Rossana de Aguiar Cordeiro

Characterization of the gastrointestinal yeast microbiota of cockatiels (Nymphicus hollandicus): a potential hazard to human health.

Cockatiels are the worlds second most popular psittacine pet bird, but no data characterizing their gastrointestinal microbiota have been found. Thus, the aim of this work was to characterize the yeast gastrointestinal microbiota of cockatiels and to evaluate the relevance of cockatiels as carriers of potentially pathogenic yeasts. A total of 60 cockatiels, from 15 different premises, were assessed. A thorough clinical examination was performed with each bird, and samples were collected from oral cavity, crop and cloaca. The stools were collected from cages where the birds were kept. The isolates were identified according to morphological and biochemical characteristics. Yeasts were isolated from at least one anatomical site of 65% of the birds and 64.3% of the stool samples. The oral cavity (53.3%) and the crop (58.3%) were the anatomical sites with the highest prevalence and the highest number of yeast isolates. Overall, 120 yeast isolates, belonging to 13 species, were obtained. The most frequently isolated species were Candida albicans, with 39 (32.5%) isolates, followed by Candida tropicalis (20%), Trichosporon asteroides (12.5%), Candida famata (10%) and others. Mixed yeast colonies were isolated from 23.3% of the birds and C. albicans was seldom found in association with other species (P<0.05). The results of this work demonstrated that cockatiels harbour potentially pathogenic yeasts throughout their gastrointestinal tract and in stools, and are prone to disseminating them in the environment.

Phenotypical and molecular characterization of Microsporum canis strains in north-east Brazil

Aims:  This study investigated the possible correlation between the phenotypical and genotypical characteristics of Microsporum canis isolated from cats and dogs in north‐east Brazil.

An alternative method for the analysis of melanin production in Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato

Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the assessment of melanin production and its quantification may contribute to the understanding of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neoformans sensu lato and C. gattii sensu lato. Eight C. neoformans sensu lato and three C. gattii sensu lato, identified through URA5 methodology, Candida parapsilosis ATCC 22019 (negative control) and one Hortaea werneckii (positive control) were inoculated on minimal medium agar with or without L‐DOPA, in duplicate, and incubated at 35°C, for 7 days. Pictures were taken from the third to the seventh day, under standardised conditions in a photographic chamber. Then, photographs were analysed using grayscale images. All Cryptococcus spp. strains produced melanin after growth on minimal medium agar containing L‐DOPA. C. parapsilosis ATCC 22019 did not produce melanin on medium containing L‐DOPA, while H. werneckii presented the strongest pigmentation. This new method allows the indirect analysis of melanin production through pixel quantification in grayscale images, enabling the study of substances that can modulate melanin production.

In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of azole-resistant Candida tropicalis

Abstract The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole‐resistant and one azole‐susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time‐kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 &mgr;g/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole‐resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species.

Exposure of Candida parapsilosis complex to agricultural azoles: An overview of the role of environmental determinants for the development of resistance

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance.

Combination of Phenotypic Tests as a Screening Approach for the Differentiation of Cryptic Species Candida albicans and Candida dubliniensis

Background: Candida albicans, an important pathogen for humans and animals, shares phenotypic features with Candida dubliniensis, leading to misidentification. Thus, the goal of this study was to apply a combination of phenotypic tests for the differentiation of these cryptic species. Methods and findings: Thirty-seven azole-resistant C. albicans from animals and 03 C. dubliniensis from humans were included in this study. Purity of strains was evaluated on CHROMagar Candida™, on which both species present green colonies. Then, phenotypic characterization was performed based on the growth pattern on sunflower seed agar, where C. albicans presents smooth colonies and C. dubliniensis presents rough colonies, and esterase production on Tween 80™ agar, which is positive for C. albicans, forming an opacification zone, and negative for C. dubliniensis. Molecular differentiation was performed with primers CALF/CALR for the amplification of ITS1/ITS2 regions of rDNA, yielding an amplicon of 100 bp for C. albicans, but not for C. dubliniensis. Of the 37 C. albicans, 35 showed green colonies on CHROMagar Candida Medium™, 36 presented smooth colonies on sunflower seed agar, while 34 showed an opacification zone on Tween 80™ agar. PCR yielded 100-bp-amplicons for all 37 C. albicans strains, confirming their identification. Control strains of C. dubliniensis showed the expected phenotypic features and amplicons were not obtained for the specific PCR reaction. Conclusions: The phenotypic methods used were not absolutely effective, however, the combined observation of smooth colonies on sunflower seed agar with an opacification zone on Tween 80™ agar leads to a reliable presumptive phenotypic identification of C. albicans.