RESUMO
Punicalagin is a phenolic compound extracted from Lafoensia pacari A. St.-Hil (Lythraceae) leaves. It has demonstrated interesting activity against pathogenic fungi, e.g., Cryptococcus gattii and Candida albicans, by inhibiting fungi growth in a minimum inhibitory concentration (MIC) at 4 µg/mL. However, the mechanisms behind its antifungal action are not well understood. In this study, certain parameters were investigated, by transmission electron microscopy, ergosterol synthesis inhibition, and flow cytometry analyses, to gain insight into the possible biological targets of punicalagin (4 or 16 µg/mL) against yeast cells. Data showed that, in contrast to untreated cells, punicalagin triggered severe ultrastructural changes in C. gattii and C. albicans, such as disorganization of cytoplasmic content and/or thickened cell walls. In addition, it caused a decrease in yeast plasma membrane ergosterol content in a concentration-dependent manner. However, it was unable to bring about significant fungal cell membrane rupture. On the other hand, punicalagin (16 µg/mL) significantly arrested C. albicans and C. gattii cells at the G0/G1 phase, with a consequent reduction in cells at the G2/M phase in both fungi isolates, and thereby prevented progression of the normal yeast cell cycle. However, these alterations showed no involvement of reactive oxygen species overproduction in C. albicans and C. gattii cells, although punicalagin triggered a significant loss of mitochondrial membrane potential in C. albicans. These findings suggest that punicalagin is a promising plant-derived compound for use in developing new antifungal therapies.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cryptococcus gattii/efeitos dos fármacos , Ergosterol/metabolismo , Taninos Hidrolisáveis/farmacologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/metabolismo , Cryptococcus gattii/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de TransmissãoRESUMO
INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.
Assuntos
Antifúngicos/farmacologia , Candida albicans/patogenicidade , Unhas/microbiologia , Onicomicose/microbiologia , Fatores de Virulência , Ácido Aspártico Proteases/biossíntese , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Farmacorresistência Fúngica , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Fosfolipases/biossíntese , Reação em Cadeia da PolimeraseRESUMO
Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.
Assuntos
Humanos , Candida albicans/patogenicidade , Onicomicose/microbiologia , Fatores de Virulência , Antifúngicos/farmacologia , Unhas/microbiologia , Fosfolipases/biossíntese , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Microscopia Eletrônica de Varredura , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Fúngica , Ácido Aspártico Proteases/biossíntese , HemóliseRESUMO
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
Assuntos
Acanthamoeba castellanii/metabolismo , Fungos/patogenicidade , Lectina de Ligação a Manose/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/microbiologia , Acanthamoeba castellanii/ultraestrutura , Animais , Candida albicans/patogenicidade , Candida albicans/ultraestrutura , Concanavalina A/metabolismo , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Histoplasma/patogenicidade , Histoplasma/ultraestrutura , Interações Hospedeiro-Patógeno , Larva/microbiologia , Lepidópteros/microbiologia , Manose/química , Manose/metabolismo , Lectina de Ligação a Manose/química , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Saccharomyces cerevisiae/patogenicidade , Saccharomyces cerevisiae/ultraestrutura , Fatores de Tempo , Imagem com Lapso de Tempo , Virulência , Fatores de Virulência/metabolismoRESUMO
Candida albicans is responsible for the majority of nosocomial infections affecting immunocompromised patients. Systemic antifungals may promote microbial resistance, which has led to the search for alternative treatments, such as photothermal therapy (PTT). PTT assumes that the interaction of electromagnetic radiation with a photothermal agent generates heat that can lead to the destruction of tumor cells and the death of microorganisms. Carbon nanotubes (CNTs) have the potential for applications in biomedical systems, including acting as controlled deliverers of drugs, biosensors and scaffolds for tissue engineering and regenerative medicine. Furthermore, the absorption of radiation by CNTs in the infrared region induces an increase in temperature, which makes CNTs candidates for photothermal agents. In this work, the photothermal inactivation of C. albicans was evaluated by multiple wall CNTs associated with laser radiation in the near-infrared region. The mechanisms that are involved in inactivation were evaluated through cell susceptibility studies and an analysis of microscopic images that are associated with mathematical models and fractal concepts. The results indicate that direct contact between the cells and CNTs without irradiation does not lead to cell death, whereas the laser-mediated process is effective in inactivation. The application of the laws of scale and fractal concepts indicate that in the control groups, there are two distinct regimes that are delimited by the mean diameter of the microorganisms, as described by the Eden model and by the quasi-Euclidean surface. For the irradiated groups, the surfaces present only one regime described by Kardar-Parisi-Zhang, KPZ. The analysis of the fractality of the system by mathematical models can help in the identification of new strategies for the inactivation of microorganisms.
