Alexandre José Macedo

Novel keratinase from Bacillus subtilis S14 exhibiting remarkable dehairing capabilities

ABSTRACT We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential.

Potential of medicinal plants from the Brazilian semi-arid region (Caatinga) against Staphylococcus epidermidis planktonic and biofilm lifestyles

ETHNOPHARMACOLOGICAL RELEVANCE Medicinal plants from the Caatinga, a Brazilian xeric shrubland, are used in folk medicine to treat infections. These ethnopharmacological data can contribute to obtaining new antimicrobial/antibiofilm extracts and natural product prototypes for the development of new drugs. The aim of this study was to investigate the antibiofilm and antibacterial activities of 45 aqueous extracts from 24 Caatinga plant species. MATERIALS AND METHODS The effect of aqueous extracts on planktonic cells and on biofilm formation by Staphylococcus epidermidis was studied by the OD(600) absorbance and by the crystal violet assay, respectively. Scanning electron microscopy (SEM) was used to generate comparative images of extract-treated and untreated biofilms. Chromatographic analyses were performed to characterize the active extracts. RESULTS The in vitro screening, at 0.4 mg/mL and 4.0mg/mL, showed 20 plants effective in preventing biofilm formation and 13 plants able to inhibit planktonic bacterial growth. SEM images demonstrated distinct profiles of bacterial adhesion, matrix production and cell morphology according to different treatments and surfaces. The phytochemical analysis of the selected active extracts indicates the polyphenols, coumarins, steroids and terpenes as possible active compounds. CONCLUSION This study describes the first antibiofilm and antibacterial screening of Caatinga plants against S. epidermidis. The evaluation presented in this study confirms several ethnopharmacological reports and can be utilized to identify new antibiofilm and antibacterial products against S. epidermidis from traditional Brazilian medicine.

Plant Natural Products Targeting Bacterial Virulence Factors

Decreased antimicrobial efficiency has become a global public health issue. The paucity of new antibacterial drugs is evident, and the arsenal against infectious diseases needs to be improved urgently. The selection of plants as a source of prototype compounds is appropriate, since plant species naturally produce a wide range of secondary metabolites that act as a chemical line of defense against microorganisms in the environment. Although traditional approaches to combat microbial infections remain effective, targeting microbial virulence rather than survival seems to be an exciting strategy, since the modulation of virulence factors might lead to a milder evolutionary pressure for the development of resistance. Additionally, anti-infective chemotherapies may be successfully achieved by combining antivirulence and conventional antimicrobials, extending the lifespan of these drugs. This review presents an updated discussion of natural compounds isolated from plants with chemically characterized structures and activity against the major bacterial virulence factors: quorum sensing, bacterial biofilms, bacterial motility, bacterial toxins, bacterial pigments, bacterial enzymes, and bacterial surfactants. Moreover, a critical analysis of the most promising virulence factors is presented, highlighting their potential as targets to attenuate bacterial virulence. The ongoing progress in the field of antivirulence therapy may therefore help to translate this promising concept into real intervention strategies in clinical areas.

Three Stages of a Biofilm Community Developing at the Liquid-Liquid Interface between Polychlorinated Biphenyls and Water

ABSTRACT Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.

Tannins Possessing Bacteriostatic Effect Impair Pseudomonas aeruginosa Adhesion and Biofilm Formation

Plants produce many compounds that are biologically active, either as part of their normal program of growth and development or in response to pathogen attack or stress. Traditionally, Anadenanthera colubrina, Commiphora leptophloeos and Myracrodruon urundeuva have been used by communities in the Brazilian Caatinga to treat several infectious diseases. The ability to impair bacterial adhesion represents an ideal strategy to combat bacterial pathogenesis, because of its importance in the early stages of the infectious process; thus, the search for anti-adherent compounds in plants is a very promising alternative. This study investigated the ability of stem-bark extracts from these three species to control the growth and prevent biofilm formation of Pseudomonas aeruginosa, an important opportunistic pathogen that adheres to surfaces and forms protective biofilms. A kinetic study (0–72 h) demonstrated that the growth of extract-treated bacteria was inhibited up to 9 h after incubation, suggesting a bacteriostatic activity. Transmission electron microscopy and fluorescence microscopy showed both viable and nonviable cells, indicating bacterial membrane damage; crystal violet assay and scanning electron microscopy demonstrated that treatment strongly inhibited biofilm formation during 6 and 24 h and that matrix production remained impaired even after growth was restored, at 24 and 48 h of incubation. Herein, we propose that the identified (condensed and hydrolyzable) tannins are able to inhibit biofilm formation via bacteriostatic properties, damaging the bacterial membrane and hindering matrix production. Our findings demonstrate the importance of this abundant class of Natural Products in higher plants against one of the most challenging issues in the hospital setting: biofilm resilience.

