USE OF MUELLER-HINTON BROTH AND AGAR IN THE GERM TUBE TEST

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

Identification and differentiation of Candida species from pediatric patients by random amplified polymorphic DNA

Thirty-four Candida isolates were analyzed by random amplified polymorphic DNA using the primer OPG-10:24 Candida albicans; 4 Candida tropicalis; 2 Candida parapsilosis; 2 Candida dubliniensis; 1 Candida glabrata and 1 Candida krusei. The UPGMA-Pearson correlation coefficient was used to calculate the genetic distance between the different Candida groupings. Samples were classified as identical (correlation of 100%); highly related samples (90%); moderately related samples (80%) and unrelated samples (< 70%). The results showed that the RAPD proposed was capable of classifying the isolates coherently (such that same species were in the same dendrogram), except for two isolates of Candida parapsilosis and the positive control (Netherlands, 1973), probably because they are now recognized as three different species. Concerning the only fluconazole-resistant Candida tropicalis isolate with a genotype that was different to the others, the data were insufficient to affirm that the only difference was the sensitivity to fluconazole. We concluded that the Random Amplified Polymorphic DNA proposed might be used to confirm Candida species identified by microbiological methods.

Produção de fatores de virulência in vitro por espécies patogênicas do gênero Candida

Avaliou-se, in vitro, a capacidade de crescimento em 39ºC e 42ºC, a produção de enzimas hidrolíticas e a atividade hemolítica de 21 cepas clínicas e de referência de sete espécies de Candida spp, Candida dubliniensis e Candida krusei demonstraram menor potencial de virulência e Candida albicans maior.

Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species

Aspartyl proteases are a class of enzymes that include the yeast aspartyl proteases and secreted aspartyl protease (Sap) superfamilies. Several Sap superfamily members have been demonstrated or suggested as virulence factors in opportunistic pathogens of the genus Candida. Candida albicans, Candida tropicalis, Candida dubliniensis and Candida parapsilosis harbour 10, four, eight and three SAP genes, respectively. In this work, genome mining and phylogenetic analyses revealed the presence of new members of the Sap superfamily in C. tropicalis (8), Candida guilliermondii (8), C. parapsilosis(11) and Candida lusitaniae (3). A total of 12 Sap families, containing proteins with at least 50% similarity, were discovered in opportunistic, pathogenic Candida spp. In several Sap families, at least two subfamilies or orthologous groups were identified, each defined by > 90% sequence similitude, functional similarity and synteny among its members. No new members of previously described Sap families were found in a Candida spp. clinical strain collection; however, the universality of SAPT gene distribution among C. tropicalis strains was demonstrated. In addition, several features of opportunistic pathogenic Candida species, such as gene duplications and inversions, similitude, synteny, putative transcription factor binding sites and genome traits of SAP gene superfamily were described in a molecular evolutionary context.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals.

Abstract In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland.

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre(®) YeastOne? test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Ent-kaurane diterpenoids and other constituents from the stem of Xylopia laevigata (Annonaceae)

Phytochemical investigation of the hexane extract from the stem of Xylopia laevigata led to the isolation of the ent-kaurane diterpenoids, ent-kaur-16-en-19-oic acid, 4-epi-kaurenic acid, ent-16β-hydroxy-17-acetoxy-kauran-19-al, ent-3β-hydroxy-kaur-16-en-19-oic acid, and ent-16β,17-dihydroxy-kauran-19-oic acid, as well as spathulenol and a mixture of β-sitosterol, stigmasterol and campesterol. The identification of the compounds was performed on the basis of spectrometric methods including GC-MS, IR, and 1D and 2D NMR. Potent larvicidal activity against Aedes aegypti larvae with LC50 of 62.7 µg mL-1 was found for ent-3β-hydroxy-kaur-16-en-19-oic acid. This compound also showed significant antifungal activity against Candida glabrata and Candida dubliniensis with MIC values of 62.5 µg mL-1.

