Análise fitoquímica e atividade antifúngica do óleo essencial de folhas de Lippia sidoides Cham. e do Timol contra cepas de Candida spp / Phytochemical analysis and antifungal activity of the essential oil of Lippia sidoides Cham. and of the Thymol against Candida strains

RESUMO A resistência de fungos do gênero Candida aos fármacos químicos tem lançado o desafio de se identificar novas substâncias que possuam atividade antibiótica ou venham a modular o efeito de produtos atualmente usados contra candidíase. O presente estudo avaliou a atividade antifúngica do óleo essencial de Lippia sidoides Cham. e do timol, sobre cepas de Candida. Inicialmente os produtos foram testados frente a 16 cepas fúngicas pela técnica de difusão em meio sólido, o que permitiu selecionar linhagens para continuidade da pesquisa. Com as linhagens de Candida krusei (CK LMBM 01, CK LMBM 02), Candida albicans (CA LM 62) e Candida tropicalis (CT LM 20), procedeu-se, por microdiluição em caldo, a determinação da Concentração Inibitória Mínima (CIM) e em meio sólido, a Concentração Fungicida Mínima (CFM) dos produtos foi identificada. O microcultivo das leveduras em meio empobrecido foi realizado para verificação de alterações morfológicas e, além disso, uma análise da composição química do óleo foi realizada por Cromatografia Gasosa acoplada à espectrometria de massas (CG-EM). Nesta análise, o constituinte majoritário foi o timol (84,95%), seguido de compostos como p-cimeno e Éter metil carvacrol, entre outros. A CIM do óleo essencial de Lippia sidoides Cham. frente às cepas variou entre 64 e 256 μg/mL, enquanto a CIM do timol foi estabelecida entre 32 e 64 μg/mL. A CFM do óleo essencial foi determinada entre 128 e 512 μg/mL e para o timol foram encontrados valores entre 64 e 128 μg/mL. Em relação à análise micromorfológica, verificada nas concentrações de CIM e CIM x 2, o óleo essencial inibiu o dimorfismo das cepas CK 01 e CT 20 na CIM e quando foi ensaiado o timol, este, na CIM, impediu a transição morfológica das cepas CK 01 e CA 62. Uma redução da morfogênese também foi obsevada na cepa CT 20, porém apenas em CIM x 2 e de forma mais discreta. Os resultados enaltecem o potencial antifúngico de L. sidoides e de seu composto majoritário timol tanto no combate à Candida quanto na neutralização de um dos fatores de virulência, a capacidade invasiva por formação de hifas e pseudohifas verificado na condição patogênica da candidíase. Estes dados são promissores e poderão incentivar futuras pesquisas sobre os aspectos fitoquímicos, toxicológicos e farmacológicos tanto do óleo essencial de Lippia sidoides como também de seus componentes químicos.

ABSTRACT The resistance of the Candida against drugs has been a challenge to the discovery of new substances with antimicrobial or modulatory effects that could be used against the cadidiasis. This work evaluated the antifungal activity of the essential oil of Lippia sidoides Cham. and of the Thymol against Candida strains. The products were tested towards 16 strains of Candida using the diffusion method, which allowed to select the strains in order to proceed with the research. The strains of Candida krusei (CK LMBM 01, CK LMBM 02), Candida albicans (CA LM 62) and Candida tropicalis (CT LM 20) were assayed by the microdilution method so that the Minimal Inhibitory Concentration (MIC) and the Minimal Fungicide Concentration (MFC) could be determined. The morphogenesis of the Candida was evaluated using poor environment in order to observe morphological changes. The composition of the essential oil was determined by GC-MS. The main compound observed was the thymol (84.95%). The MIC of the essential oil of L. sidoides and Thymol ranged between 64 to 256 μg/mL, and between 32 to 64 μg/mL respectively. The MFC of the essential oil and the thymol varied between 128 to 512 μg/mL and 64 to 128 μg/mL respectively. The morphogenesis of different Candida strains was inhibited in the MIC and MICx2 to the essential oil and thymol. The results indicated the antifungal potential of the L. sidoides and of the Thymol due to the inhibition of the invasive capacity, one of the most important virulence factors for the candidiasis` development. These results are promising to new researches about the phytochemical, toxicological and pharmacological aspects of the essential oil of L. sidoides and its phytochemical compounds.