Assuntos
Candida albicans/efeitos da radiação , Fractais , Luz , Modelos Teóricos , Nanotubos de Carbono/química , Temperatura , Candida albicans/ultraestrutura , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos da radiaçãoRESUMO
The pomegranate (Punica granatum) sarcotesta contains a chitin-binding lectin (PgTeL) with antibacterial activity against human pathogenic species. In this work, the structural stability of PgTeL was evaluated by fluorimetric analysis and the lectin was evaluated for cytotoxicity to human peripheral blood mononuclear cells (PBMCs) and antifungal activity against Candida albicans and Candida krusei. PgTeL folding was impaired when lectin was incubated at pH≥6.0. On the other hand, the lectin did not undergo unfolding even when heated at 100°C. PgTeL (1, 10, and 100µg/mL) was not cytotoxic to PBMCs. Antifungal activity was detected for C. albicans (MIC: 25µg/mL; MFC: 50µg/mL) and C. krusei (MIC and MFC of 12.5µg/mL). Treatment of yeast cells with PgTeL resulted in decrease of intracellular ATP content even at sub-inhibitory concentrations (½MIC and »MIC) and induced lipid peroxidation. In addition, PgTeL damaged the integrity of fungal cell wall of both species, with more pronounced effects in C. krusei. The lectin showed significant antibiofilm activity on C. albicans at sub-inhibitory concentrations (0.195 and 0.39µg/mL). In conclusion, PgTeL is an anti-Candida agent whose action mechanism involves oxidative stress, energetic collapse, damage to the cell wall and rupture of yeast cells.
Assuntos
Candida albicans/efeitos dos fármacos , Candida/efeitos dos fármacos , Lectinas/farmacologia , Lythraceae/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/metabolismo , Candida/ultraestrutura , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Parede Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Lectinas/química , Lectinas/isolamento & purificação , Peroxidação de Lipídeos/efeitos dos fármacos , Testes de Sensibilidade Microbiana , TemperaturaRESUMO
Vulvovaginal candidiasis (VVC) in HIV-infected (HIV+) women is a serious public health problem. However, little is known about the virulence mechanisms of vaginal Candida albicans from HIV+ women in the post-highly active antiretroviral therapy (HAART) era. Here, we report a comparative analysis of the expression of key virulence factors and genetic variability of 26 vaginal C. albicans strains isolated from HIV+ women undergoing HAART and 18 from HIV-uninfected (HIV-) women. In general, we observed that C. albicans from HIV+ women receiving HAART showed lower expression of virulence factors compared with C. albicans from HIV- women, except for the proteinase activity which is highly expressed. The results in HIV-women further suggest that virulence factors appear to be expressed in response to the yeast stress, in the presence of an adequate immune response. Furthermore, the RAPD results showed a high heterogeneity among isolates from both groups of women. These findings in HIV+ women using HAART will help to improve the monitoring of vaginal yeast infections and the quality of life of patients.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase Vulvovaginal/microbiologia , Fatores de Virulência/genética , Terapia Antirretroviral de Alta Atividade , Biofilmes/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Candida albicans/ultraestrutura , Feminino , Variação Genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Microscopia Eletrônica de VarreduraRESUMO
The aim of this study was to increase the solubility of gallic acid (GA) for the treatment of Candida albicans biofilm, which is very difficult to treat and requires high drug concentrations. Cyclodextrins (CDs) were used for this purpose. Complexes were evaluated by phase-solubility studies, prepared by spray drying and characterized by drug loading, scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The complexes were tested on C. albicans biofilm using in vitro and in vivo models. HPßCD formed soluble inclusion complexes with GA. The percentage of GA in GA/HPßCD was 10.8 ± 0.01%. The SEM and DSC analyses confirmed the formation of inclusion complexes. GA/HPßCD maintained the antimicrobial activity of the pure GA. GA/HPßCD was effective on C. albicans biofilms of 24 and 48h. The in vivo results showed an anti-inflammatory activity of GA/HPßCD with no difference in invading hypha counting among the groups. This study encourages the development of new antifungal agents.
Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Ácido Gálico/química , Ácido Gálico/farmacologia , beta-Ciclodextrinas/química , 2-Hidroxipropil-beta-Ciclodextrina , Varredura Diferencial de Calorimetria , Candida albicans/ultraestrutura , Ciclodextrinas/química , Microscopia Eletrônica de Varredura , SolubilidadeRESUMO
Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.
Assuntos
Biofilmes , Candida albicans/fisiologia , Candida albicans/ultraestrutura , Microscopia Eletrônica de Varredura , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Criopreservação/métodos , Cromatografia Gasosa-Espectrometria de Massas , Microscopia Eletrônica de Varredura/métodos , TemperaturaRESUMO
Candida albicans biofilms play a key role in denture stomatitis, one of the most common oral pathologies in elderly people. Because biofilms are highly resistant to antifungals, new pharmacological strategies are needed. Aspirin and nitric oxide-donor molecules have both shown antibiofilm effects on C. albicans, making them promising candidates for treatment. In this study, we evaluated the antifungal/antibiofilm effect of a nitric-oxide releasing aspirin (NO-ASA) on C. albicans isolates from denture stomatitis patients in vitro. Disk diffusion assays showed that while NO-ASA had no antifungal effect, the drug potentiated fluconazole inhibition zone diameters, increasing the effect of fluconazole by 20-30% (p<0.05). The effect of NO-ASA on the morphogenesis of C. albicans was evaluated using light microscopy after inducing hyphae formation. For all clinical strains assayed, 125 µM NO-ASA significantly decreased the number of filamentous cells present (p<0.01). Adhesion to abiotic surfaces, a critical event for biofilm formation, was evaluated in 96-well polystyrene plates using crystal violet assay; 125 µM NO-ASA significantly inhibited adhesion. Biofilms were observed with scanning electron microscopy (SEM) and quantified using XTT reduction assay. NO-ASA decreased biofilm formation (IC50 ranging from 300 µM to 700 µM), consistent with SEM findings of altered biofilm microarchitecture. PGE2 and carboxy-PTIO (an NO scavenger) both blocked the antibiofilm effects of NO-ASA, suggesting that the efficacy of NO-ASA may be associated with both inhibition of PGE2 synthesis and release of NO. NO-ASA is a promising novel antibiofilm agent for treating fluconazole-resistant strains of C. albicans.
Assuntos
Aspirina/análogos & derivados , Biofilmes/efeitos dos fármacos , Candida albicans/isolamento & purificação , Nitrocompostos/farmacologia , Estomatite sob Prótese/microbiologia , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspirina/farmacologia , Aspirina/uso terapêutico , Aderência Bacteriana/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Dinoprostona/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Fluconazol/uso terapêutico , Sequestradores de Radicais Livres/farmacologia , Humanos , Concentração Inibidora 50 , Viabilidade Microbiana/efeitos dos fármacos , Nitrocompostos/uso terapêutico , Estomatite sob Prótese/tratamento farmacológicoRESUMO
CONTEXT: Buchenavia tetraphylla (Aubl.) RA Howard (Combretaceae: Combretoideae) is an ethnomedicinal plant with reported antifungal action. OBJECTIVE: This study evaluates the antimicrobial activity of B. tetraphylla leaf extracts against clinical isolates of Candida albicans. The morphological alterations, combinatory effects with fluconazole and the cytotoxicity of the active extract were analyzed. MATERIALS AND METHODS: Extracts were obtained using different solvents (hexane: BTHE; chloroform: BTCE; ethyl acetate: BTEE; and methanol: BTME). Antimicrobial activity was determined by the broth microdilution method using nine strains of C. albicans isolated from vaginal secretions and one standard strain (UFPEDA 1007). RESULTS: All extracts showed anti-C. albicans activity, including against the azole-resistant strains. The MIC values ranged from 156 to 2500 µg/mL for the BTHE; 156 to 1250 µg/mL for the BTCE; 625 to 1250 µg/mL for the BTME and 625 µg/mL to 2500 µg/mL for the BTEE. BTME showed the best anti-C. albicans activity. This extract demonstrated additive/synergistic interactions with fluconazole. Scanning electron microscopy analysis suggested that the BTME interferes with the cell division and development of C. albicans. BTME showed IC50 values of 981 and 3935 µg/mL, against J774 macrophages and human erythrocytes, respectively. This extract also enhanced the production of nitric oxide by J774 macrophages. DISCUSSION AND CONCLUSION: Buchenavia tetraphylla methanolic extract (BTME) is a great source of antimicrobial compounds that are able to enhance the action of fluconazole against different C. albicans strains; this action seems related to inhibition of cell division.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Combretaceae/química , Extratos Vegetais/farmacologia , Vagina/microbiologia , Animais , Antifúngicos/isolamento & purificação , Antifúngicos/toxicidade , Candida albicans/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Candida albicans/ultraestrutura , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Fúngica , Quimioterapia Combinada , Feminino , Fluconazol/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Testes de Sensibilidade Microbiana , Óxido Nítrico/metabolismo , Fitoterapia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/toxicidade , Plantas Medicinais , Solventes/química , Vagina/metabolismoRESUMO