Oral colonization by Candida species in AIDS pediatric patients

The aim of this study was to assess the prevalence of factors associated with oral colonization by Candida spp. in pediatric patients with AIDS. The sample comprised of 117 children. Clinical status, medicines in use, and laboratory findings were obtained from hospital records; sociodemographic data were given by relatives. A dental examination assessed the prevalence of dental caries. The prevalence of oral colonization by Candida was 62%. Only seven children presented clinical manifestation of oral candidosis despite their high viral load index and low-for-age CD4 count. Candida colonization was directly associated with frequent use of antibiotics (prevalence ratio [PR] = 1.44), sulfa drugs (PR = 1.23), alteration in the oral mucosa (PR = 1.55), and untreated dental caries (PR = 1.93). It was inversely associated with the use of antiretroviral therapies (PR = 0.65). Candida albicans was the most frequently detected species (80%); phenotypic tests did not detect C. dubliniensis strains. This study observed a low prevalence of Candida-related oral lesions in these patients, which is compatible with the hypothesis that antiretroviral medicines may have contributed to reducing oral manifestations from Candida infection. The high prevalence of Candida colonization in HIV+/AIDS children with untreated dental caries reinforces the importance of oral health care in interdisciplinary health units that assist these patients.

Alkaloids from the Bark of Guatteria hispida and Their Evaluation as Antioxidant and Antimicrobial Agents

Phytochemical investigation of the bark of Guatteria hispida afforded three new alkaloids, 9-methoxy-O-methylmoschatoline (1), 9-methoxyisomoschatoline (2), and isocerasonine (3), along with 10 known alkaloids, 8-oxopseudopalmatine (4), O-methylmoschatoline (5), lysicamine (6), liriodenine (7), 10-methoxyliriodenine (8), nornuciferine (9), anonaine (10), xylopine (11), coreximine (12), and isocoreximine (13). The major compounds, 2, 6, 12, and 13, showed significant antioxidant capacity in the ORAC(FL), assay. Compounds 5, 6, and 7 were active against S. epidermidis and C. dubliniensis, with MIC values in the range 12.5-100 mu g mL(-1).

Identificação e fatores de virulência de Candida spp isoladas da cavidade bucal de transplantados renais do hospital universitário Onofre Lopes em Natal-RN

Despite Candida species are often human commensals isolated from various oral sites such as: tongue, cheek and palatal mucosa plus subgingival region, there are some properties linked to the organism commonly known as virulence factors which confer them the ability to produce disease. Oral candidiasis is one of the main oral manifestations reported in literature related to kidney transplant patients. The objectives of the present study were to identify and investigate virulence factors of yeasts isolated from the oral cavity of kidney transplant recipients admitted at the Hospital Universitário Onofre Lopes, in Natal RN. Seventy Candida species isolated from 111 kidney transplant recipients were investigated in this study. Identification of the isolates was performed by using the evidence of germ tube formation, hypertonic broth, tolerance to grow at 42°C, micromorphology and biochemical profiles. We observed a high rate of isolation of yeasts from the oral cavity of kidney transplant recipients (63.1%) being C. albicans was the most prevalent species. Oral candidiasis was diagnosed in 14.4% of transplant recipients. We evaluated virulence properties of the isolates regarding to: biofilm formation on polystyrene microplates as well as XTT reduction, adherence to acrylic resin and human buccal epithelial cells and proteinase activity. Most isolates were able to form biofilm by the method of adhesion to polystyrene. All isolates of Candida spp. remained viable during biofilm formation when analyzed by the method of XTT reduction. The number of CFU attached to the acrylic resin suggested high adherence for C. parapsilosis. C. albicans isolates showed higher median adherence to human buccal epithelial cells than non-C. albicans Candida isolates. Nevertheless, this difference was not statistically significant. C. dubliniensis showed low ability to adhere to plastic and epithelial cells and biofilm formation. Proteolytic activity was observed for all the isolates investigated, including the unique isolate of C. dubliniensis. There was a statistically significant association between proteinase production and the presence of oral candidiasis. Studies related to oral candidiasis in renal transplant recipients are limited to clinical and epidemiological data, but investigations concerning Candida spp. virulence factor for this group of individuals are still scarce. We emphasize the importance of studies related to virulence factors of yeasts isolated from this population to contribute to the knowledge of microbiological aspects of oral candidiasis

