IDENTIFICATION OF A PUTATIVE MEMBRANE-INSERTED SEGMENT IN THE ALPHA-TOXIN OF STAPHYLOCOCCUS-AUREUS

To gain a fuller understanding of the regions of the Staphylococcus aureus alpha-toxin important in pore formation, we have used Forster dipole-dipole energy transfer to demonstrate that a central glycine-rich region of alpha-toxin (the so-called ''hinge'' region) inserts deeply into the bilayer on association of toxin with liposomes. Mutant alpha-toxins with unique cysteine (C) residues at positions 69 and 130 [Palmer, M., et al. (1993) J. Biol. Chem. 268, 11959) were reacted with the C-specific fluorophore acrylodan, which acted as an energy donor. The chosen acceptor was N-(7-nitrobenz-2-oxa-13-diazol-4-yl)-1,2-bis(hexadecanoyl) -sn-glycero-3-phosphoethanolamine (NBD-PE). Measurement of the degree of donor quenching with increasing NBD-PE in the inner bilayer leaflet enables the distance of closest approach between donor and acceptor to be estimated. For toxin labeled with acrylodan at position 130 (in the hinge region), this distance is approximately 5 +/- 2 Angstrom, showing that the probe is close to the inner surface of the liposomes. A second probe labeled at position 69 (in the N-terminal domain) shows negligible energy transfer, indicating a distance of closest approach >40 Angstrom. This implies that this N-terminal region remains ''outside'' the liposome. We propose a model in which the central region of the alpha-toxin inserts into the membrane and possibly participates in forming the wall of the pore.

Análise fenotípica e molecular da produção de biofilmes por estirpes de staphylococcus aureus isoladas de casos de mastite subclínica bovina

Biofilm formation is considered an advantage for Staphylococcus aureus mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix known as exopolysaccharide. The objective of this study was to evaluate the ability of Staphylococcus aureus isolated from bovine subclinical mastitis for formation of biofilms. A total of 94 Staphylococcus aureus strains obtained from milk samples of cows suffering from subclinical mastitis in dairy herds on two properties in the state of São Paulo were evaluated. These strains were characterized by in vitro biofilm formation, and by the presence of icaA and icaD genes which are responsible for intercellular adhesion. The results revealed that 98.9% of the isolates produced biofilm in vitro by adherence in sterile 96-well "U" bottom polystyrene tissue culture plates; 95.7% of the isolates possessed the icaA and icaD genes. These bacterial isolates biofilm producers may impair eradication of chronic mastitis, rendering antibiotherapy less effective. The detection of biofilm forming ability in mastitis isolates may provide useful information for more adequate therapeutic regimen and for preventive actions in the control of those bacterial isolates in bovine herds.

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of São Paulo, Brazil, using pulsed-field gel electrophoresis

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

Population analysis of susceptibility to propolis in reference strains of Staphylococcus aureus and Escherichia coli

Susceptibility to several ethanolic extracts of propolis (EEP) concentrations was tested with the population analysis technique in reference strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 35218). The results of these tests showed that all bacteria were killed by EEP concentrations approximately equal or higher (2.0% v/v - S. aureus; 10.0% v/v - E. coli) than the respective minimal inhibitory concentrations (MIC). Regarding the susceptibility to propolis, there was a homogeneity of data with the respective time kill curves showing a clear bactericidal effect during 6 to 9 h of exposition.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

Atividade anti Staphylococcus aureus de extratos de própolis (EP) de Apis mellifera preparados com diferentes concentrações de etanol

Propolis has been used in folk medicine and possesses a broad spectrum of biological activities, specially antibacterial activity. Studies have demonstrated that the composition of propolis extract may have influence in such activity. The goal of this study was to investigate the antibacterial activity of eleven propolis extracts (PE) against sixty one Staphylococcus aureus strains, isolated from newborn clinical specimens. The PE from Apis mellifera were prepared by using pure water and mixtures of water with ethanol at different concentrations (from 0 to 100%), 25g of propolis in 100 mL of solvent, and three days of maceration followed by filtration. Determination of Minimal Inhibitory Concentration (MIC) by agar dilution method was performed and serial concentrations from each PE were achieved (%v/v) in plates containing Mueller Hinton agar. It was possible to verify that the anti S. aureus activity was directly proportional to ethanol concentration and no significant differences were observed among PE with ethanol concentration from 70 to 100%. The MIC 90% values ranged from 0.4 to 0.6% (v/v) and the 70% ethanolic extract were the most efficient to inhibit bacterial growth (MIC 90%=0.42%, v/v). In conclusion, our results suggest that the EP composition and, consequently, the concentration of ethanol used as solvent may influence the antibacterial activity of propolis from A. mellifera.

Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Aderência e formação do biofilme por Staphylococcus aureus ao polietilenotereftalato (PET) usado como embalagem para acondionamento de medicamentos e cosméticos

The polyethylene terephthalate (PET) is used for package drugs and cosmetics. The aim of this research was examine by scanning electron microscopy (SEM) a Staphylococcus aureus attachment and biofilm formation on a polyethylene terephthalate (PET) surface and hydrophobicity of S. aureus by adherence to hydrocarbons. A suspension of S. aureus was prepared in Mueller-Hinton broth and, coupons of polyethylene terephthalate were incubated for 30 minutes, two, 24 and 48 hours, 15 and 30 days. Afteron the coupons were removed and prepared for scanning electron microscope analysis. The attachment and biofilm formation was observed on the surfaces of PET. The SEM revealed adhesion and biofilm formation on PET surfaces. The hydrophobicity test classified S. aureus as hydrophobic.

Propolis: anti-Staphylococcus aureus activity and synergism with antimicrobial drugs

Propolis is a natural resinous substance collected by bees from tree exudates and secretions. Its antimicrobial activity has been investigated and inhibitory action on Staphylococcus aureus growth was evaluated The in vitro synergism between ethanolic extract of propolis (EEP) and antimicrobial drugs by two susceptibility tests (Kirby and Bauer and E-Test) on 25 S. aureus strains was evaluated Petri dishes with sub-inhibitory concentrations of EEP were incubated with 13 drugs using Kirby and Bauer method and synergism between EEP and five drugs [choramphenicol (CLO), gentamicin (GEN), netilmicin (NET), tetracycline (TET), and vancomycin (VAN)] was observed. Nine drugs were assayed by the E-test method and five of them exhibited a synergism [CLO, GEN, NET, TET, and clindamycin (CLI)]. The results demonstrated the synergism between EEP and antimicrobial drugs, especially those agents that interfere on bacterial protein synthesis.

Photodegradation of sparfloxacin and isolation of its degradation products by preparative HPLC

Sparfloxacin, a third generation fluoroquinolone derivative, is a potent antibacterial agent active against a wide range of Gram-positive and Gram-negative organisms including Streptococcus pneuinoniae, Staphylococcus aureus, methicillin resistant S. aureus, Legionella spp., Mycoplasina spp., Chlamydia spp. and Mycobacterium spp. A drawback of fluoroquinolones is their photoreactivity. Sparfloxacin has been studied in terms of therapeutic activities. However, there are few published of analytical methods being applied to sparfloxacin. The aim in this study was to determine the photodegradation products of sparfloxacin, when submitted to UV light, and to characterize two of these products, designated SPAX-PDP1 and SPAX-PDP2. An accelerated study of stability in methanol solution was carried out by exposing a solution of sparfloxacin to UV light (peak wavelength 290 nm) for 36 hours at room temperature. The products were analyzed by NMR spectrophotometry, IR spectrometry and mass spectrophotometry. The results suggest that the products isolated here could be used to estimate the degradation of sparfloxacin in a stability study. However, the low activity exhibited by UV-irradiated sparfloxacin is a source of concern that demands further investigation of the mechanism of its photodegradation mechanism.

