Efeito antibiofilme e citotoxicidade do hidróxido de cálcio associado ao óleo essencial de Melaleuca alternifolia / Antibiofilm effect and cytotoxicity of calcium hydroxide associated with the Melaleuca alternifolia essential oil

Objetivo: Avaliar e comparar os efeitos antimicrobiano e antibiofilme, e a citotoxicidade promovida pela associação do hidróxido de cálcio ao óleo essencial de Melaleuca alternifolia (MA), em diferentes concentrações, e ao propilenoglicol (PG). Métodos: As seguintes medicações compuseram os grupos experimentais: G1) HC/MA 1%; G2) HC/MA 5%; G3) HC/MA 10%; G4) HC/MA 20%; e G5) HC/PG. Solução salina 0,85% e meio DMEM serviram como controle nos testes antimicrobianos e de citotoxidade em fibroblastos do ligamento periodontal humano (FbLP), respectivamente. A atividade antimicrobiana (n = 12) foi avaliada por meio do teste de difusão em ágar. O efeito antibiofilme (n = 12) imediato das medicações foi avaliado por meio do teste de viabilidade bacteriana em biofilmes de 72 horas de E. faecalis, formados sobre discos de dentina e tratados por sete dias com as medicações. Após a coleta microbiológica do biofilme remanescente, os discos de dentina foram imersos em meio estéril e armazenados por mais sete dias, para a análise do efeito antibiofilme residual das medicações, quando nova coleta microbiológica foi realizada. A atividade metabólica de FbLP foi avaliada por meio do ensaio colorimétrico MTS (n = 9). Os valores médios dos halos de inibição, em mm, das unidades formadoras de colônia, e o percentual de atividade metabólica celular foram analisados pelos testes Kruskal-Wallis e post hoc Dunn (α = 5%). Resultados:Todas as medicações experimentais apresentaram superior ação antimicrobiana e antibiofilme comparadas ao controle, solução salina (p < 0,05), e mantiveram viáveis os FbLP, semelhante ao controle DMEM (p > 0,05). Conclusão: A associação do óleo essencial de Melaleuca alternifolia, nas concentrações de 1%, 5%, 10% e 20%, ao hidróxido de cálcio promoveu excelente ação antimicrobiana, antibiofilme e biocompatibilidade com fibroblastos, de forma semelhante à associação com propilenoglicol.

Aim:To evaluate and compare the antimicrobial and antibiofilm effect, as well as the cytotoxicity of calcium hydroxide (CH) associated with the Melaleuca alternifolia (MA)essential oil, in different concentrations, and with propylene glycol. Methods: The following medications composed the experimental groups: G1) CH/MA 1%; G2) CH/MA 5%; G3) CH/MA 10%; G4) CH/MA 20%; and G5) CH/PG. Saline solution and culture medium DMEM were used as a control in antimicrobial and cytotoxicity tests in human periodontal ligament fibroblasts (PDLF), respectively. The antimicrobial activity (n = 12) was evaluated by the disk-diffusion agar method. The immediate antibiofilm effect (n = 12) of the medications was evaluated for bacterial viability in 72 hours-biofilms of E. faecalis, formed on the dentin disc surface and treated for seven days with medications. After microbiological sampling of the remaining biofilm, the dentin discs were immersed in sterile culture medium and stored for another seven days, for analysis of the residual antibiofilm effect of the medications, when a new microbiological sampling was performed. PDLF viability was evaluated by MTS colorimetric assay (n = 9). The mean values of the inhibition halos, in mm, the colony forming units, and the metabolic cell activity percentage were analyzed by means of Kruskal-Wallis and post hoc Dunn (α = 5%) tests. Results:All of the experimental medications presented higher antimicrobial and antibiofilm effects, when compared to the saline solution control (p < 0.05), and maintained the PDLF feasible, similar to the DMEM control (p > 0.05). Conclusions:The association of the Melaleuca alternifolia essential oil, at concentrations of 1%, 5%, 10%, and 20%, with calcium hydroxide promoted an excellent antimicrobial and antibiofilm activity, and biocompatibility with fibroblasts, similarly to the association with propylene glycol.

Computational analysis suggests that virulence of Chromobacterium violaceum might be linked to biofilm formation and poly-NAG biosynthesis

Groups of genes that produce exopolysaccharide with a N-acetyl-D-glucosamine monomer are in the genome of several pathogenic bacteria. Chromobacterium violaceum, an opportunistic pathogen, has the operon hmsHFR-CV2940, whose proteins can synthesize such polysaccharide. In this work, multiple alignments among proteins from bacteria that synthesize such polysaccharide were used to verify the existence of amino acids that might be critical for pathogen activity. Three-dimensional models were generated for spatial visualization of these amino acid residues. The analysis carried out showed that the protein HmsR preserves the amino acids D135, D228, Q264 and R267, considered critical for the formation of biofilms and, furthermore, that these amino acids are close to each other. The protein HmsF of C. violaceum preserves the residues D86, D87, H156 and W115. It was also shown that these residues are also close to each other in their spatial arrangement. For the proteins HmsH and CV2940 there is evidence of conservation of the residues R104 and W94, respectively. Conservation and favorable spatial location of those critical amino acids that constitute the proteins of the operon indicates that they preserve the same enzymatic function in biofilm synthesis. This is an indicator that the operon hmsHFR-CV2940 is a possible target in C. violaceum pathogenicity.

Extension of the IsaViz software for the representation of metabolic and regulatory networks

In this work we developed an extension of IsaViz software, a RDF (Resource Description Framework) authoring tool, designed to be a graphical environment to build models of metabolic and regulatory networks. This environment, called Metabolic IsaViz, was linked to a genomic library of types and was modeled on the basis of ontologies. Biochemical pathways included data at sequence level (e.g., the amino acid sequence of enzymes), besides kinetic and thermodynamic parameters for the reactions. Models created with Metabolic IsaViz could be exported to pathways simulators through SBML (Systems Biology Markup Language), which allowed to analyze the pathway dynamics of target chemicals.