Arachidonic acid affects biofilm formation and PGE2 level in Candida albicans and non-albicans species in presence of subinhibitory concentration of fluconazole and terbinafine

Candida albicans utilizes arachidonic acid (AA) released during the course of infection (Candidiasis) from phospholipids of infected host cell membranes and synthesizes extracellular prostaglandin(s) which play an important role in hyphae formation and host cell damage. C. albicans biofilms secrete significantly more prostaglandin(s) and evidence suggests that Candida biofilms have dramatically reduced susceptibility to majority of antifungal drugs. AA influences the saturation level of lipids and fluidity of yeast cell membranes. Therefore the aim of this study was to evaluate the effect of AA alone or in combination with antifungal agents on biofilm formation and production of prostaglandin (PGE2) in C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. albicans amphotericin B resistant strain (AmBR). Maximum biofilm formation was found to be in the case of C. albicans compared to C. non-albicans species. However, among the non-albicans species C. tropicalis exhibited highest biofilm formation. Treatment with AA in combination with subinhibitory concentrations of fluconazole and terbinafine separately exhibited significant (p < 0.05) reduction in biofilm formation against C. glabrata, C. parapsilosis, C. tropicalis and AmBR as compared to their individual effect. Further, these two antifungal agents in combination with AA caused an increase in production of prostaglandin from fungal cell itself which was significant (p < 0.05) in case of all the strains tested.

Application of MALDI-TOF MS for requalification of a Candida clinical isolates culture collection

Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.

Productividad y selectividad del medio de cultivo a partir de guayaba agria (psidium araca) en el crecimiento de levaduras nativas del género candida sp / Productivity and selectivity of culture medium from the sour guava (psidium araca) in the growth of native yeast candida sp

El presente trabajo se llevó a cabo para evaluar la eficiencia del medio de cultivo a partir de guayaba agria (Psidium araca) frente a medios comerciales en el crecimiento de tres cepas nativas: Candida guillermondii, Candida famita y Candida sp. Se evaluó el crecimiento microbiano a diferentes concentraciones de fruta, 5, 10, 25 y 50% p/v, tomando como control los medios comerciales: Malta, Sabouraud y agar papa dextrosa (PDA). La productividad y selectividad del medio de guayaba agria fue determinada mediante el método Ecométrico en un tiempo de 48 horas. Los análisis estadísticos aplicados para evaluar y comparar el crecimiento de las cepas en los medios comerciales y en el medio de guayaba agria a diferentes concentraciones demostraron lo siguiente: Candida guillermondii presentó crecimiento mayor o igual a 25 y 50% p/v comparado con los medios comerciales; Candida famata y Candida sp presentaron mejores crecimientos al 5% p/v, con respecto a los diferentes medios comerciales. Los resultados demostraron que el medio de cultivo es altamente productivo y no selectivo, lo que representa una alternativa en la conservación, el mantenimiento y el desarrollo de las levaduras estudiadas.

This work was carried out to evaluate the efficiency of the culture medium from sour guava (Psidium araca) against commercial media in the growth of three native strains: Candida guillermondii, Candida famata and Candida sp. Microbial growth was evaluated at different concentrations of fruit, 5, 10, 25, 50% w /v, using as control the commercial media: Malta, Sabouraud and PDA (Potato Dextrose Agar). The productivity and selectivity of the sour guava medium was determined by the Ecometric method in a time of 48 hours. The applied statistical analysis to evaluate and compare growth of strains in commercial culture medium and in the medium from sour guava at different concentrations showed: Candida guillermondii grew greater than or equal to 25 and 50% w / v compared with commercial medium, Candida famata and Candida sp showed better growth at 5% w / v, with respect to commercial medium. The results showed that the medium is highly productive and non-selective representing an alternative to the conservation, maintenance and development of the yeasts.

Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis / 대한진단검사의학회지

BACKGROUND: Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species. METHODS: The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C. haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates. RESULTS: The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3+/-18.5, 126.9+/-6.6, 88.5+/-18.5, 50.8+/-7.0, and 38.1+/-3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF. CONCLUSIONS: This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.

