Evaluation of leguminous lectins activities against bacterial biofilm formation

Biofilms are composed by microbial cells that are irreversibly associated with a surface and enclosed in a matrix of polymeric material. Lectins are sugar binding proteins of non immune origin that agglutinate cells and ⁄ or precipitate glycoconjugate molecules. Due to their capacity to bind and recognize specific carbohydrates, lectins can be a potent tool in biofilm studies. The search for potential phytochemicals as anti-biofilm agents has become an active area of research, and these proteins can bind to the bacteria or prevent the interaction with the surface and consequently decrease biofilm formation. Thus, the present work aims to evaluate in vitro the antibacterial activity of plant lectinsfrom Canavalia genus against a panel of bacteria of medical relevance, and to inspect their capacity to interfere on the initial adhesion events and biofilm formation. The assays were carried out using different concentrations of leguminous lectins, isolated from Canavalia ensiformis (ConA), C. maritima (ConM) and C. boliviana (ConBol). The effect of lectins was tested on Klebsiella oxytoca ATCC13182, Pseudomonas aeruginosa ATCC10145, Staphylococcus epidermidis CECT231 and Staphylococcus aureus. The bacterial planktonic growth in the presence of the lectins was determined trough absorbance measurement at 640 nm. Adhesion and biofilm assays were performed in polystyrene plates, and chalenged with the three lectins. The biomass accumulated was quantified using crystal violet staining. The results showed that ConA emerged as the most promising lectin since it clearly reduced the bacterial plankctonic growth, specially of the Gram+ strains, with MIC values ranging between 30 and 125 μg/mL. ConA also disturbed the initial adhesion events of all bacteria and disturbed the biofilm formation ability of the Staphylococcus species for all the concentrations tested. Concerning Gram- bacteria, its biofilm formation ability was only prejudiced with higher concentrations of the lectin. Therefore, the results seem to highlight that the antimicrobial activity of ConA was more noticeable in the disturbance of bacterial adhesion and biofilm formation than impairing planktonic growth. In conclusion, our results show that lectins, an important class of natural products, possess promising antibiofilm activity, suggesting that they may have therapeutic potential for the pharmacological treatment of biofilm-associated infections.IBB-CEB, FCT (PTDC/SAU-ESA/64609/2006) and CAPE

Effect of cashew (Anacardium occidentale L.) peduncle bagasse extract on Streptococcus mutans and its biofilm

This study investigated the antimicrobial activity of cashew (Anacardium occidentale L.) peduncle bagasse extract. Cashewpeduncle bagasse extract was prepared, and its minimal inhibitory concentration (MIC) and minimal bactericidal concentration(MBC), and effect on biofilm formation were determined against the strain Streptococcus mutans UA159. The bagasse extractshowed MIC at a concentration of 500 μg/mL and MBC at 1000 μg/mL against S. mutans. At 250 μg/mL, the extract significantlyreduced the biofilm formation of S. mutans, possibly associated with its effect on planktonic cells. The results of this study showedthat cashew peduncle residue has biomedical potential as an antimicrobial agent against an important pathogen responsible forthe formation of biofilm and subsequent dental caries.(Efeito do extrato do bagasso do pedúnculo do caju (Anacardium occidentale L.) sobre Streptococcus mutans e seubiofilme). Este estudo investigou a atividade antimicrobiana do extrato do bagaço do pedúnculo do caju (Anacardium occidentaleL.). O extrato do bagasse do caju foi preparado e sua concentração inibitória mínima (CIM), concentração bactericida mínima(CBM) e seu efeito sobre a formação de biofilme foram determinadas sobre a estirpe Streptococcus mutans AU 159. O extratodo bagaço apresentou CIM a uma concentração de 500 μg/mL e CBM a 1000 μg/mL contra S. mutans. A 250 μg/mL, o extratoreduziu significantemente a formação de biofilme de S. mutans, possivelmente associado ao seu efeito sobre células planctônicas.Os resultados desse estudo mostraram que o resíduo do pedúnculo do caju tem potencial biomédico como agente antimicrobianocontra um importante patógeno responsável pela formação de biofilme e subsequentemente carie dental

Expression of recombinant buck (Capra hircus) spermadhesinin a prokaryotic system

