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.

Poly-ε-caprolactone Nanoparticles Loaded with 4-Nerolidylcatechol (4-NC) for Growth Inhibition of Microsporum canis

Dermatophyte fungal infections are difficult to treat because they need long-term treatments. 4-Nerolidylcatechol (4-NC) is a compound found in Piper umbellatum that has been reported to demonstrate significant antifungal activity, but is easily oxidizable. Due to this characteristic, the incorporation in nanostructured systems represents a strategy to guarantee the compound's stability compared to the isolated form and the possibility of improving antifungal activity. The objective of this study was to incorporate 4-NC into polymeric nanoparticles to evaluate, in vitro and in vivo, the growth inhibition of Microsporum canis. 4-NC was isolated from fresh leaves of P. Umbellatum, and polymer nanoparticles of polycaprolactone were developed by nanoprecipitation using a 1:5 weight ratio (drug:polymer). Nanoparticles exhibited excellent encapsulation efficiency, and the antifungal activity was observed in nanoparticles with 4-NC incorporated. Polymeric nanoparticles can be a strategy employed for decreased cytotoxicity, increasing the stability and solubility of substances, as well as improving the efficacy of 4-NC(AU).

Quantitative and structural analyses of the in vitro and ex vivo biofilm-forming ability of dermatophytes.

PURPOSE: The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment. METHODOLOGY: Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy. RESULTS: Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species. CONCLUSION: This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

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.

Antifungal and marker effects of Talisia esculenta lectin on Microsporum canis in vitro.

AIMS: The purpose of this study was to demonstrate the usefulness of lectin obtained from Talisia esculenta (TEL) seeds as a tool to recognize and study Microsporum canis. For this purpose, we investigated the antifungal and marker action of this lectin and the relationship of these effects with the presence of carbohydrates on the structure of this fungus. METHODS AND RESULTS: The in vitro antifungal activity of TEL was analysed by broth microdilution assay. In addition, TEL was assessed against the arthroconidia present on hairs obtained from infected dogs and cats. The affinity of fluorescein isothiocyanate (FITC)-labelled TEL for macroconidia and arthroconidia of M. canis was also tested. The effects of TEL on the growth of the M. canis strains began with 0.125 mg ml(-1), and 100% inhibition was obtained with a concentration of 2 mg ml(-1). The addition of carbohydrates, especially N-acetyl-glucosamine and d-mannose, inhibited these antifungal effects. TEL was able to inhibit the growth of arthroconidial chitin-rich forms of M. canis obtained from hairs of infected animals and strains cultured in Sabouraud agar. FITC-labelled TEL efficiently marked macroconidial and arthroconidial forms of M. canis, as shown by fluorescent microscopy. CONCLUSIONS: These results show that the inhibitory effects of TEL on M. canis growth may be related to the interaction of lectin with the carbohydrates present at the micro-organism's surface, mainly D-mannose and N-acetyl-glucosamine. SIGNIFICANCE AND IMPACT OF THE STUDY: Talisia esculenta can be used as an important tool in the biochemical study of M. canis or as a molecule to recognize this dermatophyte in infected tissue.

Dermatophytes growth curve and in vitro susceptibility test: a broth micro-titration method.

The introduction of systemic antifungal drugs which act upon different targets is the main issue of the in vivo antifungal resistance control. Different factors, such as growth curve phase, quality of the specimen, quantity of the inoculum, temperature, pH, culture medium composition, incubation duration and solvent, are believed important factors affecting minimum inhibitory concentration (MIC) value to most of the antifungal agents. We assayed an in vitro susceptibility test with 40 isolates of dermatophytes: Microsporum canis, Trichophyton rubrum, Trichophyton mentagrophytes and Epidermophyton floccosum against griseofulvin, fluconazole, itraconazole and terbinafine, using the guidelines of the M38-P document approved by the NCCLS. We determined the growth curves, to estimate the specific growth rate (mu max) and the generation time (G) of each dermatophyte, using dry weight and spectrophotometry methods. We demonstrate that, at 192 h, all fungi tested had a constant growth curve and we considered this as the optimal time for MIC determination. Terbinafine, griseofulvin and itraconazole possessed the highest antifungal activity against the four groups of dermatophytes studied. Fluconazole demonstrated no efficacy. Our MIC results differ from other authors and this difference is due to the timing of the MIC determination based on the growth curve of each fungi tested.

Evaluation of Microsporum canis in different methods of storage.

