Virulence of Cryptococcus sp. Biofilms In Vitro and In Vivo using Galleria mellonella as an Alternative Model.

Cryptococcus neoformans and C. gattii are fungal pathogens that are most commonly found in infections of the central nervous system, which cause life-threatening meningoencephalitis and can grow as a biofilm. Biofilms are structures conferring protection and resistance of microorganism to the antifungal drugs. This study compared the virulence of planktonic and biofilm cells of C. neoformans and C. gattii in Galleria mellonella model, as well as, the quantification of gene transcripts LAC1, URE1, and CAP59 by real time PCR. All three of the genes showed significantly increased expressions in the biofilm conditions for two species of Cryptococcus, when compared to planktonic cells. C. neoformans and C. gattii cells in the biofilm forms were more virulent than the planktonic cells in G. mellonella. This suggests that the biofilm conditions may contribute to the virulence profile. Our results contribute to a better understanding of the agents of cryptococcosis in the host-yeast aspects of the interaction.

A flow cytometry method for testing the susceptibility of Cryptococcus spp. to amphotericin B.

BACKGROUND: Human fungal infections have increased at an alarming rate in recent years, particularly in immunocompromised individuals. Cryptococcosis is the second most prevalent systemic fungal infection worldwide, and the most prevalent systemic infection in immunocompromised individuals, representing more than 70% of cases. The incidence of cryptococcosis is high in people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. AIMS: The aim of this research was to develop a rapid flow cytometric antifungal susceptibility test and to compare the results with the standard methods. METHODS: A reference strain and clinical isolates of Cryptococcus neoformans and Cryptococcus gattii were tested for susceptibility to amphotericin B by flow cytometry using propidium iodide as indicator of viability. Flow cytometry (FC) results were compared with the minimum inhibitory concentration (MIC) values determined by microdilution. RESULTS: The antifungal activity of amphotericin B ranged from MICs of 0.06 to 2µg/ml for the 11 isolates studied. The same results were found by FC. CONCLUSIONS: The FC method allows same-day results, assisting in the selection of appropriate antifungal therapies. These results demonstrate an excellent correlation between FC and the classic methods of testing for susceptibility to antifungal agents. This rapid diagnosis method makes it possible to quickly administer effective therapeutic interventions, often saving lives.

Antifungal Activity of Decyl Gallate against Several Species of Pathogenic Fungi.

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action.

Antimicrobial activity of Pterogyne nitens Tul., Fabaceae, against opportunistic fungi / Atividade antimicrobiana de Pterogyne nitens Tul., Fabaceae, contra fungos oportunistas

As part of our ongoing research on antifungal agents from Brazilian flora, eight extracts and twelve fractions from Pterogyne nitens Tul., Fabaceae, were screened for antimicrobial activity against four opportunistic fungi species (Candida albicans, Candida krusei, Candida parapsilosis and Cryptococcus neoformans) using a broth microdilution method. The present investigation reveals that P. nitens extracts and fractions were more effective against C. krusei and C. parapsilosis than against C. neoformans. The growth of C. albicans was moderately affected by all tested extracts and fractions. The strongest effects were observed for n-butanol fractions from branches (MIC = 15.6 μg/mL) and roots (MIC = 31.2 μg/mL) against C. krusei. Additionally, the chromatographic fractionation of the n-butanol fraction from branches afforded four guanidine alkaloids; N-1,N-2,N-3-triisopentenylguanidine (1), described for the first time in the Fabaceae family, and nitensidines A-C (2-4), which showed moderate activity towards C. krusei (MIC = 62.5 μg/mL) and C. parapsilosis (MIC = 31.2 μg/mL).

No contexto de nossas pesquisas por novos agentes antifúngicos obtidos da flora brasileira, oito extratos e doze frações de Pterogyne nitens Tul., Fabaceae, foram submetidos ao ensaio antifúngico pelo método de microdiluição, contra quatro espécies de fungos oportunistas, Candida albicans, Candida krusei, Candida parapsilosis e Cryptococcus neoformans. Este trabalho revelou que os extratos e frações de P. nitens foram mais ativos contra C. krusei e C. parapsilosis quando comparados a C. neoformans, sendo que o crescimento de C. albicans foi moderadamente afetado por todos os extratos e frações. As atividades mais potentes foram observadas para as frações n-butanólica dos galhos (CIM = 15,6 μg/mL) e raízes (CIM = 31,2 μg/mL) contra C. krusei. Adicionalmente, a fração n-butanólica dos galhos foi submetida ao fracionamento cromatográfico, resultando no isolamento de quatro alcaloides guanidínicos, sendo N-1,N-2,N-3-tri-isopentenilguanidina (1), descrito pela primeira vez em espécies da família Fabaceae e nitensidinas A-C (2-4), os quais apresentaram atividade antifúngica moderada contra C. krusei (CIM = 62,5 μg/mL) e C. parapsilosis (CIM = 31,2 μg/mL).