The low purification efficiency and the incomplete characterization of buck spermadhesins (Bdhs) prompted us to establish an effective system to produce recombinant Bdhs (rBdhs). The Bdh-4 cDNA was inserted in a prokaryotic expression plasmid pTrcHis TOPO to produce a His6 fusion protein in E. coli Top10 cells. The recombinant clones were selected by growth in ampicillin-containing medium, PCR amplifications and nucleotide sequencing. The recombinant protein synthesis was monitored by SDS-PAGE followed by immunoblotting using a monoclonal anti-His antibody. The expression of the rBdh-4 was achieved at 0.1 to 2.0 mM IPTG after 2 to 6 h of induction. A greater production of rBdh-4 (P < 0.001) was obtained with 0.1 mM IPTG after 2 h of induction. The apparent molecular weight of rBdh-4 was 15.85 ± 0.09 kDa. This result agrees with the theoretical molecular weight of 16.5 kDa predicted from the nucleotide sequence. In conclusion, an effective rBdh-4 expression system was established in order to provide a good tool for studying the biofunctions of buck spermadhesins.(Expressão da espermadesina recombinante de bode (Capra hircus) em sistema procariótico). A baixa eficiência de purificação e a incompleta caracterização das espermadesinas de bode (Bdhs) nos levou a estabelecer um sistema efetivo para produzir as Bdhs recombinantes (rBdhs). O cDNA da Bdh-4 foi inserido no plasmídeo de expressão procariótico pTrcHis TOPO para produzir uma proteína de fusão His6 em células de E.coli Top10. Os clones recombinantes foram crescidos em meio contendo ampicilina, amplificação por PCR e sequenciamento de nucleotídeos. A síntese da proteína recombinante foi monitorada por SDS-PAGE seguida por imunobloting usando anticorpo monoclonal anti-His. A expressão da rBdh-4 foi conseguida de 0,1 a 2,0 mM de IPTG e depois de 2 a 6 H de indução. A maior produção da rBdh-4 (

Effect of algae and plant lectins on planktonic growth and biofilm formation in clinically relevant bacteria and yeasts

This study aimed to evaluate the abilities of plant and algae lectins to inhibit planktonic growth and biofilm formation in bacteria and yeasts. Initially, ten lectins were tested on Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella oxytoca, Pseudomonas aeruginosa, Candida albicans, and C. tropicalis at concentrations of 31.25 to 250 μg/mL. The lectins from Cratylia floribunda (CFL), Vatairea macrocarpa (VML), Bauhinia bauhinioides (BBL), Bryothamnion seaforthii (BSL), and Hypnea musciformis (HML) showed activities against at least one microorganism. Biofilm formation in the presence of the lectins was also evaluated; after 24 h of incubation with the lectins, the biofilms were analyzed by quantifying the biomass (by crystal violet staining) and by enumerating the viable cells (colony-forming units). The lectins reduced the biofilm biomass and/or the number of viable cells to differing degrees depending on the microorganism tested, demonstrating the different characteristics of the lectins. These findings indicate that the lectins tested in this study may be natural alternative antimicrobial agents; however, further studies are required to better elucidate the functional use of these proteins.This study was supported by the CAPES (Brazil) under the BEX NT 2052/11NT3 Project, by the IBB-CEB and FCT (Portugal), by the European Community Fund FEDER, and by the COMPETE Program under the auspices of the PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480 Project. Kyria Santiago Nascimento, Alexandre Holanda Sampaio, Benildo Sousa Cavada, and Edson Holanda Teixeira are Senior Fellows of CNPq. Mr. David Martin helped with the English editing of the paper that was also revised by AJE (American Journal Experts)

Prophylactic outcomes of casbane diterpene in Candida albicans and Candida glabrata biofilms

Biofilms are surface associated communities of microorganisms embedded within a self-produced extracellular matrix and adhered on inert and biotic surfaces. These biological consortia are considered the most prevalent growth form of microorganisms. Biofilm formation is a potent virulence factor for a number of Candida species, as it confers significant tolerance to antimicrobial therapy, primarily by limiting the penetration of substances through the biofilm matrix. Casbane Diterpenes (CD) belongs to the class of diterpenoids isolated from few species of plants from Euphorbiaceae family with important anticancer and antibacterial activities. So, the goal of this study was to assess the antibiofilm effect of a Casbane Diterpene isolated from the stalks of Croton nepetaefolius against Candida albicans and Candida glabrata. Biofilms were developed within the 96- well microtiterplates in the presence of the CD. After 24 hours of growth, 100 μL of cells suspensions (1 x 106 cells ml-1 in Nutrient Broth) and 100 μL of solution of CD (500 - 31.5 μg/mL) were pipetted into each well and incubated for 24 h at 37ºC in an orbital shaker at 120 rpm. Biofilms formation was characterized by total biomass, through crystal violet (CV), and number of viable cells, expressed as log CFU per cm2. CD showed to be able to reduce the biofilm formation of C. albicans and C. glabrata. CD reduced C. albicans biomass in 82, 64, 57 and 27 % at the concentrations of 500, 250, 125 and 62.5 μg/mL, respectively. C. glabrata biomass was reduced in 68 and 26 % at 500 and 250 μg/mL. Regarding the number of viable cells embedded in the yeast biofilms, CD at 500 and 250 μg/mL reduced 2 and 1 log of C. albicans biofilm CFUs, and 2.5 and 1 log for C. glabrata, respectively. Regarding the high resistance and recalcitrance of Candida biofilms to the traditional therapies, CD emerges as a good prophylactic alternative to be used alone or in combination with other traditional drugs

