Occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in 'coalho' cheese produced in Brazil.

The experiments reported in this research communication analysed the presence of methicillin-resistant Staphylococcus aureus (MRSA) in 112 samples of 'coalho' cheese, from 56 dairy producing farms in 28 cities in all mesoregions of the State of Ceará, Brazil. To assess antimicrobial resistance we also examined the presence of genes encoding enterotoxins and toxic shock syndrome toxin, as well as the presence of the blaZ gene for ß-lactamases, and resistance to oxacillin. The research found 69 isolates of S. aureus, of which 13.04% had the mecA gene encoding the penicillin-binding protein, which confers resistance to methicillin, in cheese samples from 6 different cities. This included the state capital, Fortaleza, which had the largest prevalence (23.19%) of mecA positive isolates. It was also found that 55.07% of the isolates of S. aureus had the blaZ gene, and 7.25% demonstrated resistance to oxacillin in the plate disc diffusion tests. We did not show the presence of isolates carrying toxigenic genes. The findings suggest that strict supervision of production processes in the dairy industry is necessary in all production scale processes, thus preventing contamination and possible problems for consumers.

Lipid transfer protein isolated from noni seeds displays antibacterial activity in vitro and improves survival in lethal sepsis induced by CLP in mice.

In the present study, we aimed to evaluate the antibacterial activity of a lipid transfer protein isolated from Morinda citrifolia L. seeds, named McLTP1, and to investigate its effect in the cecal ligation and puncture (CLP) mouse sepsis model. Antimicrobial assays revealed that McLTP1 (12.5-800 µg/mL) significantly reduced Staphylococcus aureus (ATCC 6538P and ATCC 14458) and Staphylococcus epidermidis (ATCC 12228) planktonic growth, reaching maximal inhibition of approximately 50% and 98%, respectively. Furthermore, McLTP1 inhibited biofilm formation of both S. aureus strains, achieving percentages ranging from 39.1% to 69.1% (200-800 µg/mL) for ATCC 6538P and 34.4%-63% (12.5-800 µg/mL) for ATCC 14458. A synergistic interaction between McLTP1 and oxacillin against S. aureus and S. epidermidis was also observed, as determined by fractional inhibitory concentration indices of 0.18 and 0.38, respectively. McLTP1 showed no significant inhibitory effect against Gram-negative bacteria. In the in vivo experiments, sepsis was lethal to 83% of the animals, 72 h after CLP. In contrast, 100% of the animals treated with McLTP1 (8 mg/kg) before (intraperitoneal injection or oral dose) or after (oral dose) CLP were still alive 3 days later. In addition, oral or intraperitoneal administration of McLTP1 (8 mg/kg) significantly reduced the body weight loss, fever, leukocytosis, organ damage, and the level of inflammatory serum cytokines induced by sepsis. In conclusion, McLTP1 could be exploited for its antimicrobial properties, and can be considered a potential therapeutic candidate for the management of clinical sepsis.

Antimicrobial Effects of Violacein against Planktonic Cells and Biofilms of Staphylococcus aureus.

Violacein is an indole compound, produced by Chromobacterium violaceum, a bacteria present in tropical and subtropical areas. Among its numerous biological activities, its antimicrobial potential stands out. This study aims to determine the antimicrobial activity of VIO on S. aureus in planktonic culture and biofilms. VIO showed excellent antimicrobial activity in inhibiting and killing S. aureus in planktonic cultures and biofilm formation. The minimum bactericidal concentration (5 µg/mL) of VIO caused the death of S. aureus after 3-4 h of exposure and the minimum inhibitory concentration (1.25 µg/mL) of VIO inhibited bacterial growth within the first 8 h of contact. Biofilm formation was also strongly inhibited by VIO (1.25 µg/mL), in contrast to the higher resistance verified for S. aureus in mature biofilm (40 µg/mL). The high bacterial metabolic activity favored VIO activity; however, the good activity observed during phases of reduced metabolism indicates that VIO action involves more than one mechanism. Thus, VIO is a promising molecule for the development of an antimicrobial drug for the eradication of S. aureus infections.

Antibacterial and antiparasitic effects of Bothropoides lutzi venom.

The therapeutic potential of toxins has aroused great interest in the scientific community. Microbial resistance is a serious current public health problem, in part because of the wide use of antimicrobial drugs. Furthermore, there are several problems in the treatment of parasitic diseases such as leishmaniosis and Chagas' disease, including the low efficacy in some clinical phases of the diseases and the loss of effectiveness of benzonidazole in the chronic phase of Chagas' disease. In this context, the aim of this work was to study the antimicrobial and antiparasitic effects of Bothropoides lutzi total venom (BltTV). The venom exerted an antibacterial effect on S. aureus, with MIC=MLC=200 microg/mL. The inhibitory effects of BltTV on promastigote forms of Leishmania amazonensis and L. chagasi were assessed by counting of viable cells after incubation with BltTV. IC50 values of 234.6 microg/mL and 61.2 microg/mL, were obtained, respectively. Furthermore, the venom repressed epimastigote forms of Trypanosoma cruzi growth. Finally, BltTV was verified to affect murine peritoneal macrophages, causing a cytotoxic effect at the highest concentrations (100 and 50 microg/mL). In conclusion, Bothropoides lutzi venom demonstrated antibacterial and antiparasite effects, suggesting that the venom contains some substance(s) of therapeutic value.

