Corrosion protection of AZ31 alloy and constrained bacterial adhesion mediated by a polymeric coating obtained from a phytocompound.

The prevention of microbial biofilm formation on a biomaterial surface is crucial in avoiding implants failures and the development of antibiotic resistant bacteria. It was reported that biodegradable Mg alloys may show antimicrobial effects due to the alkalinization of the corroding area. However, this issue is controversial and deserves a detailed study, since the processes occurring at the [biodegradable metal/biological medium] interface are complex and varied. Results showed that bacterial adhesion on AZ31 was lower than that of the titanium control and revealed that was dependent on surface composition, depicting some preferential sites for bacterial attachment (C-, P-, O-containing corrosion products) and others that are particularly avoided (active corrosion sites). As a key challenge, a strategy able to improve the performance of Mg alloys by both, reducing the formation of corrosion products and inhibiting bacterial adhesion was subsequently developed. A polymeric layer (polyTOH) was obtained by electropolymerization of thymol (TOH), a phytophenolic compound. The polyTOH can operate as a multifunctional film that improves the surface characteristics of the AZ31 Mg alloy by enhancing corrosion resistance (ions release was reduced to almost the half during the first days) and create an anti-adherent surface (bacterial attachment was 30-fold lower on polyTOH-AZ31 than on non-coated Mg alloy and 200-fold lower than Ti control and was constrained to specific regions). This anti-adherent property implies an additional advantage: enhancement of the efficacy of antibiotic treatments.

Assay for transposase-accessible chromatin and circularized chromosome conformation capture, two methods to explore the regulatory landscapes of genes in zebrafish.

Accurate transcriptional control of genes is fundamental for the correct functioning of organs and developmental processes. This control depends on the interplay between the promoter of genes and other noncoding sequences, whose interaction is mediated by 3D chromatin arrangements. Thus, the detailed description of transcriptional regulatory landscapes is essential to understand the mechanisms of transcriptional regulation. However, to achieve that, two important challenges have to be faced: (1) the identification of the noncoding sequences that contribute to gene transcription and (2) the association of these sequences to the respective genes they control. In this chapter, we describe two protocols that allow overcoming these important challenges: the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and circularized chromosome conformation capture (4C-seq). ATAC-seq is a very efficient technique that, using a very low number of cells as starting material, allows the identification of active chromatin regions genome wide, whereas 4C-seq detects the subset of sequences that interact specifically with the promoter of a given gene. When combined, both techniques provide a comprehensive snapshot of the regulatory landscapes of developmental genes. The protocols we present here have been optimized for teleost fish samples, zebrafish and medaka, allowing the in-depth study of transcriptional regulation in these two emerging animal models. Given the amenability and easy genetic manipulation of these two experimental systems, we anticipate that they will be important in revealing general principles of the vertebrate regulatory genome.

The importance of 2D aggregates on the antimicrobial resistance of Staphylococcus aureus sessile bacteria.

Biofilms formed on implanted devices are difficult to eradicate. Adhesion mechanism, high bacterial density, aggregation, induction of persisters and stressed bacteria are some of the factors considered when the antimicrobial resistance of these biofilms is analyzed. The aim of this work was to provide an alternative approach to the understanding of this issue by using a specially designed experimental set up that includes the use of microstructured (MS) surfaces (potential inhibitors of bacterial aggregation) in combination with antimicrobial agents (streptomycin and levofloxacin) against Staphylococcusaureus attached cells. Biofilms formed on smooth surfaces were used as plain controls (biofilmed-PC) characterized by the formation of dense 2D bacterial aggregates. Results showed bacterial persistence when streptomycin or levofloxacin were applied to PC-biofilms. The antimicrobial activity of both antibiotics was enhanced when bacteria were attached on MS, where single cells or small aggregates were observed. Thus, dense 2D aggregates of bacteria seem to be crucial as a required previous stage to develop the antimicrobial resistance.

Synergistic antimicrobial effect against early biofilm formation: micropatterned surface plus antibiotic treatment.

The detrimental effects of biofilms are a cause of great concern in medical, industrial and environmental areas. In this study, we proposed a novel eradication strategy consisting of the combined use of micropatterned surfaces and antibiotics on biofilms to reduce the rate of bacterial colonisation. Pseudomonas fluorescens biofilms were used to perform a comparative evaluation of possible strategies to eradicate these biological layers. First, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration of planktonic cultures were determined. Subsequently, adhesion of bacteria on microstructured gold surfaces (MS) with patterned features that were similar to the bacterial diameter as well as on smooth nanostructured gold (NS) was assessed. As expected, lower bacterial attachment as well as inhibition of bacterial aggregation were observed on MS. The effect of streptomycin treatment (ST) in the concentration range 1-4 mg/L (0.25-1× MIC) on biofilms grown on MS and NS was also evaluated. The combined strategy involving the use of micropatterned surfaces and antibiotic treatment (MS+ST) to eradicate Pseudomonas biofilms was then investigated. Results showed a synergistic effect of MS+ST that yielded a reduction of ≥1000-fold in the number of surviving biofilm bacteria with respect to those obtained with single ST or MS. The combined strategy may be a significant contribution to the eradication of biofilms from different environments. In addition, the important role of early monolayer bacterial aggregates in increasing resistance to antimicrobial agents was demonstrated.