Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Item Safety evaluation of the antimicrobial peptide bovicin HC5 orally administered to a murine model(BMC Microbiology, 2013) Paiva, Aline Dias; Fernandes, Kenner Morais; Dias, Roberto Sousa; Rocha, Alípio dos Santos; Oliveira, Leandro Licursi de; Neves, Clóvis Andrade; Paula, Sérgio Oliveira de; Mantovani, Hilário CuquettoBovicin HC5 is an antimicrobial peptide that shows a broad spectrum of activity and potential for biotechnological and therapeutic applications. To gain insight about the safety of bovicin HC5 application, the histological and immunostimulatory effects of orally administrated bovicin HC5 to BALB/c mice were evaluated. BALB/c mice were divided into three groups: negative control (NC group); mice given purified bovicin HC5 (Bov group); mice given ovalbumin (positive control, PC group; a murine model of enteropathy). The mice were initially pre-sensitized, and PBS, bovicin HC5 or ovalbumin were administered for 30 days by daily gavages. Histological and morphometric analysis were performed and the relative expression of cytokines was analyzed by real-time RT-PCR. The oral administration of bovicin HC5 to BALB/c mice reduced weight gain and caused alterations in the small intestine, although absorptive changes have not been detected. The number of total goblet cells and the mucopolysaccharides production were not affected by bovicin HC5 administration. A hypertrophy of Paneth cells and an increase in the number of mitotic cells were observed in Bov group, while the number of mast cells remained unaltered. Increased expression of TNF-α, INF-γ and IL-12 was observed in the small intestine upon bovicin HC5 administration. Bovicin HC5 has only minor effects on intestinal permeability and did not elicit an allergenic response upon oral administration to animal models. Considering the low in vivo toxicity of bovicin HC5, it might be a good candidate for enteral applications.Item Production of edible mushroom and degradation of antinutritional factors in jatropha biodiesel residues(LWT - Food Science and Technology, 2013-03) Luz, José Maria Rodrigues da; Paes, Sirlaine Albino; Torres, Denise Pereira; Nunes, Mateus Dias; Silva, Juliana Soares da; Mantovani, Hilário Cuquetto; Kasuya, Maria Catarina MegumiThe elimination of antinutritional factors of the Jatropha curcas L. seed cake is important for decreasing environmental damage and adding economic value to this residue of the biodiesel industry. In this study, we analyzed the ability of Pleurotus ostreatus to degrade antinutritional factors and produce edible mushrooms using different proportions of the J. curcas seed cake as substrate. After 60 d of incubation at 25 °C, we observed 95% phytic acid and 85% tannins reductions, and high mushrooms productivity. There was no evidence of tannins or phytic acid in these mushrooms. Furthermore, the phorbol ester concentration observed in these mushrooms was around 1000-fold lower than that found in the non-toxic variety of J. curcas. Thus, P. ostreatus can degrade the antinutritional factors found in J. curcas seed cake. The jatropha seed cake can potentially be used for mushroom production, with high nutritional value, and animal ration, after treated by P. ostreatus, adding economic value to the biodiesel residue and avoiding inadequate disposal in the environment.Item Temperature and pH influence the susceptibility of Salmonella Typhimurium to nisin combined with EDTA(Food Control, 2016-03) Prudêncio, Cláudia Vieira; Mantovani, Hilário Cuquetto; Cecon, Paulo Roberto; Prieto, Miguel; Vanetti, Maria Cristina Dantasantibiotics, such as nisin, generally act on Gram-positive bacteria, whereas Gram-negative bacteria exhibit natural resistance to the action of these bacteriocins. However, Gram-negative bacteria can become susceptible to the action of these antimicrobial peptides with the use of chelating agents, such as EDTA. The activity of bacteriocins can be influenced by several factors, such as environmental conditions and chemical composition of the medium. Moreover, the susceptibility of target microorganism can be altered, due to changes in cellular composition induced by environmental conditions. In this study, we examined the combined effect of variations in temperature and pH on the susceptibility of Salmonella Typhimurium to the action of nisin associated with EDTA. The reduction in the number of viable cells of Salmonella Typhimurium varied according to the temperature and pH, and major reduction in logarithmic cycles of viable cells occurred with a temperature increase. Under low temperature and low pH, a