Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, 1828

The antimicrobial, hemagglutinating and toxic activities of the purple fluid of the sea hare Aplysia dactylomela are described. Intact or dialyzed purple fluid inhibited the growth of species of Gram-positive and Gram-negative bacteria and the action was not bactericidal but bacteriostatic. The active factor or factors were heat labile and sensitive to extreme pH values. The fluid preferentially agglutinated rabbit erythrocytes and, to a lesser extent, human blood cells, and this activity was inhibited by the glycoprotein fetuin, a fact suggesting the presence of a lectin. The fluid was also toxic to brine shrimp nauplii (LD50 141.25 µg protein/ml) and to mice injected intraperitoneally (LD50 201.8 ± 8.6 mg protein/kg), in a dose-dependent fashion. These toxic activities were abolished when the fluid was heated. Taken together, the data suggest that the activities of the purple fluid are due primarily to substance(s) of a protein nature which may be involved in the chemical defense mechanism of this sea hare.

Monoassociation with Lactobacillus acidophilus UFV-H2b20 stimulates the immune defense mechanisms of germfree mice

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the host's nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 108 cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the host's macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1% of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic.

In vitro antimicrobial activity of a new series of 1,4-naphthoquinones

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 µg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 µg/ml. All compounds presented a minimal bactericidal concentration higher than 500 µg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 µg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

Antioxidant and antimicrobial activities of Bauhinia racemosa L. stem bark

The present study was carried out to evaluate the antioxidant and antimicrobial activities of a methanol extract of Bauhinia racemosa (MEBR) (Caesalpiniaceae) stem bark in various systems. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical, superoxide anion radical, nitric oxide radical, and hydroxyl radical scavenging assays were carried out to evaluate the antioxidant potential of the extract. The antioxidant activity of the methanol extract increased in a concentration-dependent manner. About 50, 100, 250, and 500 µg MEBR inhibited the peroxidation of a linoleic acid emulsion by 62.43, 67.21, 71.04, and 76.83%, respectively. Similarly, the effect of MEBR on reducing power increased in a concentration-dependent manner. In DPPH radical scavenging assays the IC50 value of the extract was 152.29 µg/ml. MEBR inhibited the nitric oxide radicals generated from sodium nitroprusside with an IC50 of 78.34 µg/ml, as opposed to 20.4 µg/ml for curcumin. Moreover, MEBR scavenged the superoxide generated by the PMS/NADH-NBT system. MEBR also inhibited the hydroxyl radical generated by Fenton's reaction, with an IC50 value of more than 1000 µg/ml, as compared to 5 µg/ml for catechin. The amounts of total phenolic compounds were also determined and 64.7 µg pyrocatechol phenol equivalents were detected in MEBR (1 mg). The antimicrobial activities of MEBR were determined by disc diffusion with five Gram-positive, four Gram-negative and four fungal species. MEBR showed broad-spectrum antimicrobial activity against all tested microorganisms. The results obtained in the present study indicate that MEBR can be a potential source of natural antioxidant and antimicrobial agents.

Differential activity of a lectin from Solieria filiformis against human pathogenic bacteria

A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium). The lectin (500 µg/mL) stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 µg/mL but the lectin (10-1000 µg/mL) had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline) were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 µg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 µg/mL), its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37ºC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 µg/mL) and S. aureus (MIC = 500 µg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 µg/mL). Fractions I and II were inactive against standard strains at concentrations of <=1000 µg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 µg/mL) and S. aureus (MIC = 250 µg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 µg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 µg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 µg/mL) and S. aureus (MIC = 31.3 µg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 µg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 µg/mL) and E. faecalis (MIC = 50 µg/mL), with EC50 of 8 and 2.5 µg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Antioxidant and antimicrobial activities of bitter and sweet apricot (Prunus armeniaca L.) kernels

Abstract The present study describes the in vitro antimicrobial and antioxidant activity of methanol and water extracts of sweet and bitter apricot (Prunus armeniaca L.) kernels. The antioxidant properties of apricot kernels were evaluated by determining radical scavenging power, lipid peroxidation inhibition activity and total phenol content measured with a DPPH test, the thiocyanate method and the Folin method, respectively. In contrast to extracts of the bitter kernels, both the water and methanol extracts of sweet kernels have antioxidant potential. The highest percent inhibition of lipid peroxidation (69%) and total phenolic content (7.9 ± 0.2 µg/mL) were detected in the methanol extract of sweet kernels (Hasanbey) and in the water extract of the same cultivar, respectively. The antimicrobial activities of the above extracts were also tested against human pathogenic microorganisms using a disc-diffusion method, and the minimal inhibitory concentration (MIC) values of each active extract were determined. The most effective antibacterial activity was observed in the methanol and water extracts of bitter kernels and in the methanol extract of sweet kernels against the Gram-positive bacteria Staphylococcus aureus. Additionally, the methanol extracts of the bitter kernels were very potent against the Gram-negative bacteria Escherichia coli (0.312 mg/mL MIC value). Significant anti-candida activity was also observed with the methanol extract of bitter apricot kernels against Candida albicans, consisting of a 14 mm in diameter of inhibition zone and a 0.625 mg/mL MIC value.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