Compositional analysis of cashew (Anacardium occidentale L.) peduncle bagasse ash and its in vitro antifungal activity against Fusarium species

Cashew (Anacardium occidentale L.) is a plant with a highly social and economic importance in the Northeast Region of Brazil. Cashew peduncle bagasse is one of the greatest sources of residues (90–94%) produced by the cashew agronomic industry. In this study, we prepared cashew peduncle bagasse ash and submitted it to compositional analysis and in vitro tests for antifungal activity against Fusarium species. This analysis indicated a crystallinity of around 73%, corresponding to the following soluble phases: potassium bicarbonate - KHCO3 (39.54%), potassium sulfate - K2SO4 (24.87%), and struvite-K - MgKPO4·6H2O (8.59%). The amorphous phases (around 27%) were identified as the insoluble fraction of the ash. The solution showed high antifungal activity against F. oxysporum, F. moniliforme and F. lateritium. The activity of this product was greater than that of Cercobin® (thiophanate-methyl), indicating that this material could possibly be used as a non-toxic antifungal agent.(Análise da composição das cinzas do bagaço do pedúnculo do cajú (Anacardium occidentale L.) e sua atividade antifúngica in vitro contra espécies de Fusarium.). O Cajueiro (Anacardium occidentale L.) é uma planta com uma grande importância social e econômica no Nordeste do Brasil. O bagaço do pedúnculo do caju é uma das maiores fontes de resíduos (90-94%) produzidos pela indústria cajueira. Neste estudo, foram preparadas cinzas do bagaço e submetidas à análise da composição e a testes de atividade antifúngica in vitro contra espécies de Fusarium. Esta análise indicou uma cristalinidade em torno de 73%, correspondendo às seguintes fases solúveis: bicarbonato de potássio - KHCO3 (39,54%), sulfato de potássio - K2SO4 (24,87%), e estruvita-K - MgKPO4 • 6H2O (8,59%). As fases amorfas (cerca de 27%) foram identificadas como a fração insolúvel de cinzas. A solução apresentou alta atividade antifúngica contra F. oxysporum, F. moniliforme e F. lateritium. Sua ação foi maior do que o Cercobin® (tiofanato metílico), indicando uma possível utilização como um agente antifúngico não tóxico

Antibacterial and antioxidant activities of Derriobtusone A isolated from Lonchocarpus obtusus

This study evaluated the effect of derriobtusone A, a flavonoid isolated from Lonchocarpus obtusus, on two important pathogenic bacteria, Staphylococcus aureus and Escherichia coli, as well as its antioxidant activity and toxicity. Planktonic growth assays were performed, and the inhibition of biofilm formation was evaluated. In addition, antioxidant activity was assessed by DPPH radical scavenging assay, ferrous ion chelating assay, ferric-reducing antioxidant power assay, and β-carotene bleaching assay. Toxicity was evaluated by the brine shrimp lethality test. Results showed that derriobtusone A completely inhibited the planktonic growth of S. aureus at 250 and 500 μg/mL; however, it did not have the same activity on E. coli. Derriobtusone A reduced the biomass and colony-forming unit (cfu) of S. aureus biofilm at concentrations of 250 and 500 μg/mL. In various concentrations, it reduced the biofilm biomass of E. coli, and, in all concentrations, it weakly reduced the cfu. Derriobtusone A showed highly efficient antioxidant ability in scavenging DPPH radical and inhibiting β-carotene oxidation. The compound showed no lethality to Artemia sp. nauplii. In conclusion, derriobtusone A may be an effective molecule against S. aureus and its biofilm, as well as a potential antioxidant compound with no toxicity.This study was supported by CAPES (Brazil) through the BEX NT 2052/11NT3 Project and by IBB-CEB and FCT (Portugal) and European Community Fund FEDER, through Program COMPETE, in the ambit of Project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480. Otilia Deusdenia Loiola Pessoa, Benildo Sousa Cavada, and Edson Holanda Teixeira are Senior Fellows of CNPq. Mr. David Martin helped with the English editing of the paper