considerable reduction in the viability was also observed after prolonged exposure to bacteriocin, regardless of the presence of EDTA. According to results, temperature and pH conditions influence on the susceptibility of Salmonella Typhimurium to combination of nisin and EDTA and thus should be considered to establish optimal conditions for nisin use.Item Morphological changes in Salmonella Typhimurium caused by the lantibiotic bovicin HC5 in association with EDTA(Annals of Microbiology, 2015-07-03) Prudêncio, Cláudia Vieira; Ferreira, Sukarno Olavo; Mantovani, Hilário Cuquetto; Vanetti, Maria Cristina DantasBacteriocins, particularly those produced by Gram-positive bacteria, have in recent years been considered promising antimicrobial agents to inhibit bacterial growth in food, and thus are potential food preservatives. These peptides generally exhibit a spectrum of action limited to Gram-positive bacteria. However, their action can be extended to Gram-negative bacteria through association with chelating agents. In the present study, we evaluated the occurrence of morphological changes on the cell envelope of Salmonella Typhimurium cells treated with bovicin HC5—a lantibiotic from Streptococcus bovis HC5—in association with EDTA. The morphological changes of the cells were visualized by atomic force microscopy (AFM), and the increase in cell membrane permeability was confirmed by the leakage of potassium ions (K+). The images displayed changes in the cell envelope, with increased surface roughness and a decreased cell volume. These changes indicate that EDTA plays a role in the destabilization of the outer membrane, allowing bovicin HC5 to act on the cytoplasmic membrane through the formation of pores, which was confirmed by the detection of potassium in the cell supernatant. These results suggest that bovicin HC5 combined with EDTA has potential for use on Salmonella cells.Item A central composite rotatable design (CCRD) approach to study the combined effect of antimicrobial agents against bacterial pathogens(World Journal of Microbiology and Biotechnology, 2015-06-17) Santos, Fernanda Godoy; Mendonça, Layanne Andrade; Mantovani, Hilário CuquettoThe combination of antimicrobial agents has been proposed as a therapeutic strategy to control bacterial diseases and to reduce the emergence of antibiotic-resistant strains in clinical environments. In this study, the interaction between the lantibiotic bovicin HC5 with chloramphenicol, gentamicin, nisin, lysostaphin and hydrogen peroxide against Staphylococcus aureus O46 was evaluated by MIC assays. The central composite rotatable design (CCRD), a robust and economic statistical design, was used to combine concentration levels of different antimicrobials agents with distinct mechanisms of action and the presence of significant interactions among the antimicrobials was determined by regression analysis. According to the adjusted model, there were no significant interactions between bovicin HC5 and gentamicin, lysostaphin, nisin or hydrogen peroxide. However, bovicin HC5 showed a significant interaction (P < 0.02) with chloramphenicol. This is the first study applying the CCRD approach to evaluate the combined effect of antimicrobials against S. aureus. Based on our results, this approach is an effective strategy to determine synergistic interactions between antimicrobial agents applied in human and veterinary medicine against bacterial pathogens.Item Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows(World Journal of Microbiology and Biotechnology, 2011-07-17) Santana, Henrique Freitas; Barbosa, Ana Andréa Teixeira; Ferreira, Sukarno Olavo; Mantovani, Hilário CuquettoStaphylococcus aureus is an important pathogen for both humans and animals, and it has been an ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. Elimination of S. aureus with classic antibiotics is difficult, and the current study aimed to evaluate the efficacy of ethanolic extracts of propolis (EEP) against S. aureus cultivated in complex media or milk. EEP (0–0.5 mg ml^−1) decreased growth of S. aureus in BHI media and 1 mg ml^−1 was bactericidal against washed cell suspensions (10^7 CFU ml−1). Propolis extracts also killed S. aureus cells resuspended in milk, but the bactericidal dose was at least 20-fold greater. Cultures that were transferred for at least 60 generations with sub-lethal doses of propolis did not change much their sensibility to EEP. Atomic force microscopy images revealed changes in morphology and cell size of S. aureus cells exposed to EEP (0.5 mg ml^−1). Our results indicate that propolis extracts might be effective against