RESUMO
Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) have been frequently associated with bacterial resistance mechanisms. These mechanisms, in turn, restrict a range of therapeutic opportunities for the treatment of infections caused by these micro-organisms. Faced with this problem, the present study aims to isolate and characterize molecules with antimicrobial activity derived from the fungus Penicillium citrinum isolated from Cerrado soil. Furthermore, we also tested possible antibacterial potential alone and in combination with commercial antimicrobial agents. In this context, citrinin was isolated and characterized by nuclear magnetic resonance and electrospray ionization. Functional analyses showed MIC of 128 µg ml-1 against S. aureus ATCC 25923, E. faecalis ATCC 29212 and a clinical isolate of vancomycin-resistant E. faecium (VRE01). However, for a clinical strain of methicillin-resistant S. aureus (MRSA01), the MIC was 256 µg ml-1. In order to avoid such high concentrations and reduce the collateral effects, additive effects were evidenced by combining citrinin with cefoxitin against MRSA01 (FIC index=0.5) and also citrinin with vancomycin toward VRE01 (FIC index=0.5). In vivo studies with BALB/c-tipe mice (MRSA assay) demonstrated a clinical ineffectiveness of cefoxitin associated with citrinin (9.8 mg kg-1 of cefoxitin +0.2 mg kg-1 of citrinin), with this combination being inefficient to increase animal survival. However, the combination used in the treatment of VRE (23.5 mg kg-1 of citrinin +1.5 mg kg-1 of vancomycin) sepsis model was extremely promising, leading to an animal survival rate of 80 percent. In summary, our data show, for the first time, the possible successful use of citrinin associated with vancomycin for pathogenic bacteria control.
Assuntos
Antibacterianos/farmacologia , Citrinina/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Penicillium/química , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Vancomicina/farmacologia , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/toxicidade , Cefoxitina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citrinina/isolamento & purificação , Citrinina/toxicidade , Sinergismo Farmacológico , Feminino , Dose Letal Mediana , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Penicillium/citologia , Células RAW 264.7 , Testes de ToxicidadeRESUMO
A comparative study on the lipase-producing fungus Penicillium simplissicimum, grown on a tray type solid-state fermentation (SSF) bioreactor, was performed using stereoscopy, focus Z-stacking stereoscopic images, field emission scanning electron microscopy (FE-SEM) and environmental scanning electron microscopy (ESEM) to better characterize the morphology of filamentous fungi in SSF and their distribution over the solid matrix. The imaging of live fungal samples using a stereomicroscope with focus Z-stacking showed differences in colonization between a static SSF and an intermittent agitated SSF. A comparison of the stereomicroscopy, hi-vacuum and environmental scanning electron microscopy results obtained using different protocols for sample processing showed that fixation with osmium tetroxide vapor and subsequent hydrated imaging is the best combination of sample preparation and imaging conditions for keeping the arrangement of the aerial hyphae and conidia morphology closer to the natural state. These combined methodologies can be applied in the SSF of fungal growth to characterize the formation of conidiophores over time, the conidia morphology and the spatial organization after their release from conidiophores. Mycelium colonization over the matrix, which is an important characteristic related to the production of different biotechnological products, could be observed and provide more knowledge about fungal physiology behavior during SSF.
Assuntos
Fermentação , Fungos/crescimento & desenvolvimento , Fungos/ultraestrutura , Penicillium/crescimento & desenvolvimento , Penicillium/ultraestrutura , Fungos/citologia , Fungos/fisiologia , Hifas/ultraestrutura , Microscopia/instrumentação , Microscopia/métodos , Microscopia Eletrônica de Varredura/métodos , Micélio/ultraestrutura , Penicillium/citologia , Penicillium/fisiologia , Esporos Fúngicos/ultraestrutura , Propriedades de SuperfícieRESUMO
ß-carbolines (ßCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six ßCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic ßCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-ßCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.