Mastite subclínica causada por Staphylococcus aureus: Custo-benefício da antibioticoterapia de vacas em lactação

Economic evaluation of the treatment bovine subclinical mastitis caused by S. aureus was evaluated. Two hundred and seventy udder quarters with or without subclinical mastitis were distributed into four groups, in conformity to lactational stage and treatments. Group 1 included animals treated between 10 and 60 days of lactation; group 2 included animals treated from 61 days of lactation to two months before drying; group 3 included animals no treated between 10 and 60 days of lactation; group 4 included animals no treated from 61 days of lactation to two months before drying. Treatment with gentamicin (150mg) was accomplished by intramammary doses, once a day, after performing sensitivity tests. The mammary quarters were re-evaluated after 30 days. The costs with the treatment were calculated considering a S. aureus prevalence of 5% as well as expenses with antibiotic, milk disposal, tests of drug sensitivity and workload. There was loss of income of 2% and 14% in the groups 1 and 2, respectively, when compared with the values before the treatment. In such case, the treatment of bovine subclinical mastitis caused by S. aureus in the lactation was economically unviable.

Subclinical mastitis caused by staphylococcus aureus: Analysis cost benefit of antibiotic therapy in lactating cows

The cost benefit analysis of treatment of bovine subclinical mastitis caused by S. aureus was evaluated. Two hundred and seventy udder quarters with subclinical mastitis and healthy were selected in four groups, in conformity to lactational stage and with the treatment or not. Group 1 included treated animals 10 to 60 days in milk; group 2 included treated animals 61 days in milk until two months before the end of lactation; group 3 included animals not treated 10 to 60 days in milk; group 4 included animals not treated from 61 days in milk until two months before the end of lactation. Treatment with gentamicin (150 mg) was accomplished by intramammary doses, once a day, after sensitivity tests. The mammary quarters were evaluated after 30 days again. The costs with the treatment were calculated considering a S. aureus prevalence of 5%, expenses with antibiotic, loss in milk, tests of sensitivity and workload. There was loss of income of 2% and 14% in the groups 1 and 2, respectively, when compared with the incomes before treatment. In such case, the treatment of bovine subclinical mastitis by S. aureus in the lactation was economically not practicable.

Quercetin reduces Staphylococcus aureus interaction with neutrophils

Flavonoids, including quercetin, have been reported to modulate the ability of Staphylococcus aureus to adhere to host tissue without exhibiting direct antibacterial activity. In the present study, we evaluated the interaction of S. aureus pretreated with 40 μg/mL of quercetin with neutrophils to assay oxidative burst stimulation, using luminol-amplified chemiluminescence. S. aureus pre-incubated with subinhibitory concentration of quercetin induced significantly less light emission by neutrophils than did untreated bacteria. The results of the present study demonstrate that quercetin decreases S. aureus uptake by neutrophils.

Atividade antimicrobiana in vitro de extrato de Punica granatum L. sobre staphylococcus aureus isolado em leite bovino

Bovine mastitis is considered important disease causing major economic losses in dairy herds. Antimicrobial drug can promote resistance, chemical residues in food and environmental contamination. The purpose of this study was to evaluate the antibacterial activity in vitro of pomegranate extract on Staphylococcus aureus isolated from bovine milk, evaluate its antioxidant activity, and quantify levels of total phenols and fravonoides of the different extracts used. Aqueous extracts were used in nature and dry, from the peel of the fruit (EAC) and leaves (EAF). Additionally, it was evaluated the antioxidant activity (AA%), total phenols and flavonoids. Milk samples were inoculated, incubated, and the colonies were characterized as Staphylococcus aureus were adjusted to 1.0x106 UFC/mL in the 6 standard of the MacFarland scale. The sensitivity of the microbial isolates were determined in quintuplicate, in disk diffusion test. The minimum inhibitory concentration was determined by visible inhibition zone greater than 15mm. The results were evaluated by ANOVA, Tukey 5%, using the program SISVAR 5.3 - DEX/UFLA. Results implied that the aqueous extract from the bark of dried fruit was capable of inhibiting bacterial growth at concentrations of 3%. The other treatments only showed this activity, from the concentrations of 15%, 20% and 30% for dry EAF, in nature EAC and in nature EAF, respectively. Regarding the action antioxidant of dry EAC, was not correlated with total phenols and flavonoids. Probably other alkaloids substances present in the extract studied, may have been responsible for this activity. It is concluded that extracts of Punica granatum L., especially those obtained by the shell of the fruit, showed inhibitory activity against S. aureus, indicating your use potential for the control of bovine mastitis.