mastitis-causing S. aureus strains in vivo, but milk constituents affect the inhibitory activity of propolis. Considering that propolis-resistance appears to be a phenotype not easily selected, the use of EEP combined or not with other antimicrobial agents might be useful for mastitis control in vivo.Item Role of lipid II and membrane thickness in the mechanism of action of the lantibiotic bovicin HC5(Antimicrobial Agents and Chemotherapy, 2011-08-19) Paiva, Aline Dias; Breukink, Eefjan; Mantovani, Hilário CuquettoLantibiotics are antimicrobial peptides produced by Gram-positive bacteria, nisin being the most well known member. Nisin inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon interaction with lipid II, the essential bacterial cell wall precursor. Bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II binding motif, and we investigated the mode of action of bovicin HC5 using both living bacteria and model membranes, with special emphasis on the role of lipid II. Bovicin HC5 showed activity against Staphylococcus cohnii and Staphylococcus warneri, but bovicin HC5 hardly interfered with the membrane potential of S. cohnii. In model membranes, bovicin HC5 was not able to cause carboxyfluorescein release or proton influx from DOPC vesicles containing lipid II. Bovicin HC5 blocked lipid II-dependent pore formation activity of nisin, and a high-affinity interaction with lipid II was observed (apparent binding constant [K a ] ⴝ 3.1 ⴛ 10 6 M ⴚ1 ), with a 1:1 stoichiometry. In DOPC vesicles containing lipid II, bovicin HC5 was able to assemble with lipid II into a prepore-like structure. Furthermore, we observed pore formation activity of bovicin HC5, which was stimulated by the presence of lipid II, in thin membranes. Moreover, bovicin HC5 induced the segregation of lipid II into domains in giant model membrane vesicles. In conclusion, bovicin HC5 has a primary mode of action similar to that of nisin, but some differences regarding the pore-forming capacity were demonstrated.Item Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity(Microbiology, 2012-09-03) Paiva, Aline Dias; Oliveira, Michelle Dias de; Paula, Sérgio Oliveira de; Baracat-Pereira, Maria Cristina; Breukink, Eefjan; Mantovani, Hilário CuquettoBacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC50 of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC50 obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.Item Chemical structure, antiproliferative and antioxidant activities of a cell wall α-d-mannan from yeast Kluyveromyces marxianus(Carbohydrate Polymers, 2017-02-10) Galinari, Éder; Sabry, Diego Araújo; Sassaki, Guilherme Lanzi; Macedo, Gorete Ribeiro; Passos, Flávia Maria Lopes; Mantovani, Hilário Cuquetto; Rocha, Hugo Alexandre OliveiraCell wall polysaccharides from filamentous fungi and yeasts have been reported as antioxidant and antiproliferative polymers. Thus, we evaluated these activities from cell wall polysaccharides from Kluyveromyces marxianus CCT7735. By using a centrifugal filter, a 203 kDa α-d-mannan (KMM-5) was obtained. KMM-5 exhibited no effect on HeLa cells and a weak antiproliferative activity against Hep-G2 cells. In addition, at higher concentrations, it presented a cytotoxicity to the normal cell line, 3T3. However, KMM-5 showed copper- and iron-chelating abilities, the latter of which presented improved activity. By using 2D-NMR COSY, HSQC edited and HMBC experiments, a structure arrangement was proposed. The main chain was formed by 6)-α-d-Manp-(1 → 6) units substituted at the 2-O-position by non-reducing terminals α-d-Manp-(1 → 2) and by a branched tetrasaccharide. The latter was formed by an internal 2)-α-d-Manp-(1 → 2) unit with linked to it a 2,3)-α-d-Manp-(1 → 2) unit substituted at the 2-O-position by a non-reducing terminal α-d-Manp-(1 → 2), and at the 3-O-position by a non-reducing terminal α-d-Manp-(1 → 3). In conclusion, we considered K. marxianus CCT7735 a source of natural and renewable polysaccharides with pharmacological properties.Item Acyl homoserine lactone-based quorum sensing stimulates biofilm formation by Salmonella Enteritidis in anaerobic conditions(Archives of Microbiology, 2016-11-12) Almeida, Felipe Alves de; Pimentel-Filho, Natan de Jesus; Pinto, Uelinton Manoel; Mantovani, Hilário Cuquetto; Oliveira, Leandro Licursi de; Vanetti, Maria Cristina DantasQuorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12-HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37 °C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50 nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36 h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.