Standardization of Galleria mellonella as an Infection Model for Malassezia furfur and Malassezia pachydermatis.

Galleria mellonella larva has been widely exploited as an infection model for bacteria and fungi. Our laboratory uses this insect as a model for fungal infection caused by the genus Malassezia, in particular, systemic infections caused by Malassezia furfur and Malassezia pachydermatis, which are poorly understood. Here, we describe the G. mellonella larva inoculation process with M. furfur and M. pachydermatis and the posterior assessment of the establishment and dissemination of the infection in the larvae. This assessment was done through the evaluation of larval survival, melanization, fungal burden, hemocytes populations, and histological changes. This methodology allows for the identification of virulence patterns between Malassezia species and the impact of inoculum concentration and temperature.

Galleria mellonella as a Novelty in vivo Model of Host-Pathogen Interaction for Malassezia furfur CBS 1878 and Malassezia pachydermatis CBS 1879.

Malassezia furfur and Malassezia pachydermatis are lipophilic and lipid dependent yeasts, associated with the skin microbiota in humans and domestic animals, respectively. Although they are commensals, under specific conditions they become pathogens, causing skin conditions, such as pityriasis versicolor, dandruff/seborrheic dermatitis, folliculitis in humans, and dermatitis and otitis in dogs. Additionally, these species are associated with fungemia in immunocompromised patients and low-weight neonates in intensive care units with intravenous catheters or with parenteral nutrition and that are under-treatment of broad-spectrum antibiotics. The host-pathogen interaction mechanism in these yeasts is still unclear; for this reason, it is necessary to implement suitable new host systems, such as Galleria mellonella. This infection model has been widely used to assess virulence, host-pathogen interaction, and antimicrobial activity in bacteria and fungi. Some advantages of the G. mellonella model are: (1) the immune response has phagocytic cells and antimicrobial peptides that are similar to those in the innate immune response of human beings; (2) no ethical implications; (3) low cost; and (4) easy to handle and inoculate. This study aims to establish G. mellonella as an in vivo infection model for M. furfur and M. pachydermatis. To achieve this objective, first, G. mellonella larvae were first inoculated with different inoculum concentrations of these two Malassezia species, 1.5 × 106 CFU/mL, 1.5 × 107 CFU/mL, 1.5 × 108 CFU/mL, and 11.5 × 109 CFU/mL, and incubated at 33 and 37°C. Then, for 15 days, the mortality and melanization were evaluated daily. Finally, the characterization of hemocytes and fungal burden assessment were as carried out. It was found that at 33 and 37°C both M. furfur and M. pachydermatis successfully established a systemic infection in G. mellonella. M. pachydermatis proved to be slightly more virulent than M. furfur at a temperature of 37°C. The results suggest that larvae mortality and melanization is dependent on the specie of Malassezia, the inoculum concentration and the temperature. According to the findings, G. mellonella can be used as an in vivo model of infection to conduct easy and reliable approaches to boost our knowledge of the Malassezia genus.

Antifungal activity of Mongolian medicinal plant extracts.

The in vitro antifungal activity of extracts obtained from 14 medicinal plants of the mongolian flora were investigated by measuring their minimal inhibitory concentration (MIC) against fungi cause of cutaneous diseases such as Candida species, dermatophytes and Malassezia furfur. Among the species examined, Stellaria dichotoma L., Scutellaria scordifolia L. Aquilegia sibirica Fisch. Et Schrenk. and Hyoscyamus niger L. extracts demonstrated antifungal activity against all studied fungi. In particular, S. scordifolia L. methanol extract, obtained at room temperature, showed the best activity against Candida spp., Malassezia furfur and dermatophytes with GMMIC50 values of 22 µg/mL, 64 µg/mL and 32 µg/mL, respectively. The flavones, luteolin and apigenin, identified in S. scordifolia extracts, and rutin identified in S. dichotoma and Hyoscyamus niger L. extracts, could be responsible of the observed antifungal activity.

