Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile.

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37ºC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.

Variability in UVB tolerances of melanized and nonmelanized cells of Cryptococcus neoformans and C. laurentii.

Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. Inactivation by UVA and UVB radiation is especially important for the control of fungi that disperse infective units through the air, including fungi such as Cryptococcus spp. that infect their vertebrate hosts by inhalation. Cryptococcus neoformans produces melanin in the presence of certain exogenous substrates such as l-3,4 dihydroxyphenylalanine and melanization may protect the fungus against biotic and abiotic environmental factors. In the present study, we investigated the effect of exposure to an UVB irradiance of 1000 mW m(-2) (biologically effective weighted irradiance) on the survival of melanized and nonmelanized cells of four strains of C. neoformans and four strains of C. laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of cells grown 2, 4, 6 or 8 days on medium with or without L-dopa was determined after exposure to UVB doses of 1.8 and 3.6 kJ m(-2). Both the irradiance spectrum and the intensities of those doses are environmentally realistic, and, in fact, occur routinely during summer months in temperate regions. Differences in tolerance to UVB radiation were observed between the C. neoformans and C. laurentii strains. The C. neoformans strains were more susceptible to UVB radiation than the C. laurentii strains. In C. neoformans, differences in tolerance to radiation were observed during development of both melanized and nonmelanized cells. For most treatments (strain, time of growth and UVB dose), there were virtually no differences in tolerances between melanized and nonmelanized cells, but when differences occurred they were smaller than those previously observed with UVC. In tests with two strains of C. laurentii, there was no difference in tolerance to UVB radiation between melanized and nonmelanized cells during 8 days of culture; and in tests with four strains for less culture time (4 days) there were no significant differences in tolerance between melanized and nonmelanized cells of any strain of this species.

Bioactive chemical constituents and comparative antimicrobial activity of callus culture and adult plant extracts from Alternanthera tenella.

Crude extracts of a callus culture (two culture media) and adult plants (two collections) from Alternanthera tenella Colla (Amaranthaceae) were evaluated for their antibacterial and antifungal activity, in order to investigate the maintenance of antimicrobial activity of the extracts obtained from plants in vivo and in vitro. The antibacterial and antifungal activity was determined against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. Ethanolic and hexanic extracts of adult plants collected during the same period of the years 1997 and 2002 [Ribeirão Preto (SP), collections 1 and 2] and obtained from plant cell callus culture in two different hormonal media (AtT43 and AtT11) inhibited the growth of bacteria, yeasts and dermatophytes with inhibition halos between 6 and 20 mm. For the crude extracts of adult plants bioassay-guided fractionation, purification, and isolation were performed by chromatographic methods, and the structures of the isolated compounds were established by analysis of chemical and spectral evidences (UV, IR, NMR and ES-MS). Steroids, saponins and flavonoids (aglycones and C-glycosides) were isolated. The minimum inhibitory concentration (MIC) of the isolated compounds varied from 50 to 500 microg/mL.

Lactoferrin, a marker for periodontal disease.

This study evaluated the salivary concentrations of lactoferrin (Lf) in HIV-seropositive and -seronegative subjects correlating these levels with the incidence of periodontal disease, quantity of Candida spp and systemic condition of the HIV-seropositives (viral load and T lymphocytes CD-4+ count and antiretroviral therapy). Whole saliva samples were obtained from 109 subjects who were divided into four groups according to the extent of their HIV infection and their periodontal condition. The salivary Lf concentrations were determined by a standard enzyme-linked immunosorbent assay and the quantification of Candida spp. was obtained from all subjects. Among the HIV- participants, higher concentrations of Lf were found in individuals with periodontal diseases (p<0.0001). A similar result was found for HIV+ participants (p<0.0001). No correlation was found between the concentration of salivary Lf and the quantification of Candida spp or between the Lf concentration and the systemic condition of the HIV+ subjects. The existence of periodontal diseases can modulate an early inflammatory process in the oral mucosa by increasing the expression of Lf, where Lf can act as an antibacterial peptide in HIV- and HIV+ patients. These results suggest that Lf is a possible marker for periodontal diseases in immunocompetent and immunocompromised subjects.

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirão Preto, São Paulo, Brazil.

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeirão Preto, São Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors.

