Luciana Furlaneto-Maia

Production and regulation of cuticle-degrading proteases from Beauveria bassiana in the presence of Rhammatocerus schistocercoides cuticle.

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.

In Vitro Evaluation of Putative Virulence Attributes of Oral Isolates of Candida spp. Obtained from Elderly Healthy Individuals

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

In vitro antifungal activity of the flavonoid baicalein against Candida species.

The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC(50) (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 µg ml(-1). For the three species tested, exposure to baicalein at the MIC(50) concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC(50) (fractional inhibitory concentration index = 0.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC(50) produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

INTRODUCTION In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

Interaction of Candida parapsilosis isolates with human hair and nail surfaces revealed by scanning electron microscopy analysis

Candida parapsilosis is found frequently as commensal organism on epithelial tissues, and is also an increasing cause of nosocomial infection. Scanning electron microscope (SEM) observations were used to analyse the capability of C. parapsilosis cells to adhere and grow as biofilm on human natural substrates and to compare the adherence pattern of isolates exhibiting distinct phenotypes. Cells from the crepe phenotype are predominantly elongated and form pseudohyphae whereas cells from the smooth phenotype are yeast-shaped, either in liquid cultures or on human nail and hair surfaces. The electron micrographs revealed that C. parapsilosis cells from the smooth phenotype adhered in higher number to both surfaces compared to the observed for the crepe phenotype. SEM analysis of human hair surface revealed that cells from the smooth phenotype appear as clumped blastoconidia of uniform morphology embedded in a flocculent extracellular material forming biofilm. The extracellular material and biofilm were seeing in a less extension in the crepe phenotype. A distinct adherence pattern was observed when human nail was used as substrate. Here C. parapsilosis cells seem to be linked to surface structures of human nail plate. Fibrillar extracellular material was observed connecting neighbouring cells as well as nail surface.

Ultrastructural architecture of colonies of different morphologies produced by phenotypic switching of a clinical strain of Candida tropicalis and biofilm formation by variant phenotypes.

Candida tropicalis has been identified as one of the most prevalent pathogenic yeast species of the Candida-non-albicans (CNA) group. Study of switching in C. tropicalis has not been the subject of extensive research. Therefore, we investigated switching event and characterized the ultrastructural architecture of different phenotypes and biofilm produced in a C. tropicalis clinical strain. Cells switched heritably, reversibly, and at a high frequency between four phenotypes readily distinguishable by the shape of colonies formed on agar at 25°C. SEM analysis was used to verify the architecture of whole Candida colonies at ultrastructural level. The smooth phenotype (parental phenotype) colony showed a hemispherical shape character, while the semi-smooth was characterized by the presence of shallow marginal depressions. The ring and rough phenotypes exhibited more complex architecture and were characterized by the presence of deep central and peripheral depressions areas. The biofilm-forming ability varied among the switch phenotypes. After 12h incubation, the smooth phenotype formed less biofilm compared to the other phenotypes (P<0.05). The electron microscopy analysis revealed that filamentation (pseudohyphae) was associated with ring and rough colonies. The ultrastructural analysis allowed the observation of the arrangement of individual cells within the colonies. At the deep central and peripheral depressions areas of the ring and rough colonies extracellular material was seen in different arrangements. The data presented here open new avenues to study a possible role for extracellular material in the formation and maintenance of the architecture of switch phenotypes in C. tropicalis. It is therefore essential that more strains be investigated to determine the biological significance of extracellular material in C. tropicalis phenotypic switching phenomenon.

Phenotypic switching in Candida tropicalis: association with modification of putative virulence attributes and antifungal drug sensitivity.

Although Candida tropicalis has become an increasingly important human pathogen, little is known regarding its potential to cause disease. In this study we evaluated the phenotypic switching ability of C. tropicalis and analyzed the effect of switching on biological properties related to virulence factors. We demonstrated that C. tropicalis switched spontaneously, reversibly and at high frequency (10(-1) to 10(-3)) when grown on yeast extract-peptone-D-glucose (YPD) agar medium. Phenotypic switching in five clinical isolates of C. tropicalis resulted in colonies exhibiting the following morphologies: crepe, rough, crater, irregular center, mycelial and diffuse. The majority of the variant colonies were associated with higher percentages of filamentous growth relative to their parental unswitched isolates. Significant differences (P < 0.05) in the production of hemolytic factor were found between most of the switched variants and their respective parental counterparts. Variant colonies exhibiting the crepe (derived from isolates 49.07 and 100.10) and rough phenotype (derived from isolate 49.07) had higher biofilm formation than their parental counterparts exhibiting a smooth dome surface (P < 0.05). Our data revealed that switching was correlated with changes in the in vitro minimum inhibitory concentrations (MICs) of a subset of the switched variants phenotypes to itraconazole. While the MIC to itraconazole was higher for crepe variant compared with its parental isolate 49.07, the rough variant of 100.10 had a lower MIC to this antifungal agent. The presented data support the role of phenotypic switching in promoting changes in phenotypic expression of putative virulence traits and itraconazole susceptibility of clinical isolates of C. tropicalis.