Dichotomous response of Malassezia-infected macrophages to Malassezia pachydermatis and Malassezia furfur.

Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-γ and tumor necrosis factor [TNF]-α) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-α and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.

Dichotomous response of Malassezia-infected macrophages to Malassezia pachydermatis and Malassezia furfur

Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-gama and tumor necrosis factor [TNF]-a) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-a and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.

[In vitro antifungal activity of azoles and amphotericin B against Malassezia furfur by the CLSI M27-A3 microdilution and Etest® methods]. / Actividad antifúngica in vitro de azoles y anfotericina B frente a Malassezia furfur por el método de microdilución M27-A3 del CLSI y Etest®.

BACKGROUND: Malassezia furfur is a human skin commensal yeast that can cause skin and opportunistic systemic infections. Given its lipid dependant status, the reference methods established by the Clinical and Laboratory Standards Institute (CLSI) to evaluate antifungal susceptibility in yeasts are not applicable. AIMS: To evaluate the in vitro susceptibility of M. furfur isolates from infections in humans to antifungals of clinical use. METHODS: The susceptibility profile to amphotericin B, itraconazole, ketoconazole and voriconazole of 20 isolates of M. furfur, using the broth microdilution method (CLSI M27-A3) and Etest®, was evaluated. RESULTS: Itraconazole and voriconazole had the highest antifungal activity against the isolates tested. The essential agreement between the two methods for azoles antifungal activity was in the region of 60-85% and the categorical agreement was around 70-80%, while the essential and categorical agreement for amphotericin B was 10%. CONCLUSIONS: The azoles were the compounds that showed the highest antifungal activity against M. furfur, as determined by the two techniques used; however more studies need to be performed to support that Etest® is a reliable method before its implementation as a routine clinical laboratory test.

Comportamiento de malassezia furfur en medios de cultivo con base en los exudados gomosos de spondias dulcis y spondias mombin: producción de lipasa extracelular / Growth of malassezia furfur in media with spondias dulcis and spondias mombin gum exudates: production of extracellular lipase

El desarrollo de las levaduras del género Malassezia requiere condiciones especiales, estos hongos que producen afecciones en la piel, son generalmente cultivados en el medio Dixon. Se ensayaron los exudados gomosos de Spondias dulcis y Spondias mombin como sustratos para Malassezia furfur. Se evaluó también la producción de lipasa. Se determinó la cinética de crecimiento a un determinado intervalo de tiempo (0-168h), y a diferente concentración (0,5; 1%) y pH (4,5; 6,0). La biomasa obtenida para la levadura probada demostró que los sustratos preparados con los exudados gomosos son adecuados para su desarrollo. La mayor actividad de lipasa extracelular se observó al tiempo inicial de estudio (18h) en ambos sustratos, en las condiciones usadas: concentración (0,5 y 1%) y pH(4,5 y 6,0). Estas especies botánicas, ampliamente localizadas en Venezuela, especialmente en los Estados Zulia y Falcón, producen abundante goma. Este hecho, y los resultados obtenidos podrían ser útiles en la obtención de un nuevo sustrato, que pueda competir con el medio Dixon para el aislamiento y la caracterización de especies de Malassezia, y para la producción de lipasa

The development of genus Malassezia yeasts requires special conditions. This fungus, which produces skin diseases, is generally cultivated in the Dixon medium. Gum exudates from Spondias dulcis and Spondias mombin were tested as substrates for Malassezia furfur. Lipase production was also evaluated. The growth kinetic was determined for a given time range (0-168h) at different concentrations (0.5; 1%) and pH levels (4.5; 6.0). The biomass obtained for the tested yeast showed that substrates prepared with S. dulcis and S.mombin gum exudates are suitable for its development. The highest extracellular lipase activity was observed at 18h on both substrates at given concentrations (0.5; 1%) and pH (4.5; 6.0). These botanical species, widely located in Venezuela, especially in the States of Zulia and Falcon, yield abundant gum. Findings may be useful for obtaining new substrates that could compete with the Dixon medium for isolation and characterization of Malassezia species and for lipase production