Genetic diversity and antifungal susceptibility testing of Trichosporon asahii isolated of Intensive Care Units patients / Diversidade genética e suscetibilidade a antifúngicos de Trichosporon asahii isolado de pacientes de Unidades de Terapia Intensiva

Trichosporon asahii is an opportunistic pathogen, associated with a high mortality rate in immunocompromised patients. In this study, ten isolates, recovered from oral cavity and urine of patients in Intensive Care Units (ICU) over six months, were identified by classical and molecular methods, typed by RAPD and tested in vitro for susceptibility to fluconazole, itraconazole, 5-flucytosine and amphotericin B. A total agreement between the identification of Trichosporon sp by PCR based on sequences of the Internal Transcribed Spacer Regions (ITS) and on the sequences of small-subunit (SSU) ribosomal DNA (rDNA) was found. Randomly amplified of polymorphic DNA (RAPD), with primers P6 and M13, was used to determine the genomic profiles. The dendogram analysis indicated that almost all strains showed similarity >0.9 among them and all strains were multidrug-resistant. This study brings new results on the identification and genotyping of T. asahii isolated from Brazilian ICU patients and information about their antifungal drugs susceptibility.

Trichosporon asahii é um patógeno oportunista que apresenta altos índices de mortalidade em pacientes imunocomprometidos. No presente trabalho, dez cepas foram isoladas da cavidade bucal e urina de pacientes internados na Unidade de Terapia Intensiva (UTI) por seis meses. Todos os isolados foram identificados por métodos clássicos e moleculares, tipados por RAPD e testados in vitro quanto à sensibilidade ao fluconazol, itraconazol, 5-fluorocitosina e anfotericina B. Houve concordância total entre a identificação de Trichosporon sp por PCR baseado na seqüência da região ITS (Internal Transcribed Spacer) e na seqüência da subunidade menor do DNA ribossômico (rDNA). Os perfis genéticos foram determinados por RAPD utilizando dois iniciadores P6 e M13. A análise do dendrograma mostrou que a maioria das amostras apresentou alta similaridade entre elas (>0.9) e todas foram multidrogas resistentes. Este estudo traz novos resultados em relação à identificação e genotipagem de isolados brasileiros de T. asahii em pacientes internados em UTI, bem como dados sobre o perfil de sensibilidade aos antifúngicos.

Genetic diversity and antifungal susceptibility testing of Trichosporon asahii isolated of Intensive Care Units patients.

Trichosporon asahii is an opportunistic pathogen, associated with a high mortality rate in immunocompromised patients. In this study, ten isolates, recovered from oral cavity and urine of patients in Intensive Care Units (ICU) over six months, were identified by classical and molecular methods, typed by RAPD and tested in vitro for susceptibility to fluconazole, itraconazole, 5-flucytosine and amphotericin B. A total agreement between the identification of Trichosporon sp by PCR based on sequences of the Internal Transcribed Spacer Regions (ITS) and on the sequences of small-subunit (SSU) ribosomal DNA (rDNA) was found. Randomly amplified of polymorphic DNA (RAPD), with primers P6 and M13, was used to determine the genomic profiles. The dendogram analysis indicated that almost all strains showed similarity >0.9 among them and all strains were multidrug-resistant. This study brings new results on the identification and genotyping of T. asahii isolated from Brazilian ICU patients and information about their antifungal drugs susceptibility.

The use of standard methodology for determination of antifungal activity of natural products against medical yeasts Candida sp and Cryptococcus sp

Methodology for testing natural compounds for determination of antifungal activity had been developed with adaptations. The most used are bioautography and agar diffusion with a complex and no defined media. In this study, different methods for determination of antifungal activity of natural products are discussed and the use of M27-A2 microdilution test from CLSI (Clinical and Laboratory Standards Institute, 2002), as general standard methodology for testing plant extracts activity is recommended.

Metodologias para determinar atividade antifúngica foram desenvolvidas com adaptações para avaliar produtos naturais. As mais usadas são bioautografia e o método de difusão em agar, que empregam meios de cultura complexos, não definidos. Neste estudo são discutidos os métodos para determinação de atividade antifúngica de produtos naturais e é recomendado o uso do micrométodo modificado segundo o documento M27A2 do CLSI.