Walicyranison P. Silva-Rocha

Evaluation of Virulence Factors In vitro, Resistance to Osmotic Stress and Antifungal Susceptibility of Candida tropicalis Isolated from the Coastal Environment of Northeast Brazil

Several studies have been developed regarding human health risks associated with the recreational use of beaches contaminated with domestic sewage. These wastes contain various micro-organisms, including Candida tropicalis. In this context, the objective of this study was to characterize C. tropicalis isolates from the sandy beach of Ponta Negra, Natal, Rio Grande do Norte, Brazil, regarding the expression of in vitro virulence factors, adaptation to osmotic stress and susceptibility to antifungal drugs. We analyzed 62 environmental isolates and observed a great variation among them for the various virulence factors evaluated. In general, environmental isolates were more adherent to human buccal epithelial cells (HBEC) than C. tropicalis ATCC13803 reference strain, and they also showed increased biofilm production. Most of the isolates presented wrinkled phenotypes on Spider medium (34 isolates, 54.8%). The majority of the isolates also showed higher proteinase production than control strains, but low phospholipase activity. In addition, 35 isolates (56.4%) had high hemolytic activity (hemolysis index > 0.55). With regard to C. tropicalis resistance to osmotic stress, 85.4% of the isolates were able to grow in a liquid medium containing 15% sodium chloride. The strains were highly resistant to the azoles tested (fluconazole, voriconazole and itraconazole). Fifteen strains were resistant to the three azoles tested (24.2%). Some strains were also resistant to amphotericin B (14 isolates; 22.6%), while all of them were susceptible for the echinocandins tested, except for a single strain of intermediate susceptibility to micafungin. Our results demonstrate that C. tropicalis isolated from the sand can fully express virulence attributes and showed a high persistence capacity on the coastal environment; in addition of showing high minimal inhibitory concentrations to several antifungal drugs used in current clinical practice, demonstrating that environmental isolates may have pathogenic potential.

Candida species distribution, genotyping and virulence factors of Candida albicans isolated from the oral cavity of kidney transplant recipients of two geographic regions of Brazil

BackgroundCandida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. This investigation aimed to study the prevalence of Candida spp. and to analyze the ABC genotypes of 76 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil.MethodsWe typed 48 strains with ABC genotyping and Microsatelitte using primer M13 and tested three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils phagocytosis.ResultsC. albicans was the most prevalent species (86.4%), followed by C. tropicalis (4.5%). C. albicans genotype A was the most prevalent (58 isolates; 76.4%), followed by genotype C (15 isolates; 19.7%) and genotype B (3 isolates; 3.9%). When Microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. The majority of Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed also better ability to combat PMNs phagocytosis.ConclusionsWe found a high rate of C. albicans genotype C strains isolated from the oral cavity of this group of patients. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute to a better understanding of the pathogenesis of oral candidiasis.

An Update on Candida tropicalis Based on Basic and Clinical Approaches

Candida tropicalis has emerged as one of the most important Candida species. It has been widely considered the second most virulent Candida species, only preceded by C. albicans. Besides, this species has been recognized as a very strong biofilm producer, surpassing C. albicans in most of the studies. In addition, it produces a wide range of other virulence factors, including: adhesion to buccal epithelial and endothelial cells; the secretion of lytic enzymes, such as proteinases, phospholipases, and hemolysins, bud-to-hyphae transition (also called morphogenesis) and the phenomenon called phenotypic switching. This is a species very closely related to C. albicans and has been easily identified with both phenotypic and molecular methods. In addition, no cryptic sibling species were yet described in the literature, what is contradictory to some other medically important Candida species. C. tropicalis is a clinically relevant species and may be the second or third etiological agent of candidemia, specifically in Latin American countries and Asia. Antifungal resistance to the azoles, polyenes, and echinocandins has already been described. Apart from all these characteristics, C. tropicalis has been considered an osmotolerant microorganism and this ability to survive to high salt concentration may be important for fungal persistence in saline environments. This physiological characteristic makes this species suitable for use in biotechnology processes. Here we describe an update of C. tropicalis, focusing on all these previously mentioned subjects.