The main objective of this investigation was to evaluate different methods of storage for Microsporum canis based on materials and equipment that are readily available in developing countries. We tested 32 strains of M. canis at - 20 degrees C in potato dextrose agar (PDA) in its plain condition, or amended with 10% dimethyl sulfoxide or with 10% glycerol. In addition, we tested 25 degrees C storage of isolates in plain saline (0.9% NaCl) and in saline covered with a mineral-oil layer. After 9 months of storage, none of the M. canis strains frozen in PDA supplemented with glycerol survived, while only 16 and 6%, respectively, of the isolates in plain and DMSO medium lost viability. Nine month's storage in saline with or without mineral oil increased the amount of pleomorphic development of sterile hyphae; this phenomenon occurred at a significantly higher level than was seen in isolates stored at -20 degrees C. The physiological characteristics of M. canis were not affected by the different storage tests. The results suggest that, in order to ensure optimal viability, purity and pristine isolate condition, each M. canis isolate maintained should be held in at least two methods of storage, namely, PDA at -20 degrees C and saline with a mineral-oil layer at 25 degrees C.

Fluorescent method for studying the morphogenesis and viability of dermatophyte cells.

The dermatophyte Microsporum canis is commonly isolated from human and animal infection. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorescein Diacetate and Ethidium Bromide. To this end, 50 microl dermatophyte suspension were transferred onto cellophane wrapping esterilized discs (2.5 cm of diameter) placed over the surface of Sabouraud dextrose agar on Petri dishes and incubated at 25 degrees C for 30 days. Every 60 minutes during the first 24 hours and every 12 hours for next 29 days, one disc was transferred onto glass slide, covered with equal volumes of freshly prepared fluorescein diacetate (FDA) and ethidium bromide (EB) solution, mounted with a coverslip and incubated in the dark for 30 minutes, at 25 degrees C. Each preparation was then examined on a fluorescent microscope. M. canis presented well defined growth stages: (1) tumescence of cells; (2) germination; (3) development of hyphae; (4) production of conidia and (5) tumescence and formation of arthroconidiae. Using the fluorescent method, non viable cells showed a light bright red coloration and viable cells presented green fluorescence. The principal morphological changes have occurred between the 3rd until the 18th day of culture. The method is very useful to demonstrate the dermatophyte growth stages as well as the perfect differentiation between viable and non viable cells.

Caracterizaçäo de amostras de Microsporum gypseum quanto ao seu perfil eletroforético e produçäo de enzimas extracelulares / The characterization of samples of Microsporum gypseum about its electrophoetic patterns and extracellular enzyme production

Objetivou-se caracterizar as amostras de M. gyseum isoladas de diferentes fontes quanto ao seu perfil eletroforético e à produçäo de enzimas extracelulares. A partir de fonte ambiental, animal e humana foram isoladas outo amostras de M. gypseum. Estas amostras foram avaliadas por curva de crescimento, caracterizaçäo de perfis eletroforéticos quanto ao número e intensidade de bandas e a produçäo de enzimas extracelulares empregando-se método em placas para avaliaçäo da presença de fosfolipase e proteinase e por eletroforese em gel-substrato (caseína e gelatina), além da determinaçäo quantitativa da atividade azocolítica e caseinolítica. Diferenças morfológicas näo foram observadas entre as amostras. As curvas de crescimento de duas amostras, uma de solo e outra de paciente foram bastante semelhantes, principalmente no período do 12§ ao 20§ dias de incubaçäo. O 19§ dia foi escolhido como o ideal para a obtençäo dos filtrados de uma cultura de todas as amostras. Os filtrados apresentaram concentraçäo protéica média de 2,15 mg/mL. Quanto ao perfil eletroforético, a análise revelou existir diferentes padröes protéicos entre as amostras de M. gypseum. A produçäo de enzimas extracelulares em placas foi baixa e näo permitiu observar diferenças entre as amostras nem tampouco relacioná-la com a patogenicidade. Os filtrados de cultura das diferentes amostras apresentaram variaçöes no número de enzimas expressas. As proteinases observadas em gel de gelatina variaram de 180 a 27 kDa e também variaram dependendo da amostra. As diferentes amostras de M. gypseum expressaram componentes protéicos com e sem atividade enzimática. As enzimas proteolíticas foram expressas sem uso de substratos indutores, significando que sua produçäo é independente da fonte protéica e portanto podem ser proteinases de amplo espectro de atividade

Dermatoficia por microsporum canis en un recién nacido / A case of tinea by microsporum canis in a newborn

Se comunica el caso de un recién nacido que presentó una dermatoficia por microsporum canis en el cuero cabelludo. Se observaron múltiples lesiones eritematoanulares que se agrupan en las áreas parietotemporales. El niño había tenido contacto con felinos una semana antes de la consulta. Se realizó tratamiento tópico con buen resultado. Se ha registrado un incremento en la frecuencia de las microsporias y el gato es el agente trasmisor más importante

Dermatoficia por microsporum canis en un recién nacido / A case of tinea by microsporum canis in a newborn

Se comunica el caso de un recién nacido que presentó una dermatoficia por microsporum canis en el cuero cabelludo. Se observaron múltiples lesiones eritematoanulares que se agrupan en las áreas parietotemporales. El niño había tenido contacto con felinos una semana antes de la consulta. Se realizó tratamiento tópico con buen resultado. Se ha registrado un incremento en la frecuencia de las microsporias y el gato es el agente trasmisor más importante

[Isolation of Microsporum ferrugineum in Cuba. Presentation of a case]. / Informe del aislamiento de Microsporum ferrugineum en Cuba. Presentación de 1 caso.