Assuntos
Botrytis/efeitos dos fármacos , Carbolinas/farmacologia , Fungicidas Industriais/farmacologia , Penicillium/efeitos dos fármacos , Botrytis/citologia , Botrytis/ultraestrutura , Citrus/microbiologia , Frutas/microbiologia , Germinação/efeitos dos fármacos , Harmina/análogos & derivados , Harmina/farmacologia , Testes de Sensibilidade Microbiana , Micélio/efeitos dos fármacos , Penicillium/citologia , Penicillium/ultraestrutura , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/fisiologiaRESUMO
Self-assembly of nanoparticles on living biotemplate surfaces is a promising route to fabricate nano- or microstructured materials with high efficiency and efficacy. We used filamentous fungi to fabricate microtubules of gold nanoparticles through a novel approach that consists of isolating the hyphal growth from the nanoparticle media. This improved methodology resulted in better morphological control and faster adsorption kinetics, which reduced the time needed to form homogeneous microtubules and allowed for control of microtubule thickness through successive additions of nanoparticles. Differences in the adsorption rates due to modifications in the chemical identity of colloidal gold nanoparticles indicated the influence of secondary metabolites and growth media in the fungi metabolism, which demonstrated the need to choose not only the fungus biotemplate but also the correct medium to obtain microtubules with superior properties.
Assuntos
Fungos/citologia , Ouro/química , Nanopartículas Metálicas/química , Microtúbulos , Aspergillus/química , Aspergillus/citologia , Fungos/química , Microscopia Eletrônica de Transmissão e Varredura , Microtúbulos/química , Penicillium/química , Penicillium/citologia , Difração de Raios X , Xylariales/química , Xylariales/citologiaRESUMO
The effect of different carbon sources on morphology and cellulase and xylanase production of Penicillium echinulatum was evaluated in this work. Among the six carbon sources studied, cellulose and sugar cane bagasse were the most suitable for the production of filter paper activity, endoglucanases, xylanases, and ß-glucosidases. However, sucrose and glucose showed ß -glucosidase activities similar to those obtained with the insoluble sources. The polyacrylamide gels proved the enzymatic activity, since different standards bands were detected in the media mentioned above. Regarding morphology, it was observed that the mycelium in a dispersed form provided the greatest enzymatic activity, possibly due to greater interaction between the substrate and hyphae. These data are important in understanding the physiology of fungi and could contribute to obtaining enzyme with potential application in the technology of second generation ethanol.
Assuntos
Carbono/química , Celulase/biossíntese , Meios de Cultura/química , Endo-1,4-beta-Xilanases/biossíntese , Proteínas Fúngicas/biossíntese , Penicillium/citologia , Penicillium/enzimologia , Celulose/química , Saccharum/químicaRESUMO
A number of parameters, including culture medium pH, affect growth and enzyme production by microorganisms. In the present study, the production and secretion of pectin lyase (PL) and polygalacturonase (PG) by recombinant strains of Penicillium griseoroseum cultured in mineral-buffered media (MBM; initial pH 6.8) and mineral-unbuffered medium (MUM; initial pH 6.3) were evaluated. Under these culture conditions, no change in the transcriptional levels of plg1 and pgg2 was observed. However, the levels of secreted total protein ranged from 7.80 ± 1.1 to 3.25 ± 1.50 µg ml(-1) in MBM and MUM, respectively, and were evaluated by SDS-PAGE. PL and PG enzymatic activities decreased 6.4 and 3.6 times, respectively, when P. griseoroseum was cultivated under acidic pH conditions (MUM). Furthermore, differences were observed in the hypha and mycelium morphology. These findings suggest that acidic growing conditions affect PL and PG secretion, even though the transcription and translation processes are successful. The data obtained in this study will help to establish optimal culture conditions that increase production and secretion of recombinant proteins by filamentous fungi.