In Vitro Antioxidant, Anticoagulant and Antimicrobial Activity and in Inhibition of Cancer Cell Proliferation by Xylan Extracted from Corn Cobs

Xylan is one of most abundant polymer after cellulose. However, its potential has yet to be completely recognized. Corn cobs contain a considerable reservoir of xylan. The aim of this work was to study some of the biological activities of xylan obtained from corn cobs after alkaline extraction enhanced by ultrasonication. Physical chemistry and infrared analyses showed 130 kDa heteroxylan containing mainly xylose:arabinose: galactose:glucose (5.0:1.5:2.0:1.2). Xylan obtained exhibited total antioxidant activity corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. Furthermore, xylan displayed high ferric chelating activity (70%) at 2 mg/mL. Xylan also showed anticoagulant activity in aPTT test. In antimicrobial assay, the polysaccharide significantly inhibited bacterial growth of Klebsiella pneumoniae. In a test with normal and tumor human cells, after 72 h, only HeLa tumor cell proliferation was inhibited (p < 0.05) in a dose-dependent manner by xylan, reaching saturation at around 2 mg/mL, whereas 3T3 normal cell proliferation was not affected. The results suggest that it has potential clinical applications as antioxidant, anticoagulant, antimicrobial and antiproliferative compounds.

Application of a feasible method for determination of biofilm antimicrobial susceptibility in staphylococci

Antunes ALS, Trentin DS, Bonfanti JW, Pinto CCF, Perez LRR, Macedo AJ, Barth AL. Application of a feasible method for determination of biofilm antimicrobial susceptibility in staphylococci. APMIS 2010; 118: 873–7.

A quantitative resazurin assay to determinate the viability of Trichomonas vaginalis and the cytotoxicity of organic solvents and surfactant agents

Trichomonas vaginalis causes trichomonosis, the most common, non-viral sexually transmitted disease. To test anti-Trichomonas agents, usually many with low water solubility, organic solvents and surfactant agents should be used. Therefore, the minimal inhibitory concentration (MIC) of acetone, methanol, ethanol, isopropanol, DMSO, Tween 20, Tween 80, and Triton X-100 was determined against T. vaginalis isolates using the quantitative resazurin method. Our results showed that solvents and surfactant agents can be employed as vehicles to test bioactive compounds at lower concentrations than MIC values and we suggest acetone and DMSO as preferential. Moreover, a new methodology is established to substitute or to complement the counting of viable trophozoites. The amount of resazurin reduced by T. vaginalis can be quantified by fluorescence spectroscopy, making the test a quantitative determination of cell viability. These results contribute for pharmacological investigations of bioactive compounds that need the use of solvents as solubilization vehicles to test anti-Trichomonas activity.

Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from fresh water and human blood. As determined by analyses of 16S rRNA gene sequences, the prosthecate strain FWC 38T was affiliated to the alphaproteobacterial genus Caulobacter, with Caulobacter henricii (96.8 %) and Caulobacter fusiformis (96.8 %) as its closest relatives. The non-prosthecate strain LMG 11050T and the prosthecate strain FWC 21T both belonged to the genus Phenylobacterium with Phenylobacterium koreense (96.9 %) and Phenylobacterium immobile (96.3 %) as the closest relatives. This affiliation was supported by chemotaxonomic data (polar lipids and cellular fatty acids). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of the novel strains from all hitherto recognized species of the genera Caulobacter and Phenylobacterium. The strains therefore represent novel species, for which the names Caulobacter mirabilis sp. nov. (type strain FWC 38T=LMG 24261T=CCUG 55073T), Phenylobacterium conjunctum (type strain FWC 21T=LMG 24262T=CCUG 55074T), the first described prosthecate Phenylobacterium species, and Phenylobacterium haematophilum sp. nov. (type strain LMG 11050T=CCUG 26751T) are proposed. Marker nucleotides within the 16S rRNA genes were determined for the genera Asticcacaulis, Brevundimonas, Caulobacter and Phenylobacterium and the description of the genus Phenylobacterium is emended.

Comparative analysis of the antioxidant and DNA protection capacities of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruits.

This study aimed to explore the antioxidant and DNA protection abilities of hydroalcoholic extracts from fruits of Anadenanthera colubrina (ACHE), Libidibia ferrea (LFHE) and Pityrocarpa moniliformis (PMHE). These extracts were tested by five antioxidant methods (phosphomolibdenium and reducing power assays; superoxide, hydrogen peroxide and nitric oxide scavenging) and DNA protection capacity. Total phenolic content was measured by Folin-Ciocalteu method. ACHE exhibited the highest phenolic content (578 mg/g GAE), followed by LFHE (460 mg/g GAE) and PMHE (448 mg/g GAE). In phosphomolibdenium assay, ACHE showed 24.81% of activity in relation to ascorbic acid, whereas LFHE and PMHE had 21.08% and 18.05%, respectively. These plants showed high ability to inhibit reactive species tested with IC50 values ranged from 10.66 to 14.37 μg/mL for superoxide radical; 26.05 to 45.43 μg/mL for hydrogen peroxide; 178.42 to 182.98 μg/mL for reducing power; and 199.2 to 283 μg/mL for nitric oxide. Furthermore, these extracts had capacity to break the DNA damage induced by hydroxyl radicals. The antioxidant activity of these plants is related with their higher phenolic content and show that they may be used as source of bioactive compounds, relevant to the maintenance of oxidative stability of the food matrix, cosmetics and/or pharmaceutical preparations.