A mycologic study of skin lesions in a patient with Tines corporia was made. A fungus with a strong yellow stain color was isolated and classified as Microsporum ferrugineum, a species isolated in our country for the first time.

Influencia de herbicidas preemergentes sobre el desarrollo de dermatofitos geofílicos en suelos agrícolas / Effects of preemerging herbicides applied to farm soilson the development of geophilic dermatophytes

El objetivo de este trabajo es conocer la acción ejercida por concentraciones crecientes de herbicidas de presiembra (Metribuzin, Atrazina y Alachlor) sobre el crecimiento y la capacidad germinativa de Microsporum fulvum, Microsporum gypseum y Keratinomyces ajelloi. Comprobamos un efecto depresivo e inhibitorio de los biocidas sobre la germinación y el crecimiento de las cepas en estudio. Alachlor es el que presenta mayor poder inhibitorio y en menor grado Metribuzin y Atrazina. Además, comprobamos que eta acción ocurre sólo cuando los hongos se ponen en contacto con los herbicidas en medio de Sabouraud glucosa, que aparentemente los haría más disponibles a los propágulos fúngicos. Estos resultados nos indican la importancia que tienen, las interrelaciones entre distintas sustancias, en los efectos producidos sobre la micota de los suelos, resultando de que en estas experiencias nos aproximamos cada vez más a las condiciones presentes en los habitat naturales

Cambios en la curva de crecimiento de Microsporum canis por el uso de distintos nutrientes en los medios de cultivo / Chagnes in the growth curve of Microsporum canis for the use of different nutrients in the culture media

Las curvas de crecimiento de las colonias de dermatofitos están sujetas a diversos parámetros a veces muy difíciles de conciliar. En este trabajo se pretende, mediante el cambio de algunos nutrientes en el medio de lactrimel, verificar las fases básicas en el crecimiento de Microsporum canis: estacionaria mínima o de adaptación, exponencial, estacionaria máxima y de declinación. A su vez, se las compara con otros medios de lactrimel donde la harina de trigo es reemplazadas respectivamente por la de soja, arroz y maíz. Se pudo establecer que la fase de adaptación es similar en todos los medios, pese al cambio de harina. En cambio hubo variaciones significativas en las fases exponenciales que revelaron ventajas cuando se usaron harina de arroz y maíz, tanto en el aspecto vegetativo como por la producción de macroconidias, no así con la de soja cuyos resultados fueron inferiores en esta fase

Cambios en la curva de crecimiento de Microsporum canis por el uso de distintos nutrientes en los medios de cultivo / Chagnes in the growth curve of Microsporum canis for the use of different nutrients in the culture media

Las curvas de crecimiento de las colonias de dermatofitos están sujetas a diversos parámetros a veces muy difíciles de conciliar. En este trabajo se pretende, mediante el cambio de algunos nutrientes en el medio de lactrimel, verificar las fases básicas en el crecimiento de Microsporum canis: estacionaria mínima o de adaptación, exponencial, estacionaria máxima y de declinación. A su vez, se las compara con otros medios de lactrimel donde la harina de trigo es reemplazadas respectivamente por la de soja, arroz y maíz. Se pudo establecer que la fase de adaptación es similar en todos los medios, pese al cambio de harina. En cambio hubo variaciones significativas en las fases exponenciales que revelaron ventajas cuando se usaron harina de arroz y maíz, tanto en el aspecto vegetativo como por la producción de macroconidias, no así con la de soja cuyos resultados fueron inferiores en esta fase (AU)

Utilization of standard laboratory methods in the laboratory diagnosis of problem dermatophytes

The identification of certain dermatophytes may be simplified by using biochemical tests such as urease, nutritional requirements (with commercially available media), and the in-vitro hair penetration test. No study that combines these tests in a diagnostic scheme for identification of the common dermatophytes has been published. one to 20 isolates each of 29 species of dermatophytes (one Epidermophyton floccosum, 10 Microsporum species, and 18 Trichophyton species) were used. They were grown on Christensen's urea agar and the seven nutritional media for the differentiation of the Trichophyton species; the in-vitro hair penetration test was performed in duplicate. Patterns were developed that have been tested and proven to be useful for more than 22 months. In addition, the Microsporum species were all grown on polished rice, and color and sporulation were recorded. All dermatophytes in this study were grown on either Mycosel agar or 2% malt extract agar, and on modified potato dextrose agar and modified Sabouraud agar. Macroscopic and microscopic examinations were made to determine qualitatively the amounts of growth and sporulation on each medium.