En búsqueda del Phymatotrichopsis Omnívora, en pobladores de la Colonia Tovar, Venezuela, octubre 2007 / Phymatotrichopsis Omnivore, study search at Aragua Colonia Tovar residents, Venezuela, October 2007

Se realizó un estudio descriptivo, y de corte transversal, con el propósito de investigar la presencia del hongo Phymatotrichopsis omnívora como agente etiológico , del alerta epidemiológica denunciada en un medio de comunicación regional, la cual le atribuyo a este hongo el deterioro de la cosecha del durazno, conocida por los pobladores como secason y algunas lesiones de piel en pacientes adultos y pediátricos de los caseríos, Gabante, área urbana y el Cedral, del Municipio Tovar, Estado Aragua; Venezuela. Se examinaron 144 pacientes con lesiones de piel sospechosos de micosis superficial que acudieron a la consulta de los centros de salud de la zona. Se realizó examen de piel y faneras, examen directo; cultivos para hongos y biopsia de piel los cuales fueron procesados en el laboratorio del Hospital Central de Maracay y en el Servicio de Anatomía Patológica del Instituto Autónomo de Biomedicina, Caracas. Los datos básicos se registraron en ficha epidemiológica; se procedió al análisis estadístico; encontrándose entre las 10 primeras patologías dermatológicas: Pitiriasis alba (18,75%), Queratosis Folicular (11,1%), Quiste Millium (10,4%), Acné (6,25%),Prurigo Infantil (6,25%), Picaduras de Insectos (5,5%),Dermatitis Seborreica (4,9%), Micosis Superficial (4,16%), Urticaria (4,16%), Melasma (3.47%), otras (2.7%). De origen micótico encontramos, dos con Pitiriasis Versicolor cuyo agente etiológico fue Malassezia furfur; dos Onicomicosis, uno Tiña Corporal y uno con Tiña Inguinal; aislándose en los cultivos Trichophytum rubrum. En conclusión: de 144 personas examinadas ninguna presentó lesiones atribuidas al hongo Phymatotrichopsis omnívora.

It was made a cross-sectional descriptive, with the purpose of investigating the presence of the mushroom Phymatotrichopsis omnivore as an etiological agent, of an epidemic alert reported on a regional communications medium; which attributed to this fungus deterioration peach crop, known to residents as 'secasón' and some skin lesions in adult and paediatrics patients in the hamlets, Gabante, urban and Cedral, Tovar Municipality, Aragua; Venezuela. We examined 144 patients with skin lesions suspected of superficial fungal infections and they were seen at the health centers in the area. Examination was made of skin and appendages, direct examination, fungal cultures and skin biopsies which were processed in the laboratory of the Central Hospital of Maracay and the Pathology Department of the Autonomous Institute of Biomedicine, Caracas. Baseline data were recorded in epidemiological record, we proceeded to statistical analysis to be among the top 10 dermatological diseases: Pityriasis Alba (18.75%), follicular keratosis (11.1%), cyst Millium (10.4%) Acne (6.25%), Child Prurigo (6.25%), insect bites (5.5%), seborrheic dermatitis (4.9%) Superficial Mycoses (4.16%), urticaria (4.16 %), melasma (3.47%), other (2.7%). Of fungal origin found, two with Pityriasis Versicolor whose etiologic agent was Malassezia furfur; two onychomycosis, one Corporal and one Tinea jock itch, isolated from cultures Trichophytum rubrum. In conclusion: of 144 people tested had no injuries attributed to the fungus Phymatotrichopsis omnivorous.