Available treatments against human fungal pathogens present high levels of resistance, motivating the development of new antifungal therapies. In this context, the present work aimed to analyze direct electric current (DC) antifungal action, using an in vitro apparatus equipped with platinum electrodes. Candida albicans yeast cells were submitted to three distinct conditions of DC treatment (anodic flow-AF; electroionic flow-EIF; and cathodic flow-CF), as well as different charges, ranging from 0.03 to 2.40 C. Our results indicated C. albicans presented distinct sensibility depending on the DC intensity and polarity applied. Both the colony-forming unit assay and the cytometry flow with propidium iodide indicated a drastic reduction on cellular viability after AF treatment with 0.15 C, while CF- and EIF-treated cells stayed alive when DC doses were increased up to 2.40 C. Additionally, transmission electron microscopy revealed important ultrastructural alterations in AF-treated yeasts, including cell structure disorganization, ruptures in plasmatic membrane, and cytoplasmic rarefaction. This work emphasizes the importance of physical parameters (polarity and doses) in cellular damage, and brings new evidence for using electrotherapy to treat C. albicans pathology process. Bioelectromagnetics. 38:95-108, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Candida albicans/citologia , Eletricidade , Candida albicans/metabolismo , Candida albicans/fisiologia , Candida albicans/ultraestrutura , Adesão Celular , Linhagem Celular , Eletrodos , Células Epiteliais/citologia , Humanos , Masculino , Viabilidade Microbiana , Pessoa de Meia-Idade , Platina/química , Propídio/metabolismoRESUMO
The main secondary metabolite of Senecio nutans is 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone (4HMBA). The antifungal activity of this compound and three derivatives was assessed using Candida albicans. 4HMBA exhibited the highest antifungal activity among the assayed compounds. The Fractional Inhibitory Concentration (FIC = 0.133) indicated a synergistic fungicidal effect of 4HMBA (5 mg L(-1)) and fluconazole (FLU) (0.5 mg L(-1)) against the C. albicans reference strain (ATCC 10231). Microscopy showed that 4HMBA inhibits filamentation and reduces cell wall thickness. Our findings suggest that 4HMBA is an interesting compound to diminish resistance to commercial fungistatic drugs such as fluconazole.
Assuntos
Acetofenonas/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Acetofenonas/química , Antifúngicos/química , Candida albicans/ultraestrutura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Relação Dose-Resposta a Droga , Viabilidade Microbiana/efeitos dos fármacosRESUMO
Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.
Assuntos
Biofilmes , Candida albicans/fisiologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Interações Microbianas , Staphylococcus aureus/fisiologia , Aderência Bacteriana , Biomassa , Candida albicans/ultraestrutura , Espaço Extracelular/enzimologia , Hidrólise , Metaboloma , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Mitocôndrias/metabolismo , Staphylococcus aureus/ultraestruturaRESUMO
AIM: Confocal laser-scanning microscopy (CLSM) was carried out to investigate the exopolysaccharide matrix of Candida albicans (C. albicans) biofilms developed on denture material under dietary carbohydrate exposure. METHODS: Biofilms were developed on poly(methyl methacrylate) discs in culture media without (control) or with supplementation by glucose or sucrose for 72 h. For the CLSM analysis, biofilms were labeled with concanavalin A (ConA) during its development. Afterwards, biofilms were also labeled with SYTO-9. To confirm the results, the matrix was investigated by the phenol-sulfuric method. Data were analyzed by anova, followed by Tukey's test, with the level of significance set at 5%. RESULTS: The use of ConA during biofilm development provided effective labeling of the exopolysaccharide matrix. The exposure to sucrose resulted in biofilms with the highest exopolysaccharide matrix biovolume (P < 0.05). The characterization obtained by CLSM was confirmed by the phenol-sulfuric method. CONCLUSION: Confocal laser-scanning microscopy was found to be an effective tool for investigating the exopolysaccharide matrix of C. albicans biofilms, and exposure to sucrose resulted in increased matrix production.
