RESUMO
BACKGROUND: Candida tropicalis is an increasingly important human pathogen which usually affects neutropenic oncology patients with common hematogenous seeding to peripheral organs and high mortality rates. Candida pathogenicity is facilitated by several virulence attributes, including secretion of hydrolytic enzymes; however, little is known regarding the C. tropicalis ability to secrete them and their role in the disease. AIMS: To confirm by molecular means the identification of 187 clinical isolates (127 from blood, 52 from urine, and 8 from diverse clinical origins) phenotypically identified as C. tropicalis, and to investigate their in vitro aspartyl proteinase, phospholipase, esterase, hemolysin, DNase and coagulase activities. METHODS: The molecular confirmation was performed by ITS sequencing, and the enzymatic determinations were conducted using plate assays with specific substrates, with the exception of coagulase, which was determined by the classical tube test. RESULTS: The majority of the strains exhibited a very strong or strong activity of aspartyl proteinase, phospholipase and esterase. A 4.7% of the bloodstream isolates were hemolysin producers, and all were negative for the coagulase and DNase assays. CONCLUSIONS: Very strong activities of aspartyl proteinase, phospholipase and esterase profiles were detected, and a statistical association between phospholipase production and blood and urine isolates was found.
Assuntos
Candida tropicalis/isolamento & purificação , Candidíase/microbiologia , Líquidos Corporais/microbiologia , Candida tropicalis/enzimologia , Candida tropicalis/genética , Candidemia/microbiologia , DNA Fúngico/análise , Proteínas Fúngicas/análise , Humanos , Fenótipo , Análise de Sequência de DNA , Infecções Urinárias/microbiologiaRESUMO
Although hemolytic activity is known to be a putative virulence factor contributing to candidal pathogenesis, its production by Candida tropicalis, a species closely related to Candida albicans, is poor understood. The present study was undertaken to evaluate the hemolytic activity and the expression level of a putative haem oxygenase encoding gene by blood isolates of C. tropicalis following growth in iron deprivation, and in the presence of hemoglobin and erythrocytes. The lowest values of hemolytic activity were observed in cell-free culture supernatants of isolates growing in iron-restricted medium (RPMI medium and RPMI medium supplemented with iron chelator bathophenanthrolindisulphonic acid). Hemolysis was increased in the presence of either hemoglobin or erythrocytes. Reverse transcriptase PCR analysis showed that the putative haem oxygenase encoding gene (CtHMX1), potentially related with iron uptake, was up-regulated (p < 0.001) following growth in iron deprivation and in the presence of hemoglobin; CtHMX1 was repressed in the presence of human erythrocytes (p < 0.001). Our data suggest that hemoglobin had positive effect in the production of hemolytic factor and gene expression related to iron uptake in C. tropicalis.
Assuntos
Sangue/microbiologia , Candida tropicalis/enzimologia , Candida tropicalis/genética , Eritrócitos/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Hemoglobinas/metabolismo , Ferro/metabolismo , Candida tropicalis/crescimento & desenvolvimento , Candida tropicalis/ultraestrutura , Candidíase/sangue , Candidíase/microbiologia , Meios de Cultura , DNA Fúngico/isolamento & purificação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Fungos/crescimento & desenvolvimento , Proteínas Hemolisinas , Hemólise , Humanos , RNA Fúngico/isolamento & purificação , Regulação para Cima , Fatores de Virulência/metabolismoRESUMO
Objectives: Candidaemia is a public health problem mainly in hospitalized individuals worldwide. In Brazil, Candida albicans is the most prevalent species that causes candidaemia, followed by Candida tropicalis and Candida parapsilosis . Few data on the abundance of antifungal resistance are available for Latin America. Methods: We analysed the frequency of azole and echinocandin resistance in Candida isolates ( n = 75) collected between 2012 and 2014 at the University Hospital of Federal University of Juiz de Fora (Brazil). The primary targets erg11 (azoles) and fks1 (echinocandins) were sequenced and modelled at the protein level. Antifungal susceptibility testing was performed according to CLSI (M27-A3 and M27-S4) and according to EUCAST. Results: The three most frequent species were C. albicans (38.0%), C. tropicalis (30.0%) and Candida glabrata (17.0%). Azole resistance was observed in 27.0% of all Candida isolates, while 20.0% of all isolates were echinocandin resistant. A novel mutation in erg11 at location K143R was found to be associated with phenotypically pan-azole-resistant C. tropicalis isolates. This mutation maps near the active binding site of erg11 and is likely to confer pan-azole resistance to C. tropicalis . Conclusions: A novel point mutation (K143R) located in the erg11 gene of C. tropicalis was found in pan-azole-resistant strains. According to our protein homology model, it is very likely that the mutation K143R causes pan-azole resistance in C. tropicalis . Moreover, an up-regulation of ABC transporters was observed, which can add up to a pan-azole-resistant phenotype.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Mutação de Sentido Incorreto , Brasil , Análise Mutacional de DNA , Equinocandinas/farmacologia , Glucosiltransferases/genética , Hospitais Universitários , Humanos , Testes de Sensibilidade Microbiana , Mutação Puntual , Análise de Sequência de DNARESUMO
In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 µM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.