The antimicrobial activity of photodynamic therapy against Streptococcus mutans using different photosensitizers.

UNLABELLED: Several photosensitizers have been used against oral bacteria without standardization. Singlet oxygen ((1)O(2)) is an aggressive chemical species that can kill cells through apoptosis or necrosis. OBJECTIVE: to compare the antimicrobial activity of photodynamic therapy (PDT) with different photosensitizers at the same concentration against Streptococcus mutans. In addition, the (1)O(2) production of each photosensitizer was determined. The photosensitizers (163.5 µM) methylene blue (MB), toluidine blue ortho (TBO) and malachite green (MG) were activated with a light-emitting diode (LED; λ=636 nm), while eosin (EOS), erythrosine (ERI) and rose bengal (RB) were irradiated with a curing light (λ=570 nm). Light sources were operated at 24 J cm(-2). For each photosensitizer, 40 randomized assays (n=10 per condition) were performed under one of the following experimental conditions: no light irradiation or photosensitizer, irradiation only, photosensitizer only or irradiation in the presence of a photosensitizer. After treatment, serial dilutions of S. mutans were seeded onto brain heart infusion agar to determine viability in colony-forming units per milliliter (CFU mL(-1)). Generation of (1)O(2) was analyzed by tryptophan photooxidation, and the decay constant was estimated. Results were analyzed by one-way ANOVA and the Tukey-Kramer test (p<0.05). PDT with irradiation in the presence of the photosensitizers TBO and MG was effective in reducing S. mutans counts by 3 and 1.4 logs, respectively (p<0.01), compared to their respective untreated controls. MB generated 1.3 times more (1)O(2) than TBO, and both produced significantly higher concentrations of singlet oxygen than the other photosensitizers. Since in vitro bulk (1)O(2) production does not indicate that (1)O(2) was generated in the bacterial activity site, the bactericidal action against S. mutans cannot be related to in vitro singlet O(2) generation rate. In vitroS. mutans-experiments demonstrated TBO as the only photosensitizer that effectively reduced 99.9% of these microorganisms.

Genetic diversity of the E protein of dengue type 3 virus.

BACKGROUND: Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis. RESULTS: Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein. CONCLUSION: Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.

Latex proteins from the plant Calotropis procera are partially digested upon in vitro enzymatic action and are not immunologically detected in fecal material.

Soluble proteins from the latex of Calotropis procera (LP) were investigated in vitro and in vivo for digestibility as the latex has previously been shown to produce considerable toxic effects on animals. The latex is also an important biologically active compound that displays antiinflammatory and antidiarrhea properties. The proteins were digested by the action of trypsin, pepsin or chemotrypsin as revealed by gel filtration and SDS-PAGE analysis. Furthermore, the full LP digestion was easily achieved by protease treatment. Rabbit polyclonal antibodies raised against LP failed to detect cross-reactive molecules in fecal material of experimental rats following 35 consecutive days of LP consumption in water. Similar patterns of electrophoresis were observed for the negligible amounts of protein observed in the fecal extracts of control and test animals. No death or toxic effects were observed among animals. Taken together these results suggest that harmful and toxic effects on animals of the latex from C. procera are present in its rubber and low molecular weight fractions rather than its protein content.

Cytotoxic activity of chalcones isolated from lonchocarpus sericeus (pocr.) kunth.

In the present study, it was demonstrated that the hexanic extract obtained from the roots of Lonchocarpus sericeus and one of its major components, derricin, but not lonchocarpin, show cytotoxic activity to fertilized sea urchin eggs. Both inhibited the first cleavage of sea urchin eggs in a dose-dependent manner with an IC(50) of 30.4 (26.2-35.3) microgram/mL for the crude extract and 51.2 (42.1-62.3) microgram/mL for derricin (n = 6, in both cases). Furthermore, the hexanic extract of L. sericeus, and the isolated compounds, derricin and lonchocarpin showed cytotoxicity against CEM leukaemic cell line (IC(50) = 17.6 (13.7-22.6), 13.0 (12.0-14.0) and 10.4 (5.6-19.1) microgram/ml (n = 6), respectively). When these substances (6.25 to 125 microgram/25 microL) were examined for antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida albicans, none of them were found to be active. Neither the hexanic extract, nor the isolated compounds derricin and lonchocarpin (0.5 to 250 microgram/mL) presented hemolytic activity. These results indicated a possible antimitotic activity of L. sericeus crude extract and their major constituents.

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells.

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.