Assuntos
Biofilmes , Candida albicans/ultraestrutura , Polissacarídeos Fúngicos/ultraestrutura , Candida albicans/química , Candida albicans/metabolismo , Concanavalina A , Meios de Cultura , Materiais Dentários/química , Polissacarídeos Fúngicos/análise , Glucose/metabolismo , Humanos , Microscopia Confocal/métodos , Compostos Orgânicos , Polimetil Metacrilato/química , Distribuição Aleatória , Saliva/microbiologia , Coloração e Rotulagem , Sacarose/metabolismo , Propriedades de SuperfícieRESUMO
OBJECTIVE: The purpose of this study was to investigate the in vitro activity of terpene blends combined with tissue conditioner against Candida albicans and the effect on its morphology and sub-micro structure. STUDY DESIGN: The minimal inhibitory concentration (MIC) of terpenes, obtained from a by-product of kraft pulping, was determined using broth microdilution against C. albicans strains, and the activity of terpenes combined with Coe-Comfort tissue conditioner was assessed. Cell morphologic alterations were evaluated using scanning electronic microscopy and transmission electronic microscopy. Data was analyzed using Student's t test P < .05. RESULTS: The MIC of terpene blends fluctuated between 0.097% and 0.39% (v/v). Coe-Comfort tissue conditioner mixed with terpenes exhibited a total inhibition of C. albicans (P < .05). Terpenes induced ultrastructural alterations, even at the MIC value, including an increase in size, shape modification, cell wall damage with perforations, pronounced disconnection between cell wall and cytoplasm, and cytoplasmic vacuoles. CONCLUSIONS: Terpenes had pronounced effects against C. albicans alone and in combination with Coe-Comfort tissue conditioner, which mainly resulted in cell wall damage.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Terpenos/farmacologia , Resinas Acrílicas/farmacologia , Chile , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ácidos Ftálicos/farmacologiaRESUMO
Candida albicans is the most common fungal pathogen in humans, and recently some studies have reported the antifungal activity of silver nanoparticles (AgNPs) against some Candida species. However, ultrastructural analyses on the interaction of AgNPs with these microorganisms have not been reported. In this work we evaluated the effect of AgNPs on C. albicans, and the minimum inhibitory concentration (MIC) was found to have a fungicidal effect. The IC50 was also determined, and the use of AgNPs with fluconazole (FLC), a fungistatic drug, reduced cell proliferation. In order to understand how AgNPs interact with living cells, the ultrastructural distribution of AgNPs in this fungus was determined. Transmission electron microscopy (TEM) analysis revealed a high accumulation of AgNPs outside the cells but also smaller nanoparticles (NPs) localized throughout the cytoplasm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of intracellular silver. From our results it is assumed that AgNPs used in this study do not penetrate the cell, but instead release silver ions that infiltrate into the cell leading to the formation of NPs through reduction by organic compounds present in the cell wall and cytoplasm.