Effect of the crude extract of Eugenia uniflora in morphogenesis and secretion of hydrolytic enzymes in Candida albicans from the oral cavity of kidney transplant recipients

BackgroundCandida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. Yeasts virulence factors contribute for both the maintenance of colonizing strains in addition to damage and cause tissue invasion, thus the establishment of infection occurs. The limited arsenal of antifungal drugs for the treatment of candidiasis turn the investigation of natural products mandatory for the discovery of new targets for antifungal drug development. Therefore, tropical countries emerge as important providers of natural products with potential antimicrobial activity. This study aimed to investigate morphogenesis and secretion of hydrolytic enzymes (phospholipase and proteinase) in the presence of the CE of Eugenia uniflora.MethodsThe isolates were tested for their ability to form hyphae in both solid and liquid media under three different conditions: YPD + 20% FBS, Spider medium and GlcNac and the ability to secrete phospholipase and proteinase in the presence of 2000 μg/mL of E. uniflora.ResultsThe CE of E. uniflora inhibited hypha formation in both liquid and solid media tested. It also impaired hydrolytic enzymes production.ConclusionsThis was the first study to describe the interaction of a natural product with the full expression of three different factors in C. albicans. E. uniflora may be an alternative therapeutic for oral candidiasis in the future.

Effect of the Ethyl Acetate Fraction of Eugenia uniflora on Proteins Global Expression during Morphogenesis in Candida albicans

Candida albicans is able to switch from yeast to hyphal growth and this is an essential step for tissue invasion and establishment of infection. Due to the limited drug arsenal used to treat fungal infections and the constant emergence of resistant strains, it is important to search for new therapeutic candidates. Therefore, this study aimed to investigate by proteomic analysis the role of a natural product (Eugenia uniflora) in impairing hypha formation in C. albicans. We also tested the potential action of E. uniflora to prevent and treat oral candidiasis induced in a murine model of oral infection and the ability of polymorphonuclear neutrophils to phagocytize C. albicans cells treated with the ethyl acetate fraction of the extract. We found that this fraction greatly reduced hypha formation after morphogenesis induction in the presence of serum. Besides, several proteins were differentially expressed in cells treated with the fraction. Surprisingly, the ethyl acetate fraction significantly reduced phagocytosis in C. albicans (Mean 120.36 ± 36.71 yeasts/100 PMNs vs. 44.68 ± 19.84 yeasts/100 PMNs). Oral candidiasis was attenuated when C. albicans cells were either pre-incubated in the presence of E. uniflora or when the fraction was applied to the surface of the oral cavity after infection. These results were consistent with the reduction in CFU counts (2.36 vs. 1.85 Log10 CFU/ml) and attenuation of tissue damage observed with histopathological analysis of animals belonging to treated group. We also observed shorter true hyphae by direct examination and histopathological analysis, when cells were treated with the referred natural product. The E. uniflora ethyl acetate fraction was non-toxic to human cells. E. uniflora may act on essential proteins mainly related to cellular structure, reducing the capacity of filamentation and attenuating infection in a murine model, without causing any toxic effect on human cells, suggesting that it may be a future therapeutic alternative for the treatment of Candida infections.

Clinical improvement of chromoblastomycosis refractory to itraconazole successfully treated with high dose of terbinafine