Evaluation of the disk diffusion method for testing fluconazole susceptibility of Cryptococcus laurentii

Cryptococcus laurentii é um patógeno humano raro, ubíquo na natureza. O objetivo deste estudo foi avaliar a aplicação do método de difusão do disco para determinar a sensibilidade ao fluconazol de isolados de C. laurentii e determinar a concentração fungicida mínima (CFM) do fluconazol. Foi determinada a sensibilidade ao fluconazol pelos métodos de difusão do disco e microdiluição em caldo de 11 isolados de C. laurentii, de acordo com CLSI (Clinical and Laboratory StandardsInstitute), e a CFM pelo método de microdiluição em caldo. O método de difusão do disco mostrou quatro isolados sensíveis, três sensíveis dose-dependentes e quatro resistentes, enquanto que pelo método de microdiluição, 10 isolados foram sensíveis e um sensível dose-dependente. A concordância entre os dois métodos foi de 36,4por cento. Um isolado apresentou CFM de 8 mi g/mL e dois de 64 mi g/mL. Embora o número de isolados estudados seja pequeno, os resultados sugeremque o método de difusão do disco não deve ser usado na determinação da sensibilidade in vitro dos isolados de C. laurentii ao fluconazol, e apesar de ser uma droga fungistática, o fluconazol pode apresentar atividade fungicida in vitro para alguns isolados do complexo C. laurentii.

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91 percent) killed 50 percent or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37ºC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.

Comparative study of antibacterial and antifugal activity of callus culture and adult plants extracts from Alternanthera maritima (Amaranthaceae)

Neste estudo procedeu-se a avaliação da atividade antibacteriana e antifúngica dos extratos brutos de Alternanthera maritima (Amaranthaceae) planta in natura de duas coletas distintas e obtidos por cultura de células buscando-se averiguar a manutenção da atividade antimicrobiana dos extratos obtidos da planta in vivo e in vitro. A ação antibacteriana e antifúngica foi determinada pelo método de difusão em ágar (técnica do poço) utilizando-se trinta cepas de microrganismos indicadores (bactérias Gram-positivas e Gram-negativas, leveduras e dermatófitos). Todos os extratos obtidos com solventes orgânicos avaliados apresentaram-se bioativos com halos de inibição de 6 a 20 mm. Os extratos da planta in natura das duas coletas (Restinga de Marica (RJ), verão de 1995 e 1998) inibiram o desenvolvimento de diferentes microrganismos (bactérias, leveduras e dermatófitos). Os extratos obtidos da cultura de calos desenvolvidos em duas condições de cultivo diferentes, também se mantiveram bioativos. Assim, os resultados obtidos encorajam a realização de novos estudos com esta espécie vegetal para se determinar quais as substâncias presentes nos extratos e que contribuem para a atividade biológica, como também para entender seu mecanismo de ação e avaliar sua toxicidade, visando uma possível aplicação farmacêutica.

Leveduras do Gênero Candida na cavidade bucal com e sem lesäo: detecçäo de suscetibilidade a antifúngicos - técnica do Etest / Candida yeasts in oral cavity with and without lesions: detection of antifungal susceptibility - Etest technique

Foram avaliados 50 pacientes, de ambos os sexos e faixa etária média de 42,5 anos, objetivando-se detectar a sucetibilidade a antifúngicos de leveduras isoladas da boca com e sem lesäo. Amostras do dorso da lingua, de lesäo e saliva foram inoculadas no ágar Sabouraud-dextrose acrescido de cloranfenicol incubado a 37 graus celsius, durante três a cinco dias. Foi dectada sensibilidade aos antimicrobanos com a técnica do Etest (AB Biodisk-Solna, Suécia), com o ágar RPMI-1640 (Sigma), com L-glutamina e tampäo fosfato. A interpretaçäo da sensibilidade ou resistência in vitro das cepas foi feita de acordo com o documento M 27 A (NCCLS, 1997). Leveduras do gênero Candida foram isoladas em todos os tipos de amostras, sendo a C. albicans a espécie prevalente, detectada em 48 por cento salivas, 67,8 por cento lesöes e 14,3 por cento e 12,5 por cento linguaas, normal e fissurada e/ou saburrosa, respectivamente. Entre os pacientes com candidíase bucal isolaram-se C. tropicalis (4/8 por cento e 3/10,7 por cento), C. glabrata (2/4 por cento e 1/3,6 por cento) e C. parapsilosis (1/2 por cento e 1/3,6 por cento), respectivamente na saliva e lesäo. As 68 cepas submetidas ao Etest apresentaram 41/60,3 por cento cepas resistentes (R) a algum antifúngico. Todas as 68 amostras de Candida foram sensíveis à anfotericina B e ao fluconazol. Quanto ao cetoconazol, apenas duas C. albicans isoladas de lesäo foram resistentes. Em relaçäo ao itraconazol, 16 (55,2 por cento) e 25 (64,1 por cento) cepas, respectivamente, de lesäo e de outros nichos foram resistentes, dentre eleas, C. albicans (30), C. Tropicalis (7), Candida spp. (2), C. Krusei (1) e C. guilliermondi (1). Os testes de suscetibilidade em micologia devem ser realizados por métodos padronizados e eficientes e com maior freqüencia, para alertar os clínicos quanto à emergência de cepas resistentes in vitro aos antifúngicos