Comparación del crecimiento de Malassezia furfur y Malassezia slooffiae en los medios del exudado gomoso de spondias dulcis y dixon / Comparison of the growth of Malassezia furfur and Malassezia slooffiae on spondias dulcis gum exudate and dixon media

Las levaduras del género Malassezia son hongos que producen afecciones en la piel. El desarrollo de estos microorganismos requiere condiciones especiales. El medio Dixon es generalmente usado para su cultivo. Se ensayo el exudado gomoso de Spondias dulcis como sustrato para Malassezia furfur y Malassezia slooffiae en comparación con el medio Dixon. Se determino la cinética de crecimiento a un determinado rango de tiempo (0-120h), a diferentes concentraciones (1,2 por ciento) y pH (4,0;6,0;7,0). La relativa alta biomasa obtenida para las dos levaduras probadas demostró que el sustrato preparado con el exudado gomoso de S. dulcis es adecuado para su desarrollo. Spondias dulcis especie localizada en Venezuela produce abundante goma. Este hecho, y los resultados obtenidos podría ser útil para preparar un nuevo sustrato que pueda competir con Dixon para el aislamiento y la caracterización de especies de Malassezia.

Malassezia yeasts are fungi that produce skin affections. Growth of these microorganisms requires specific conditions. The Dixon medium has generally been used for their culture and has been tested. The use of Spondias gum as a substrate for Malassezia furfur and Malassezia slooffiae was tried and compared with the Dixon medium. The growth kinetic for a given time range(0-120 h) was determined at different concentrations (1.2 percent) and pH levels (4,0; 6,0;7,0). The relatively high biomass obtained for the two tested yeasts demonstrated that the substrate prepared with S. dulcis gum exudate is suitable for their growth. Spondias dulcis, a species located in Venezuela, yields abundant gum. This fact and the results discussed above indicate that it could be used to prepare a substrate that could compete with Dixon for isolating and characterizing the Malassezia species.

Identification and pathogenicity of Malassezia species isolated from human healthy skin and with macules

This study investigated the ocurrence of Malassezia species in clinically healthy students and with macules with a slight fawn discoloration and characterized the isolates as to the pathogenicity factors such as growth at 37ºC, lipase, phospholipase and protease detection. Clinical samples were collected from different body sites of one hundred students of different ages and both sexes. The samples, obtained by scrapping the skin surface and the scalp, were treated with potassa and cultured. Cultures were obtained in Petri dishes containing Sabouraud agar medium added of olive oil, incubated at room temperature and at 37ºC. Culture identifications were based in their morphological and physiological properties. Lipase, phospholipase and protease detection was performed in specific media on Petri dishes for formation of a zone. Globose, spherical yeast cells and hypha were investigated by direct microscopy of clinical materials. Malassezia furfur was detected in seven samples and M. sympodialis in four. All Malassezia cultures presented lipase activity, but none was phospholipase positive. Protease activity was observed in two M. furfur and two M. simpodialis isolates.

A ocorrência de espécies Malassezia em estudantes clinicamente sadios e com máculas com leve descoloração foi investigada e os isolados caracterizados quanto a fatores de patogenicidade como crescimento a 37ºC, detecção de lípase, fosfolipase e protease. Amostras clínicas de 100 estudantes de ambos os sexos, diferentes idades e sítios corpóreos foram obtidas por escarificação da superfície da pele e do couro cabeludo e examinadas com hidróxido de potássio e submetidas à cultura. As culturas foram obtidas em meio ágar Sabouraud adicionado de óleo de oliva, em placa de Petri, incubadas a temperatura ambiente e a 37ºC. A identificação foi realizada através das propriedades morfológicas e fisiológicas. A detecção de lipase, fosfolipase e protease foi analisada em meios específicos em placa de Petri pela formação de halo. O exame microscópico direto mostrou células globosas, esféricas e hifa. Malassezia furfur foi detectada em sete amostras e M. sympodialis em quatro. Todas as culturas apresentaram atividade lipásica, mas nenhuma foi fosfolipase positiva. A atividade proteásica foi observada em dois isolados de M. furfur e dois de M. simpodialis.