Assuntos
Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Nanopartículas Metálicas/administração & dosagem , Compostos de Prata/administração & dosagem , Prata , Antifúngicos/química , Antifúngicos/farmacologia , Humanos , Nanopartículas Metálicas/química , Testes de Sensibilidade Microbiana , Prata/químicaRESUMO
OBJECTIVES: The aim of this study was to evaluate the effect of SLPI on the growth and biological processes of Candida albicans. METHODS: Two C. albicans strains were used in this study, a clinical isolate resistant to fluconazole (PRI) and a reference strain ATCC 24433. The minimal inhibitory concentration (MIC) was determined according to the CLSI methodology. The influence of SLPI on secreted serine proteinase activities (SSP) was measured by the cleavage of specific substrate, and surface hydrophobicity was determined by the aqueous-hydrocarbon biphasic separation method. Flow cytometry was performed to investigate receptors for SLPI and variations in the cell wall mannoprotein expression. Interaction between yeast and epithelium was assessed using the MA-104 cells lineage. Ultrastructure was analyzed by transmission electron microscopy (TEM). RESULTS: MIC values were calculated as 18 and 18.9µM for the PRI and ATCC 24433, respectively. SSP activity was reduced by 48.8% by 18µM of SLPI and cell surface hydrophobicity increased by 11.1%. Flow cytometry suggest the existence of SLPI binding sites on the surface of the yeast. Results showed a reduction in the expression of mannoproteins in 20.8% by the cells treated with 80µM of SLPI, and 18µM reduced the adhesion of yeasts to mammalian cells in 60.1%. TEM revealed ultrastructural changes in cells treated with 80µM of SLPI, such as the presence of membrane-like structures within the cytoplasm. CONCLUSIONS: SLPI exerts a significant influence on C. albicans viability and biological processes. Considering its constitutive and physiologic features, SLPI may become a promising tool for the development of new methodologies for the treatment and control of candidiasis.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Inibidor Secretado de Peptidases Leucocitárias/farmacologia , Fenômenos Biológicos/efeitos dos fármacos , Candida albicans/ultraestrutura , Adesão Celular/efeitos dos fármacos , Farmacorresistência Fúngica , Citometria de Fluxo , Fluconazol/farmacologia , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Microscopia Eletrônica , Nistatina/farmacologiaRESUMO
Scanning electron microscope (SEM) observations were used to analyze particular morphologies of Candida albicans clinical isolate (strain 82) and mutants defective in hyphae-promoting genes EFG1 (strain HLC52) and/ or CPH1 (strains HLC54 and Can16). Transcription factors Efg1 and Cph1 play role in regulating filamentation and adhesion of C. albicans' morphologies. Comparative analysis of such mutants and clinical isolate showed that Efg1 is required for human serum-induced cell growth and morphological switching. In the study, distinct differences between ultrastructural patterns of clinical strain's and null mutants' morphologies were observed (spherical vs tube-like blastoconidia, or solid and fragile constricted septa vs only the latter observed in strains with EFG1 deleted). In addition, wild type strain displayed smooth colonies of cells in comparison to mutants which exhibited wrinkled phenotype. It was observed that blastoconidia of clinical strain exhibited either polarly or randomly located budding. Contrariwise, morphotypes of mutants showed either multiple polar budding or a centrally located single bud scar (mother-daughter cell junction) distinguishing tube-like yeast/ pseudohyphal growth (the length-to-width ratios larger than 1.5). In their planktonic form of growth, blastoconidia of clinical bloodstream isolate formed constitutively true hyphae under undiluted human serum inducing conditions. It was found that true hyphae are essential elements for developing structural integrity of conglomerate, as mutants displaying defects in their flocculation and conglomerate-forming abilities in serum. While filamentation is an important virulence trait in C. albicans the true hyphae are the morphologies which may be expected to play a role in bloodstream infections.
Assuntos
Humanos , Candida albicans/ultraestrutura , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Candidíase/microbiologia , Deleção de Genes , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/ultraestrutura , Microscopia Eletrônica de VarreduraRESUMO
Biofilms formed by Candida albicans, a human pathogen, are known to be resistant to different antifungal agents. Novel strategies to combat the biofilm associated Candida infections like multiple drug therapy are being explored. In this study, potential of chloroquine to be a partner drug in combination with four antifungal agents, namely fluconazole, voriconazole, amphotericin B, and caspofungin, was explored against biofilms of C. albicans. Activity of various concentrations of chloroquine in combination with a particular antifungal drug was analyzed in a checkerboard format. Growth of biofilm in presence of drugs was analyzed by XTT-assay, in terms of relative metabolic activity compared to that of drug free control. Results obtained by XTT-metabolic assay were confirmed by scanning electron microscopy. The interactions between chloroquine and four antifungal drugs were determined by calculating fractional inhibitory concentration indices. Azole resistance in biofilms was reverted significantly (p < 0.05) in presence of 250 µg/mL of chloroquine, which resulted in inhibition of biofilms at very low concentrations of antifungal drugs. No significant alteration in the sensitivity of biofilms to caspofungin and amphotericin B was evident in combination with chloroquine. This study for the first time indicates that chloroquine potentiates anti-biofilm activity of fluconazole and voriconazole.