Assuntos
Proteínas Fúngicas/metabolismo , Penicillium/metabolismo , Poligalacturonase/metabolismo , Proteínas Fúngicas/biossíntese , Concentração de Íons de Hidrogênio , Organismos Geneticamente Modificados , Penicillium/citologia , Penicillium/genética , Poligalacturonase/biossíntese , Polissacarídeo-Liases/biossíntese , Prostaglandinas G/genética , Prostaglandinas G/metabolismoRESUMO
There is a worldwide interest in the development of processes for colorants production from natural sources such as microorganism. The aim of this study was to optimize red colorants production by Penicillium purpurogenum DPUA 1275 and to evaluate the effect of pH, temperature, salts and polymers on the stability of these colorants. Under optimized conditions, a 78% increase in red colorants production was achieved. The best pH and temperature conditions were obtained at pH 8.0 and 70°C, respectively. In the presence of salts NaCl and Na2 SO4 , both at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 8.0), the red colorants showed good stability. In the presence of both polymers polyethylene glycol and sodium polyacrylate, the red colorants kept their color intensity. Thus, this study presents characteristics of red colorants produced by P. purpurogenum that can be applied in different industries after toxicological examination.
Assuntos
Técnicas de Cultura de Células/métodos , Penicillium/química , Penicillium/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Penicillium/citologia , Pigmentos Biológicos/análise , Cloreto de Sódio , Sulfatos , TemperaturaRESUMO
The combined effects of pH and temperature on red pigment production and fungal morphology were evaluated in a submerged culture of Penicillium purpurogenum GH2, using Czapek-Dox media with d-xylose as a carbon source. An experimental design with a factorial fix was used: three pH values (5, 7, and 9) and two temperature levels (24 and 34 °C) were evaluated. The highest production of red pigment (2.46 g/L) was reached with a pH value of 5 and a temperature of 24 °C. Biomass and red pigment production were not directly associated. This study demonstrates that P. purpurogenum GH2 produces a pigment of potential interest to the food industry. It also shows the feasibility of producing and obtaining natural water-soluble pigments for potential use in food industries. A strong combined effect (p<0.05) of pH and temperature was associated with maximal red pigment production (2.46 g/L).
Assuntos
Penicillium/química , Penicillium/metabolismo , Pigmentos Biológicos/biossíntese , Adaptação Fisiológica/fisiologia , Tamanho Celular , Concentração de Íons de Hidrogênio , Penicillium/citologia , TemperaturaRESUMO
In recent decades, beta-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 degrees C, under stationary condition for six days. The optimum activity temperature was 60 degrees C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 degrees C, with half-life of 240 and 232min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The beta-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V(max) and K(m) values was 0.48micromol PNXPmin(-1)mg(-1) protein and 0.75mM, respectively. The enzyme was xylose tolerant with a K(i) of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.
Assuntos
Penicillium/citologia , Penicillium/enzimologia , Xilosidases/biossíntese , Agricultura , Biotecnologia , Carbono/farmacologia , Meios de Cultura , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Resíduos Industriais , Cinética , Penicillium/efeitos dos fármacos , Temperatura , Fatores de Tempo , Xilose/farmacologia , Xilosidases/antagonistas & inibidoresRESUMO
We have examined beta-D-galactofuranosidase production by Penicillium fellutanum in the presence of D-galactono-1,4-lactone or 4-aminophenyl 1-thio-beta-D-galactofuranoside, two potent in vitro inhibitors of the enzyme. Activity of the enzyme in the culture filtrate was increased by 35% when glucose was replaced by D-galactose as the carbon source, and the activity diminished 80% of the control value when the inhibitors were added. Significant alterations of the culture were observed: (a) the medium became increasingly opalescent due to the secretion of a protein aggregate (PA) which contained 15% neutral sugar, mainly ribose; (b) the peptidophosphogalactomannan (pPGM) containing galactofuranose, normally produced by P. fellutanum, could not be obtained from the culture medium in the presence of the inhibitors; (c) the content of galactofuranose in the cell wall was significantly decreased in the presence of D-galactono-1,4-lactone. The influence on the mycelia growth was investigated by light microscopy (LM) and transmission electron microscopy (TEM) showing important alterations.