Assuntos
Candida albicans/enzimologia , Candida tropicalis/enzimologia , Inibidores Enzimáticos/química , Glucosiltransferases/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/química , Trealose/análogos & derivados , Candida albicans/patogenicidade , Candida tropicalis/patogenicidade , Candidíase/tratamento farmacológico , Candidíase/enzimologia , Inibidores Enzimáticos/farmacologia , Especificidade da Espécie , Fosfatos Açúcares/farmacologia , Trealose/química , Trealose/farmacologiaRESUMO
Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n = 1), as well as from aquatic mammals found dead in the environment (cetaceans, n = 5). The isolates were subjected to in vitro susceptibility testing by broth microdilution according to the CLSI M27-A3 protocol against amphotericin B, caspofungin, itraconazole, and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases, as well as biofilm formation. Resistance to itraconazole (n = 29) and fluconazole (n = 30) was detected among isolates from every source; resistance to both azoles was detected in 24 isolates, but none of them were resistant to amphotericin B and caspofungin. Protease production was detected in the majority of the isolates (n = 59), but phospholipase was produced by only a few of them (n = 6). The isolates showed different patterns in biofilm production, being considered strong producers (n = 41), moderate producers (n = 11), weak producers (n = 9) or non-producers (n = 3). In summary, C. tropicalis isolated from animals showed high rate of resistance to azoles, expressed virulence factors and therefore may represent a potential threat to human and animal health.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/enzimologia , Farmacorresistência Fúngica , Enzimas/análise , Fatores de Virulência/análise , Animais , Animais Domésticos , Animais Selvagens , Biofilmes/crescimento & desenvolvimento , Candida tropicalis/isolamento & purificação , Candida tropicalis/fisiologia , Testes de Sensibilidade MicrobianaRESUMO
There is growing interest in breeding rheas (Rhea americana) in Brazil. However, there are no data on the yeast microbiota of the gastrointestinal tract of this avian species, and the phenotypic characteristics of these yeasts are not known. Therefore, the aim of this work was to isolate Candida species from the digestive tract of rheas and to evaluate the in vitro antifungal susceptibility and secretion of phospholipases of the recovered isolates. For this purpose, 58 rheas from breeding operations in the cities of Fortaleza and Mossoró, north-eastern Brazil, were used. Samples were gathered from the oropharynx and cloaca of the animals using sterile swabs. Stool samples were collected from their pens by scraping with a scalpel blade. For the primary isolation, the material was seeded onto 2â% Sabouraud dextrose agar supplemented with chloramphenicol (0.5 g l(-1)). The isolates were identified based on morphological and biochemical features. After identification, all the strains were submitted to antifungal susceptibility testing for amphotericin B, itraconazole and fluconazole. The phospholipase activity of the Candida species isolates was also tested by culturing on egg yolk agar. Candida species were isolated from at least one anatomical site in 36/58 birds (14/17 juveniles and 22/41 adults) and in 6/10 faecal samples. Mostly, only a single species was isolated from each collection site (36/56 positive sites), with up to three species being observed only in four cases (4/56). A total of 77 isolates were obtained, belonging to the species Candida parapsilosis sensu lato (19), Candida albicans (18), Candida tropicalis (13), Candida guilliermondii (12), Candida krusei (10) and Candida famata (5). C. albicans was more prevalent in the oropharynx of the juvenile rheas when compared with adult ones (P<0.001). All tested isolates were susceptible to amphotericin B, but 16 isolates were simultaneously resistant to the two azole derivatives (11/18 C. albicans, 1/10 C. krusei, 2/19 C. parapsilosis sensu lato and 2/13 C. tropicalis). C. albicans presented a particularly high resistance rate to fluconazole (15/18) and itraconazole (13/18). Finally, 23/77 strains secreted phospholipases. In summary, healthy rheas carry potentially pathogenic Candida species in their gastrointestinal tract, including azole-resistant strains that secrete phospholipases, and are prone to disseminating them in the environment. Thus, breeding and handling these animals may have some implications for human and animal health.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida/isolamento & purificação , Farmacorresistência Fúngica , Reiformes/microbiologia , Animais , Brasil , Candida/classificação , Candida/efeitos dos fármacos , Candida/enzimologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/isolamento & purificação , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/enzimologia , Candida tropicalis/isolamento & purificação , Candidíase/microbiologia , Candidíase/transmissão , Cloaca/microbiologia , Reservatórios de Doenças , Humanos , Metagenoma , Testes de Sensibilidade Microbiana , Orofaringe/microbiologia , Fosfolipases/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND: Sterol biosynthesis is an essential pathway for fungal survival, and is the biochemical target of many antifungal agents. The antifungal drugs most widely