Aislamiento presuntivo y caracterización de cryptococcus neoformans y cryptococcus gattii desde árboles en la región de O’Higgins y Maule, Chile / Presumptive isolation and characterization of cryptococcus neoformans and cryptococcus gattii from trees in the region of O’Higgins and Maule, Chile

Introducción: la criptocococis es una micosis sistémica causada por C. neoformans y C. gattii, es frecuente y oportunista en inmunocomprometidos y patógeno primario en personas inmunocompetentes. C. neoformans tiene una distribución mundial y se ha aislado desde las excretas de palomas. C. gattii se considera restringida a regiones con clima tropical, subtropical, y templadas, se encuentra asociada frecuentemente a detritos de especies de Eucalyptus sp. La virulencia de estas levaduras le permite desarrollar patogénesis en mamíferos y supervivencia en el ambiente. Objetivo: Identificar y determinar la actividad de proteinasas y fosfolipasas, de C. neoformans y C. gattii aisladas desde las oquedades de árboles en lugares con alta afluencia de público. Materiales y Métodos: Se tomaron 200 muestras de hisopado desde distintas especies de árboles desde sectores de la región de O’Higgins y el Maule. Se siembran en ASG, se aíslan y mantienen en ASD. Identificación con tinta china, Urea de Christensen, crecimiento a 37°C, asimilación y fermentación de azucares, y siembra en medio CGB. Se mide índice de actividad enzimática Prz de proteinasas y fofolipasas. Resultados y Conclusiones: Se obtuvieron 109 cepas de C. neoformans aisladas desde las oquedades de diferentes especies arbóreas y 3 cepas presuntivas de C. gattii desde Eucalyptus sp. y Prunus cerasifera artropurpurea. El 88,1 por ciento de las cepas C. neoformans y 100 por ciento de C. gattii, presentaron alta actividad proteolítica, El 49,5 por ciento de las cepas de C. neoformans y 33,3 por ciento de C. gattii mostraron alta actividad de fosfolipasas.

Introduction: criptocococis is a systemic mycosis caused by C. neoformans and C. gattii, frequent and opportunistic in immunocompromised and primary pathogen in immunocompetent persons. C. neoformans has a worldwide distribution and has been isolated from the excreta of pigeons. C. gattii is considered restricted to regions with tropical, subtropical, and temperate, is often associated with species of Eucalyptus sp. The virulence of these yeasts develop pathogenesis allows survival in mammals and the environment. Objective: To identify and determine the activity of proteinases and phospholipases of C. neoformans and C. gattii isolated from the hollows of trees in places with high turnout. Materials and Methods: 200 swab samples were taken from different species of trees from areas of the region of O’Higgins and Maule. Planted in ASG, they are isolated and kept in ASD. Identification with ink, Urea Christensen, growth at 37 ° C, assimilation and fermentation of sugars, and planting medium CGB. Prz index proteinase enzyme activity is measured and phospholipases. Results and Conclusions: We manage to get 109 strains of C. neoformans isolated from the hollows of different tree species and 3 presumptive strains of C. gattii from Eucalyptus sp. and Prunus cerasifera artropurpurea. 88.1 percent of the strains C. neoformans and C. gattii 100 percent , they showed high proteolytic activity, 49.5 percent of the strains of C. neoformans and C. gattii 33.3 percent showed high activity phospholipases.

Actividad de fosfolipasa y susceptibilidad in vitro a fluconazol en aislamientos clínicos y aviarios de Cryptococcus neoformans / Phospholipase activity and in vitro susceptibility to fluconazol in clinical and aviars isolates of Cryptococcus neoformans

Se detectó la actividad de fosfolipasa en 19 cepas clínicas y 17 aviarias de C. neoformans var. neoformans, usando Agar Sabouraud con yema de huevo, incubándose a 37ºC por 5 días. Se determinó el índice Pz estableciéndose los siguientes rangos: Pz muy alto (0.9-1), alto (0.89-0.80), bajo (0.79-0.70) y muy bajo (<0.69). El 84 por ciento de las cepas clínicas presentaron índice Pz muy bajo, el 5 por ciento bajo y 11 por ciento muy alto. Mientras en las cepas aviarias el 82 por ciento presentaron índice muy bajo y un 18 por ciento muy alto. Los valores de Pz promedio fueron muy bajos en todos los aislamientos, sin existir diferencias significativas (p > 0,05) entre las cepas clínicas y aviarias, lo que implica una alta actividad enzimática. La susceptibilidad in vitro a Fluconazol se realizó por el método de difusión con discos y el 89,5 por ciento de las cepas clínicas fueron sensibles y el 10,5 por ciento resistentes, mientras en las cepas aviarias, el 59 por ciento fueron sensibles, 29 por ciento sensible dosis dependiente y un12 por ciento resistentes.