Yeasts isolated from a fungus-growing ant nest, including the description of Trichosporon chiarellii sp nov., an anamorphic basidiomycetous yeast

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Triagem antifúngica de extratos obtidos de espécies vegetais do nordeste brasileiro

The increase in the incidence of fungal infections due to the drug-resistance or to the number of patients with immune alterations such as AIDS, chemotherapy or organ transplantation, has done the research necesseray for new antifungal drugs. The species from Northeastern Brazil may become an important source of innovative natural molecules. To evaluate the antifungal activity of 10 medicinal plants from Northeastern Brazil, traditionally used as antimicrobial agents, 30 crude extracts (CE) were tested in vitro against four standard species of Candida spp. The CE most promising of these plants were evaluated against yeasts of the oral cavity of kidney transplant patients and through a bioassay-guided fractionation. The extracts form leaves of E. uniflora, the stem bark of L. ferrea and leaves of P. guajava showed significant activity against all yeasts evaluated, with MIC values between 15.62 and 62.5 μg/mL. E. uniflora also showed fungicidal properties against all yeasts, especially against Candida dubliniensis. In patients with immune systems compromised, such as transplanted, oral candidiasis manifests mainly due to immunosuppressive therapy, and resistance to conventional antifungals. The CE of E. uniflora presented range of MIC values between 1.95 to 1000 μg/mL, and lower MIC50 and MIC90 values were observed against C. non-albicans. Due the better results, the CE of E. uniflora was elected to performe the bioassay-guided fractionation. Thus it was possible to obtain enriched fractions, which showed good inhibitory ability against ATCC strains of Candida spp. It was also possible to perform experiments to verify the production of biofilm in two strains of C. dubliniensis and action of extracts and fractions on the same. With this, we observed a behavior between the yeast ATCC and clinical isolate. In addition, CE, fractions and subfractions of E. uniflora inhibit planktonic cells to preventing the growth of biofilm. The preliminary chemical characterization of the fractions obtained revealed the presence of polyphenols (especially flavonoids and tannins). Finally, the results suggests that among the plant species studied, E. uniflora showed a pattern very promising as regards the antifungal, requiring further study of purification and structural elucidation of compounds in order to verify that the antifungal effect found can be attributed to a specific compound or some mechanism depends on synergistic the mixture of polyphenols

Antimicrobial Properties and Cytotoxicity of an Antimicrobial Monomer for Application in Prosthodontics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Growth of Candida species on complete dentures: effect of microwave disinfection

Microwave disinfection of complete dentures has been recommended to treat denture stomatitis in non-immune compromised patients. Oral candidiasis is a frequent manifestation of HIV infection. The objective of this study is to evaluate the effectiveness of microwave irradiation on the disinfection of complete dentures inoculated with American Type Culture Collection (ATCC) and HIV isolates of five species of Candida. Fifty dentures were made, sterilised and inoculated with the tested microorganisms (C. albicans, C. dubliniensis, C. krusei, C. glabrata and C. tropicalis). After incubation (37 degrees C/48 h), dentures were microwaved (650 W/3 min). Non-irradiated dentures were used as positive controls. Replicate aliquots of suspensions were plated at dilutions 10(-1) to 10(-4) and incubated (37 degrees C/48 h). Colony counts (cfu ml(-1)) were quantified. Dentures were also incubated at 37 degrees C for 7 days. Data were analysed with 2-way anova and Tukey HSD tests (alpha = 0.05). Dentures contaminated with all Candida species showed sterilisation after microwave irradiation. All control dentures showed microbial growth on the plates. The cfu ml(-1) for C. glabrata was higher than those of C. albicans, C. dubliniensis and C. tropicalis whereas the cfu ml(-1) for C. krusei was lower. The cfu ml(-1) for clinical isolates was higher than those of ATCC yeast. Microwave irradiation for 3 min at 650 W resulted in sterilisation of all complete dentures.

Effectiveness of Photodynamic Therapy for the Inactivation of Candida spp. on Dentures: In Vitro Study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)