used to treated fungal infections are compounds that inhibit cytochrome P450-dependent C14α-demethylase (CYP51), but other enzymes of this pathway, such as squalene synthase (SQS) which catalyses the first committed step in sterol biosynthesis, could be viable targets. The aim of this study was to evaluate the antifungal activity of SQS inhibitors on Candida albicans, Candida tropicalis and Candida parapsilopsis strains. METHODS: Ten arylquinuclidines that act as SQS inhibitors were tested as antiproliferative agents against three ATCC strains and 54 clinical isolates of Candida albicans, Candida tropicalis and Candida parapsilopsis. Also, the morphological alterations induced in the yeasts by the experimental compounds were evaluated by fluorescence and transmission electron microscopy. RESULTS: The most potent arylquinuclidine derivative (3-[1'-{4'-(benzyloxy)-phenyl}]-quinuclidine-2-ene) (WSP1267) had a MIC50 of 2 µg/ml for all species tested and MIC90 varying from 4 µg/ml to 8 µg/ml. Ultrathin sections of C. albicans treated with 1 µg/ml of WSP1267 showed several ultrastructural alterations, including (a) loss of cell wall integrity, (b) detachment of the plasma membrane from the fungal cell wall, (c) accumulation of small vesicles in the periplasmic region, (d) presence of large electron-dense vacuoles and (e) significantly increased cell size and cell wall thickness. In addition, fluorescence microscopy of cells labelled with Nile Red showed an accumulation of lipid droplets in the cytoplasm of treated yeasts. Nuclear staining with DAPI revealed the appearance of uncommon yeast buds without a nucleus or with two nuclei. CONCLUSION: Taken together, our data demonstrate that arylquinuclidine derivatives could be useful as lead compounds for the rational synthesis of new antifungal drugs.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Quinuclidinas/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Candida/enzimologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/enzimologia , Candidíase/microbiologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Humanos , Testes de Sensibilidade Microbiana , Quinuclidinas/síntese química , Quinuclidinas/químicaRESUMO
Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.
Assuntos
Candida albicans , Candida tropicalis , Candidíase Bucal/microbiologia , Adulto , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/isolamento & purificação , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/enzimologia , Candida tropicalis/isolamento & purificação , Contagem de Colônia Microbiana , Farmacorresistência Fúngica , Feminino , Fluconazol/farmacologia , Humanos , Itraconazol/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfolipases/metabolismoRESUMO
MATERIAL AND METHODS: Fifty nine elders wearing complete dentures and living in retirement homes in Curitiba (southern Brazil), were divided into two groups: group #1, 26 patients with denture-induced stomatitis and group #2, 33 patients without denture-induced stomatitis. The two groups were evaluated in relation to the degree of denture-induced stomatitis, salivary fungal loads, and secretion of some histolytic enzymes. RESULTS: Patients from group #1 showed higher degrees of colonisation by Candida albicans (p = 0.031). Candida krusei, Candida tropicalis, and Candida parapsilosis were also isolated, but there were no differences between the groups (p > 0.05). Secretory aspartyl protease (Sap) and chondroitinase did not show significant differences among the isolated Candida spp. in the two groups. Phospholipase secretion rates were higher among the strains of C. albicans from group #2 (p = 0.036). The same behaviour was not detected for non-albicans Candida species. CONCLUSIONS: The results could infer that differences in the secretion rates of candidal histolytic enzymes should not be imputed as imperative for the progress of denture-induced stomatitis.
Assuntos
Candida/enzimologia , Proteínas Fúngicas/fisiologia , Instituição de Longa Permanência para Idosos , Hidrolases/fisiologia , Aposentadoria , Estomatite sob Prótese/microbiologia , Idoso , Ácido Aspártico Endopeptidases/análise , Candida/classificação , Candida albicans/enzimologia , Candida tropicalis/enzimologia , Condroitinases e Condroitina Liases/análise , Contagem de Colônia Microbiana , Prótese Total/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Hidrolases/análise , Masculino , Fosfolipases/análise , Saliva/microbiologia , Fatores Sexuais , VirulênciaRESUMO
Yeasts can metabolize xylose by the action of two key enzymes: xylose reductase and xylitol dehydrogenase. In this work, we present data concerning the cloning of the XYL2 gene encoding xylitol dehydrogenase from the yeast Candida tropicalis. The gene is present as a single copy in the genome and is controlled at the transcriptional level by the presence of the inducer xylose. XYL2 was functionally tested by heterologous expression in Saccharomyces cerevisiae to develop a yeast strain capable of producing ethanol from xylose. Structural analysis of C. tropicalis xylitol dehydrogenase, Xyl2, suggests that it is a member of the medium-chain dehydrogenase (MDR) family. This is supported by the presence of the amino acid signature [GHE]xx[G]xxxxx[G]xx[V] in its primary sequence and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD(+) and zinc ion binding domains.