Phospholipase activity was determined to 19 clinical and 17 aviars trains of C. neoformans var. neoformans, incubating the yeast for 5 days at 37º C on Sabouraud Agar supplemented with egg yolk. Pz values were determined and the following ranges were established: very high (0.9-1), high (0.89-0.80), low (0.79-0.70) and very low (<0.69). The 84 percent of the clinical isolates showed Pz values very low (5 percent) and 11 percent very high. On the other hand, the 82 percent of the aviars strains presented Pz values very low and 18 percent very high. Average Pz values were very low in all isolates , there were no statically significant differences (p>0.05) implying a high enzymatic activity. Susceptibility in vitro testing to Fluconazole was performed by a disk diffusion method (M44-A).The 89.5 percent of the clinical isolates were susceptible and 10.5 percent resistant, while in avian strains, 59 por ciento were susceptible, 29 percent susceptible dose dependent and 12 percent resistant.

Cloning and heterologous expression of Cryptococcus neoformans CnSRB1 cDNA in Saccharomyces cerevisiae.

In this study, we report the results of cloning, sequencing and functional analysis by complementation test of the putative Cryptococcus neoformans homolog CnSRB1. The nucleotide sequence revealed 63% identity, and the deduced amino acid sequence showed 66 and 64% identity to its respective homolog of Saccharomyces cerevisiae and Candida albicans, respectively. Functional complementation test indicated that the putative CnSRB1 gene could compensate the defect caused by a mutation in ScSRB1 in the S. cerevisiae srb1 mutant. Taken together, these results suggest that the putative CnSrblp is a functional homolog of ScSrb1p.

Avaliação da produção de melanina por espécies de Cryptococcus em quatro diferentes meios de cultura / Evaluation of melanin production by Cryptococcus species in four different culture media

A capacidade de Cryptococcus spp produzir melanina em meios contendo compostos fenólicos é amplamente utilizada na identificação destas espécies no laboratório. O objetivo do presente trabalho foi comparar a produção desse pigmento em quatro meios de cultura por Cryptococcus sp. Foram testadas 16 cepas de Cryptococcus neoformans, 17 de Cryptococcus albidus, 13 de Cryptococcus laurentii, e 2 de Cryptococcus uniguttulatus nos meios: ágar batata e cenoura, ágar alpiste, ágar semente de girassol e ágar L-dopa. A produção de melanina foi avaliada com base na pigmentação das colônias, e demonstrada em 5 dias de incubação por 93,8 por cento das cepas de Cryptococcus neoformans nos meios ágar batata e cenoura, ágar semente de girassol e ágar L-dopa. Dos isolados de Cryptococcus albidus, 29,4 por cento produziram o pigmento em ágar batata e cenoura e L-dopa, 11,8 por cento em ágar alpiste, e 36 por cento em ágar girassol. De Cryptococcus laurentii, 53,8 por cento produziram em batata e cenoura e em semente de girassol, 61,5 por cento em L-dopa, 84,6 por cento em ágar alpiste. Somente uma cepa de Cryptococcus uniguttulatus produziu fracamente o pigmento em ágar batata e cenoura.

The capacity of Cryptococcus spp to produce melanin in media containing phenol compounds is widely used for identifying these species in the laboratory. The aim of the present study was to compare the production of this pigment by Cryptococcus spp. in four culture media. Sixteen strains of Cryptococcus neoformans, 17 of Cryptococcus albidus, 13 of Cryptococcus laurentii and two of Cryptococcus uniguttulatus were tested in the following media: potato-carrot agar, Niger seed agar, sunflower seed agar and L-dopa agar. The melanin production was evaluated on the basis of colony pigmentation. Its production after five days of incubation was demonstrated by 93.8 percent of the strains of Cryptococcus neoformans in the media of potato-carrot agar, sunflower seed agar and L-dopa agar. From the isolates of Cryptococcus albidus, 29.4 percent produced the pigment in potato-carrot agar and L-dopa agar, 11.8 percent in Niger seed agar and 36 percent in sunflower seed agar. From Cryptococcus laurentii, 53.8 percent produced the pigment in potato-carrot agar and sunflower seed agar, 61.5 percent in L-dopa agar and 84.6 percent in Niger seed agar. Only one strain of Cryptococcus uniguttulatus presented slight production of the pigment, in potato-carrot agar.

Detection of Cryptococcus neoformans in bird excreta.

We evaluated 14 samples of bird excreta from pigeons, parrots, open billed storks and crows obtained from thirteen places in Bangkok and nearby areas between April and July 2004. These bird excreta were examined for Cryptococcus neoformans by direct plating method to inspect their ability to grow at 37 degrees C. Capsule production was examined by Indian ink preparation. They were also tested for urease and phenoloxidase enzymes. Cryptococcus neoformans var neoformans was recovered from pigeon excreta in 9.09%. This implies those having impaired immunity may get this fungus from the environment.

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.