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.

Self-assembly of 33-mer gliadin peptide oligomers.

The 33-mer gliadin peptide, LQLQPF(PQPQLPY)3PQPQPF, is a highly immunogenic peptide involved in celiac disease and probably in other immunopathologies associated with gliadin. Herein, dynamic light scattering measurements showed that 33-mer, in the micromolar concentration range, forms polydisperse nano- and micrometer range particles in aqueous media. This behaviour is reminiscent of classical association of colloids and we hypothesized that the 33-mer peptide self-assembles into micelles that could be the precursors of 33-mer oligomers in water. Deposition of 33-mer peptide aqueous solution on bare mica generated nano- and microstructures with different morphologies as revealed by atomic force microscopy. At 6 µM, the 33-mer is organised in isolated and clusters of spherical nanostructures. In the 60 to 250 µM concentration range, the spherical oligomers associated mainly in linear and annular arrangements and structures adopting a "sheet" type morphology appeared. At higher concentrations (610 µM), mainly filaments and plaques immersed in a background of nanospherical structures were detected. The occurrence of different morphologies of oligomers and finally the filaments suggests that the unique specific geometry of the 33-mer oligomers has a crucial role in the subsequent condensation and organization of their fractal structures into the final filaments. The self-assembly process on mica is described qualitatively and quantitatively by a fractal diffusion limited aggregation (DLA) behaviour with the fractal dimension in the range of 1.62 ± 0.02 to 1.73 ± 0.03. Secondary structure evaluation of the oligomers by Attenuated Total Reflection FTIR spectroscopy (ATR-FTIR) revealed the existence of a conformational equilibrium of self-assembled structures, from an extended conformation to a more folded parallel beta elongated structures. Altogether, these findings provide structural and morphological information about supramolecular organization of the 33-mer peptide, which might offer new perspectives for the understanding and treatment of gliadin intolerance disorders.

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.

Have flagella a preferred orientation during early stages of biofilm formation?: AFM study using patterned substrates.

Biofilm development involves several stages and flagellar expression of bacteria is considered an important factor in this process. However, its role in the earliest stage of biofilm development is not yet clear. In order to analyse this topic, Pseudomonas fluorescens samples were trapped on a patterned gold surface with sub-microtrenches (ST) so as to hinder their motility, and nanostructured gold with random orientation (SR) was used as control substrate. Atomic force microscopic (AFM) observations were made on untreated samples. Initially, ca. 75% of the flagella on ST and 85% of flagella on SR are oriented towards the neighbouring bacteria. Some of them made contact and surrounded the cells. Subsequently, 2-D raft structures formed on SR inert substrates with lateral curly flagella, while those at the poles of the rafts turned towards the nearest cell group. A few flagella and the formation of 3-D bacterial structures were observed on toxic substrates like copper. Results showed that patterned substrates are suitable tools to detect the orientation of flagella in the earliest stage of biofilm formation on solid opaque surfaces avoiding sample pre-treatment.

Spontaneous adsorption of silver nanoparticles on Ti/TiO2 surfaces. Antibacterial effect on Pseudomonas aeruginosa.

Titanium is a corrosion-resistant and biocompatible material widely used in medical and dental implants. Titanium surfaces, however, are prone to bacterial colonization that could lead to infection, inflammation, and finally to implant failure. Silver nanoparticles (AgNPs) have demonstrated an excellent performance as biocides, and thus their integration to titanium surfaces is an attractive strategy to decrease the risk of implant failure. In this work a simple and efficient method is described to modify Ti/TiO(2) surfaces with citrate-capped AgNPs. These nanoparticles spontaneously adsorb on Ti/TiO(2), forming nanometer-sized aggregates consisting of individual AgNPs that homogeneously cover the surface. The modified AgNP-Ti/TiO(2) surface exhibits a good resistance to colonization by Pseudomonas aeruginosa, a model system for biofilm formation.

Nano/microscale order affects the early stages of biofilm formation on metal surfaces.

The adhesion of Pseudomonas fluorescens was studied on nano/microengineered surfaces. Results show that these bacteria formed well-defined aggregates on randomly oriented nanosized granular gold substrates. These aggregates consist of aligned ensembles of bacteria, with some of them strongly elongated. This kind of biological structure was not found on ordered engineered surfaces because bacterial alignment and cell-to-cell sticking were hindered. Importantly, differences in cell morphology, length, orientation, and flagellation were observed between bacteria attached on the ordered nano/microstructures and the randomly ordered surfaces. The implications of the results are related to the design of engineered surfaces to enhance (nanostructured filters) or inhibit (medical implants and industrial biofouling) bacterial colonization on the surfaces and to the biocontrol of soil ecosystems.

Silver electrodeposition on nanostructured gold: from nanodots to nanoripples.

Silver nanodots and nanoripples have been grown on nanocavity-patterned polycrystalline Au templates by controlled electrodeposition. The initial step is the growth of a first continuous Ag monolayer followed by preferential deposition at nanocavities. The Ag-coated nanocavities act as preferred sites for instantaneous nucleation and growth of the three-dimensional metallic centres. By controlling the amount of deposited Ag, dots of approximately 50 nm average size and approximately 4 nm average height can be grown with spatial and size distributions dictated by the template. The dots are in a metastable state. Further Ag deposition drives the dot surface structure to nanoripple formation. Results show that electrodeposition on nanopatterned electrodes can be used to prepare a high density of nanostructures with a narrow size distribution and spatial order.

Stability analysis of branched silver electrodeposits: solid phase growth under a marginally stable regime.

The mass-transport controlled growth of silver deposits at the early stage of multiple bump formation, and when a silver single needle growth regime is attained, are investigated. Linear stability analysis as proposed by Barkey, Muller, and Tobias [J. Electrochem. Soc. 136, 2199 (1989)] is applied to kinetic mesoscale data. A reasonable correlation between the current density and the average amplitude of unstable perturbation is established.