Dear Editor, Chromoblastomycosis (CBM) is a chronic fungal infection that affects the subcutaneous and cutaneous tissues. A 71-yearold male agricultural worker, born in and resident of Natal, Brazil, reported that he used to work for 20 years in an endemic region of CBM in Brazil (Para State, North Region), where he developed a lower limb injury. The lesion observed on the right lower limb was classified as an erythematous desquamative plaque, on 3 March 2011 (Fig. 1a). In April 2011, a histopathological examination was performed where hyperkeratosis, pseudoepitheliomatous hyperplasia, chronic inflammatory infiltrate and microabscess in addition to sclerotic bodies were found. The patient started medical treatment in the same month, consisting of oral itraconazole 100 mg twice daily and a complementary therapy with cryotherapy once a month for 10 months. After this period, the cryotherapy sessions were performed every 2 months where hyperkeratosis and ulcerations with spontaneous healing were still observed (Fig. 1b). After 13 months of treatment with itraconazole, the lesions still exhibited dark dots. In April 2012, the patient was submitted to laboratorial investigation (Fig. 1c). The collected epidermal scales were treated with 20% KOH and subsequently visualized by direct microscopy. The scales were seeded onto seven equidistant points on the surface of Sabouraud’s dextrose agar with added chloramphenicol, and the Petri dish was incubated at room temperature (28 2°C) for 30 days. A small amount of mycelia was then subcultured on a microculture of potato dextrose agar (PDA). The Petri dish was incubated at room temperature for 10 days. The presence of muriform (sclerotic) cells with a dark, thick membrane showing internal septation in addition to dematiaceous septated hyphae were observed on direct examination (Fig. 1e). After 10 days of incubation on PDA, the colony presented a velvety to cottony texture with olive to brown-black color and no reverse pigment (Fig. 1f). The micromorphological structures were observed on optical microscopy (Fig. 1g,h). The etiological agent was identified as Fonsecaea spp. and further sent for molecular identification at the Special Mycology Laboratory, from the Division of Infectious Diseases, Federal University of Sao Paulo. The isolate was finally identified as Fonsecaea pedrosoi with 99% identity. The sequence was deposited in the GenBank database under accession no. KC357715. In April 2012, after 13 months of treatment with oral itraconazole, the patient was submitted to a change of drug therapy (a) (b)

Influence of Eugenia uniflora Extract on Adhesion to Human Buccal Epithelial Cells, Biofilm Formation, and Cell Surface Hydrophobicity of Candida spp. from the Oral Cavity of Kidney Transplant Recipients

This study evaluated the influence of the extract of Eugenia uniflora in adhesion to human buccal epithelial cells (HBEC) biofilm formation and cell surface hydrophobicity (CSH) of Candida spp. isolated from the oral cavity of kidney transplant patients. To evaluate virulence attributes in vitro, nine yeasts were grown in the presence and absence of 1000 μg/mL of the extract. Adhesion was quantified using the number of Candida cells adhered to 150 HBEC determined by optical microscope. Biofilm formation was evaluated using two methodologies: XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and crystal violet assay, and further analyzed by electronic scan microscopy. CSH was quantified with the microbial adhesion to hydrocarbons test. We could detect that the extract of E. uniflora was able to reduce adhesion to HBEC and CSH for both Candida albicans and non-Candida albicans Candida species. We also observed a statistically significant reduced ability to form biofilms in biofilm-producing strains using both methods of quantification. However, two highly biofilm-producing strains of Candida tropicalis had a very large reduction in biofilm formation. This study reinforces the idea that besides growth inhibition, E. uniflora may interfere with the expression of some virulence factors of Candida spp. and may be possibly applied in the future as a novel antifungal agent.

Characterization of virulence factors of vaginal and anal isolates of Candida albicans sequentially obtained from patients with vulvovaginal candidiasis in north-east Brazil

In order to better understand the pathogenesis of VVC, focusing on the role of C. albicans virulence factors in triggering this infirmity; we evaluated four virulence factors of 62 clinical isolates of C. albicans sequentially obtained from the vagina and anus of patients with sporadic and recurrent VVC. Virulence factors were phenotypically evaluated in vitro, including: adhesion capacity to epithelial cells obtained from healthy individuals, morphogenesis in the presence of fetal bovine serum, biofilm formation in polystyrene microtiter plates and proteinase activity using bovine serum albumin. Colonizing anal isolates were as able as infecting vaginal isolates to express the virulence factors evaluated in vitro. It was observed an association between the expression of virulence factors studied and the signs and symptoms of VVC presented by the patients. No statistically significant difference was observed in the expression of virulence factors between vaginal isolates of C. albicans obtained from patients with sporadic VVC and those obtained from patients with recurrent VVC. Our results suggest that the ability to express virulence factors is important for the pathogenesis of VVC, but it seems not to be crucial for the transition from colonization to infection.