposed in vitro to hBD-2, RNase 7, psoriasin and fluconazole. Subsequent fungal growth was measured photometrically over 168 hours. All AMPs significantly inhibited fungal growth, with the degree of inhibition dependent on the dermatophyte species and the specific AMP. E. floccosum was found to be the most susceptible species in that it was markedly suppressed by all AMPs, whereas M. canis was inhibited only by psoriasin. Overall, psoriasin was the most effective AMP and had even stronger inhibitory effects on some dermatophytes than fluconazole. Our findings show that AMPs expressed in human skin can, in principal, inhibit the growth of dermatophytes in vitro. Therefore the question whether AMPs are relevant for human protection against tineas is justified and should be addressed by investigating their role in vivo.

Difference in keratinase activity of dermatophytes at different environmental conditions is an attribute of adaptation to parasitism.

Dermatophytes are a group of morphologically and physiologically related moulds, which cause well-defined infection called dermatophytosis. The enzymatic ability of fungi to decompose keratin has long been interpreted as a key innovation in the evolution of animal dermatology. In the present study, keratinase activity profile among Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum isolated on keratin substrates such as human hair, human nail and chicken feather at variable environmental conditions of temperature, pH and metal ions was elucidated. All the above-mentioned fungal strains were isolated from soil using To-KA-Va baiting technique and keratinolytic activity was measured spectrophotometrically. In the temperature range of 30-40 °C and slightly alkaline pH (7.0-8.0), Trichophyton produced the highest activity of keratinase. It can be presumed that high enzyme production of Trichophyton species at normal body temperature range and pH could be an attribute for obligate anthropization in some dermatophytes.

Tinea capitis due to Microsporum vanbreuseghemii: report of two cases.

Two cases of dermatophytosis caused by Microsporum vanbreuseghemii are reported. A 7-year-old boy and his brother were examined for tinea capitis. Hair samples and skin scrapings were collected from each patient to microscopy and culture. Direct microscopic examination of the hairs using lactophenol revealed an ectothrix invasion. Cultures inoculated with portions of clinical material yielded M. vanbreuseghemii after 2 weeks. The identification of the fungi were based on colony morphology on mycobiotic agar, microscopic characteristic on slide cultures, biochemical reactions and hair perforation tests.

Antimicrobial and anti-inflammatory activities of leaf extract of Valeriana wallichii DC.

Valeriana wallichii DC (Valerianaceae) is one of the most widely used traditional remedies for various complications associated with nervous system and digestion. No antimicrobial and anti-inflammatory studies have so far been carried out on the aerial parts of the plant. The present work was focused to evaluate the antimicrobial (antifungal and antibacterial) and anti-inflammatory properties of V. wallichii using reported methods. Chloroform fraction (VW-2) and hexane fraction (VW-3) exhibited significant activity against S. aureus and B. subtilus, respectively. The chloroform fraction (VW-2) showed significant activity against S. aureus with 0.27 mg/ml MIC, where 0.31 mg/ml MIC was deduced for VW-3 fraction against B. subtilus. VW-3 fraction was also found to be the most potent inhibitor of M. canis, showing 70% inhibition with an MIC value of 0.19 mg/ml. Considerable inhibitory activity was also observed for VW-2 and water fraction (VW-6) against M. canis and A. flavus. A remarkable anti-inflammatory like activity was observed for the crude extract at a dose of 200 mg/kg at all observed durations. Other doses of the sample also showed excellent activity. Looking to these results it may be concluded that V. wallichii may be a potential source for activity guided isolation of natural products with antimicrobial and anti-inflammatory-like properties.

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine.

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.