Effect of methyl branching of C8H18 alkanes and water activity on lipase-catalyzed enantioselective esterification of ibuprofen

The purpose of this research was to study the effect of the methyl branching of a high log P alkane solvent and the water activity in the organic medium on the initial rate and the enantioselectivity of ibuprofen esterification catalyzed by Candida rugosa lipase. Resolution of ibuprofen is important because S-(+)-ibuprofen has the desired pharmacological activity, whereas the R-(-)-enantiomer causes much of the side effects. The Candida rugosa lipase-catalyzed reaction in isooctane at 40ºC and 0.73 water activity gave the best results, both in terms of the initial reaction rate and the enantioselectivity of the reaction. An increase in water activity allowed a higher reaction rate and enantiomeric excess in each of the four solvents. An increase in methyl branching did not necessarily increase the initial reaction rate, but it allowed a higher enantioselectivity, evidenced by an increase in the substrate enantiomeric excess.

Comparación de diferentes métodos para la identificación de especies del género Candida / Comparison of different methods for species identification of genus Candida

Las diferentes especies del género Candida producen una variedad de enfermedades, desde infecciones mucocutáneas leves a formas diseminadas graves. Tradicionalmente, la taxonomía de las levaduras se ha llevado a cabo en base a estudios morfológicos y fisiológicos, pero éstos dependen de las condiciones de cultivo de las cepas, por lo que se han observado diversas dificultades. Por tal motivo, recientemente, se han probado técnicas de biología molecular. El objetivo de este trabajo es correlacionar los estudios taxonómicos de las especies correspondientes a las principales patógenas: C. albicans, C. krusei, C. parapsilosis, C. tropicalis y C. glabrata, realizados por técnicas fenotípicas tradicionales, métodos comerciales y por PCR fingerprinting. Al comparar las técnicas que identifican Candida albicans, agar harina de maíz y formación de tubos germinativos, estadísticamente se observa que no existen diferencias significativas entre ambos métodos (valor de la estadística X2 = 0,5 p = 0,4795). Comparando los métodos que discriminan especies de Candida: pruebas fisiológicas, CHROMagar, API20C y PCR fingerprinting se observó que no existen diferencias significativas en las proporciones de resultados que identifican cualquier Candida entre las pruebas fisiológicas, API20C y PCR fingerprinting. La proporción de resultados definitivos es mayor a la obtenida usando el método CHROMagar (p< 0,001).

Different species of genus Candida can cause a wide range of pathologies, since mucocutaneous trivial infections to disseminated serious forms. Traditionally, taxonomy of yeast has been performed taking into account morphologic and physiologic studies, but they depend on the culture conditions of strains, what cause certain difficulties. Thus, recently, molecular biology methods have been tried. The aim of this work is to correlate taxonomic studies of most important pathogenic species -C. albicans, C. krusei, C. parapsilosis, C. tropicalis and C. glabrata- all of them performed by phenotypic traditional methods, commercial ones, and by a molecular method, PCR fingerprinting. Comparing useful methods for C. albicans identification, corn flour agar and germinative tube formation, no statistical differences between them are observed (X2 =0.5, p=0.4795). By comparison between methods to discriminate different Candida species, physiological tests, CHROMagar, API 20C and PCR fingerprinting we observed no significative differences in proportion of accurate results, in test that can identify any Candida species, such as physiological assays, API 20C and PCR fingerprinting. The proportion of unequivocal results is greater than the obtained performing the CHROMagar culture method (p< 0.001).

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved

Candida utilis: como sustrato de su propio sistema lítico / candida utilis: how sustrate of its lytic system

En el presente trabajo nos planteamos la interrogante sobre la manera de obtener proteínas de levaduras, con alta digestibilidad. Como posible respuesta proponemos que si candida utilis dispone de un sistema lítico capaz de degradar y resistetizar su pared durante la división celular, entonces ese mismo sistema debe degradar la pared de una levadura muerta de su misma especie. Para comprobar esta hipótesis se cultivó C.utilis #17 ATCC 9226 perteneciente al cepario del Laboratorio de Fermenataciones sobre diferentes medios de cultivo: medio salino, medio salino con glucosa y medio salino con células muertas de Cutilis como fuente de carbono. Los cultivos se incuban a 30ºC por 4 a 5 días. C.Utilis no crece en medio salino pero sí lo hace sobre células muertas de su misma especie. Se optimizaron las condiciones de incubación para obtener la máxima producción de enzimas líticas, detectándose la actividad por la disminución en la turbidez a 540 nm, de una suspensión de células muertas tratadas con el sobrenadante de un cultivo. La máxima actividad se alcanza entre 4 y 5 días de incubación