Phenotypic switching of Candida tropicalis is associated with cell damage in epithelial cells and virulence in Galleria mellonella model

Candida species are the most common causes of human fungal infection worldwide. The pathogenic status of Candida spp. is associated with modulation of virulence determinants in response to environmental changes, and impairment of host defenses. Among Candida putative virulence factors, phenotypic switching is associated with fungal adaptability to environmental changes during invasion of host organism. Switching is a biological event associated with generation of phenotypic heterogeneity that occurs in a small fraction of the population, is random, reversible and represents an epigenetic state. In yeast, phenotypic switching drives variable changes, leading to the emergence of colonies with altered morphologies. Our group has previously described a high-frequency switching of colony morphology in several clinical isolates of Candida tropicalis. The isolates could switch spontaneously, heritably and reversibly between at least six different phenotypes, not including the white-opaque transition. One of the identified switching system that include three colony phenotypes: smooth (parental phenotype) and two related switch variants (crepe and rough) was associated with in vitro changes in virulence traits, including biofilm formation, morphogenesis and hemolysis, as well as switching from itraconazole susceptibility to resistance. Soll et al. were the first to assess switch phenotypes in C. tropicalis during the course of a prolonged Candida infection in a compromised host. Further, a white-opaque phenotypic switch was described for C. tropicalis that shows some similarities to that in Candida albicans. Although these studies have begun to explore the mechanism of phenotypic switching in C. tropicalis, the role of switching in this species remains an open question. Porman et al. demonstrated that a white-opaque phenotypic switching in C. tropicalis regulates a cryptic program of sexual mating, potentially giving rise to strains with increased virulence. The white-opaque transition also is associated with sexual biofilm formation in C. tropicalis, where biofilms are formed exclusively by opaque cells. C. tropicalis together with the majority of the medically relevant Candida species belongs to the CTG clade and is genetically close to C. albicans. The frequency of infections caused by C. tropicalis has increased over the last years, particularly in tropical regions; in some settings, bloodstream infections due to C. tropicalis have been associated with higher mortality than other Candida species. In the present study, we address for the first time in the literature, if phenotypic switching affects virulence in C. tropicalis. To this end, we employed all three colony phenotypes (smooth, crepe and rough) described previously and revertants (strains that switched back from variant to parental phenotype) for the evaluation of the capacity of damaging epithelial cells and virulence in Galleria mellonella. The switched strains (crepe and rough variants) were obtained as individual subclones from a clinical isolate exhibiting a smooth colony dome. The crepe and rough variants exhibited an irregular and structured dome surface and were obtained at frequencies of 5£10¡3 and 3£10¡2, respectively. In the present study, we initially determined the reversibility of crepe and rough variants phenotypes to the smooth phenotype (parental phenotype) by visually scoring of four days old growth colonies on YPD agar medium (Gibco) at 28 C. To calculate the reversibility frequencies, a total of 6,000 colonies from each variant

Hemolysis produced by Candida tropicalis isolates from clinical samples

INTRODUCTION Yeasts belonging to the genus Candida are responsible for the majority of fungal infections in humans. Candida tropicalis has been one of most commonly isolated non-albicans species. To analyze in vitro hemolysis promoted by clinical isolates of C. tropicalis obtained from blood and other clinical samples from hospitalized patients at the University Hospital of Londrina State University, Paraná, Brazil. METHODS The hemolysis promoted by 28 clinical isolates of C. tropicalis was evaluated, and the isolates were grouped into classes according to the hemolysis levels. RESULTS The majority of the blood isolates showed weak hemolysis (+), while the classes of strong hemolysis (+++) and very strong hemolysis (++++) predominated among isolates from other clinical samples such as urine, nail lesions and tracheal secretions. However, no statistical differences were detected (p> 0.05). CONCLUSIONS Isolates of C. tropicalis obtained from different clinical samples showed a capacity to promote in vitro hemolysis.

Effects of human blood red cells on the haemolytic capability of clinical isolates of Candida tropicalis.

BackgroundCandida tropicalis is an increasingly important human pathogen associated with high mortality rates; however, little is known regarding the virulence properties of C. tropicalis, particularly the production of haemolytic factor. Although Candida spp may acquire iron from human blood red cells (RBCs) by producing a haemolytic factor that promotes cell lyses, at present there are no data regarding the effect of RBCs on the production of haemolytic molecules. The present study was undertaken to evaluate the role of human red blood cells on the production haemolytic factor by C. tropicalis; in addition, the transcription levels of a putative haemolysin-like protein gene (HLPt) were also analysed.ResultsC. tropicalis isolates produced a haemolytic factor following growth in either the absence or presence of RBCs; however, distinct levels of haemolysis were observed, with 60% of the isolates exhibiting a significant increase in the production of haemolytic factor when grown in the presence of human RBCs. All isolates in which the putative HLPt gene was up-regulated in presence of human RBCs, ranging from 1.044 to 6.965-fold, also exhibited higher haemolytic activity following growth in the presence of RBCs compared to that observed in the absence of RBCs.ConclusionsWe propose that human RBCs may induce changes in the phenotypic expression of haemolytic factor and in transcriptional levels of the putative C. tropicalis HLPt gene in an isolate-dependent fashion.