In vitro and in vivo antitumor activity of crude extracts obtained from Brazilian Chromobacterium sp isolates

Natural products produced by microorganisms have been an important source of new substances and lead compounds for the pharmaceutical industry. Chromobacterium violaceum is a Gram-negative β-proteobacterium, abundant in water and soil in tropical and subtropical regions and it produces violacein, a pigment that has shown great pharmaceutical potential. Crude extracts of five Brazilian isolates of Chromobacterium sp (0.25, 2.5, 25, and 250 µg/mL) were evaluated in an in vitro antitumor activity assay with nine human tumor cells. Secondary metabolic profiles were analyzed by liquid chromatography and electrospray ionization mass spectrometry resulting in the identification of violacein in all extracts, whereas FK228 was detected only in EtCE 308 and EtCE 592 extracts. AcCE and EtCE 310 extracts showed selectivity for NCI/ADR-RES cells in the in vitro assay and were evaluated in vivo in the solid Ehrlich tumor model, resulting in 50.3 and 54.6% growth inhibition, respectively. The crude extracts of Chromobacterium sp isolates showed potential and selective antitumor activities for certain human tumor cells, making them a potential source of lead compounds. Furthermore, the results suggest that other compounds, in addition to violacein, deoxyviolacein and FK228, may be involved in the antitumor effect observed.

Rosemary (Rosmarinus officinalis): a study of the composition, antioxidant and antimicrobial activities of extracts obtained with supercritical carbon dioxide

Rosemary leaf extracts were obtained by supercritical fluid extraction (SFE) and Soxhlet extraction. Their chemical compositions were evaluated by GC-MS. The extracts were analyzed for compounds reported in the literature as showing antimicrobial and antioxidant activities. The rosemary extracts were tested with regard to antioxidant (DPPH radical scavenging and total phenolic content - Folin-Denis reagent), antibacterial (Gram-positive bacteria - Staphylococcus aureus ATCC 25923 and Bacillus cereus ATCC 11778 - and Gram-negative bacteria - Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) and antifungal (Candida albicans) activities. Antioxidant, antibacterial and antifungal activities of the SFE extracts were confirmed.

Study of bacteria Gluconobacter sp.: isolation, purification, phenotypic and molecular identification

The importance of the study of acetic bacteria, on species of the Gluconobacter genus is based on its industrial application, as these possess the capacity of bioconversion of sorbitol to sorbose, enabling the process of vitamin C production. The study involved samples collected in industries of soft drinks, flowers, fruits and honey, followed by purification, phenotypic identification, molecular identification with the use of primer defined from Nucleotide Sequence Database consultation. Strains preserved were identified as members of the Acetobacteraceae family, Gluconobacter genus. 110 strains had been isolated of substrate: Pyrostegia venusta (ker-gawler), honey, Vitis vinifera (grape), Pyrus communis (pear), Malus sp. (apple) and in two samples of soft drinks. Of this total 57 strains had been recovered in manitol medium (manitol, yeast extract, peptone), 12 in YMG medium (glucose, manitol, yeast extract, ethanol, acetic acid), 41 in enrichment medium (De Ley and Swings) and later in the GYC medium (glucose, yeast extract and calcium carbonate). 68 strains were identified as Gram negative bacilli rods. Of these, 31 were characterized biochemically as belonging to the Acetobacteriaceae family as they were catalase positive, oxidase negative and producers of acid from glucose. The characterization of these strains was complemented with the biochemistry tests: gelatin liquefaction, nitrate reduction, indole and H2S production, oxidation of ethanol to acetic acid and molecular tests for genus identification. Only eight strains were characterized as pertaining to the Gluconobacter genus. The strains are maintained in collection cultures at the Microbiology Laboratory of the Biology Department at the São Paulo State University (UNESP) in Assis, stored in malt extract at -196 ºC.

In vitro antimicrobial properties of plant essential oils thymus vulgaris, cymbopogon citratus and laurus nobilis against five important foodborne pathogens

Several essential oils of condiment and medicinal plants possess proven antimicrobial activity and are of important interest for the food industry. Therefore, the Minimum Inhibitory Concentrations (MIC) of those oils should be determined for various bacteria. MIC varies according to the oil used, the major compounds, and the physiology of the bacterium under study. In the present study, the essential oils of the plants Thymus vulgaris (time), Cymbopogon citratus (lemongrass) and Laurus nobilis (bay) were chemically quantified, and the MIC was determined on the bacteria Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19117, Salmonella enterica Enteritidis S64, and Pseudomonas aeruginosa ATCC 27853. The essential oil of C. citratus demonstrated bacterial activity at all concentrations tested and against all of the bacteria tested. The majority of essential oil compounds were geranial and neral. The major constituent of T. vulgaris was 1.8-cineol and of L. nobilis was linalool, which presented lower antibacterial activity, followed by 1.8-cineol. The Gram-negative bacteria demonstrated higher resistance to the use of the essential oils tested in this study. E. coli was the least sensitive and was inhibited only by the oils of C. citratus and L. nobilis.