Towards a better understanding of the effect of protein conditioning layers on microbial adhesion: a focused investigation of fibronectin and bovine serum albumin layers on SiO2 surfaces.

The interaction of foreign implants with their surrounding environment is significantly influenced by the adsorption of proteins on the biomaterial surfaces, playing a role in microbial adhesion. Therefore, understanding protein adsorption on solid surfaces and its effect on microbial adhesion is essential to assess the associated risk of infection. The aim of this study is to evaluate the effect of conditioning by fibronectin (Fn) or bovine serum albumin (BSA) protein layers of silica (SiO2) surfaces on the adhesion and detachment of two pathogenic microorganisms: Pseudomonas aeruginosa PAO1-Tn7-gfp and Candida albicans CIP 48.72. Experiments are conducted under both static and hydrodynamic conditions using a shear stress flow chamber. Through the use of very low wall shear stresses, the study brings the link between the static and dynamic conditions of microbial adhesion. The results reveal that the microbial adhesion critically depends on: (i) the presence of a protein layer conditioning the SiO2 surface, (ii) the type of protein and (iii) the protein conformation and organization in the conditioning layer. In addition, a very distinct adhesion behaviour of P. aeruginosa is observed towards the two tested proteins, Fn and BSA. This effect is reinforced by the amount of proteins adsorbed on the surface and their organization in the layer. The results are discussed in the light of atomic force microscopy analysis of the organization and conformation of proteins in the layers after adsorption on the SiO2 surface, as well as the specificity in bacterial behaviour when interacting with these protein layers. The study also demonstrates the very distinctive behaviours of the prokaryote P. aeruginosa PAO1-Tn7-gfp compared to the eukaryote C. albicans CIP 48.72. This underscores the importance of considering species-specific interactions between the protein conditioning layer and different pathogenic microorganisms, which appear crucial in designing tailored anti-adhesive surfaces.

NaCl-induced modulation of species distribution in a mixed P. aeruginosa / S. aureus /B.cepacia biofilm.

Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are notorious pathogens known for their ability to form resilient biofilms, particularly within the lung environment of cystic fibrosis (CF) patients. The heightened concentration of NaCl, prevalent in the airway liquid of CF patients' lungs, has been identified as a factor that promotes the growth of osmotolerant bacteria like S. aureus and dampens host antibacterial defenses, thereby fostering favorable conditions for infections. In this study, we aimed to investigate how increased NaCl concentrations impact the development of multi-species biofilms in vitro, using both laboratory strains and clinical isolates of P. aeruginosa, S. aureus, and B. cepacia co-cultures. Employing a low-nutrient culture medium that fosters biofilm growth of the selected species, we quantified biofilm formation through a combination of adherent CFU counts, qPCR analysis, and confocal microscopy observations. Our findings reaffirmed the challenges faced by S. aureus in establishing growth within 1:1 mixed biofilms with P. aeruginosa when cultivated in a minimal medium. Intriguingly, at an elevated NaCl concentration of 145 mM, a symbiotic relationship emerged between S. aureus and P. aeruginosa, enabling their co-existence. Notably, this hyperosmotic environment also exerted an influence on the interplay of these two bacteria with B. cepacia. We demonstrated that elevated NaCl concentrations play a pivotal role in orchestrating the distribution of these three species within the biofilm matrix. Furthermore, our study unveiled the beneficial impact of NaCl on the biofilm growth of clinically relevant mucoid P. aeruginosa strains, as well as two strains of methicillin-sensitive and methicillin-resistant S. aureus. This underscores the crucial role of the microenvironment during the colonization and infection processes. The results suggest that hyperosmotic conditions could hold the key to unlocking a deeper understanding of the genesis and behavior of CF multi-species biofilms.

Ultraviolet control of bacterial biofilms in microfluidic chips.

Polydimethylsiloxane (PDMS) microfluidic systems have been instrumental in better understanding couplings between physical mechanisms and bacterial biofilm processes, such as hydrodynamic effects. However, precise control of the growth conditions, for example, the initial distribution of cells on the substrate or the boundary conditions in a flow system, has remained challenging. Furthermore, undesired bacterial colonization in crucial parts of the systems, in particular, in mixing zones or tubing, is an important factor that strongly limits the duration of the experiments and, therefore, impedes our ability to study the biophysics of biofilm evolving over long periods of time, as found in the environment, in engineering, or in medicine. Here, we develop a new approach that uses ultraviolet-C (UV-C) light-emitting diodes (LEDs) to confine bacterial development to specific zones of interest in the flow channels. The LEDs are integrated into a 3D printed light guide that is positioned upon the chip and used to irradiate germicidal UV-C directly through the PDMS. We first demonstrate that this system is successful in controlling undesired growth of Pseudomonas aeruginosa biofilm in inlet and outlet mixing zones during 48 h. We further illustrate how this can be used to define the initial distribution of bacteria to perturb already formed biofilms during an experiment and to control colonization for seven days-and possibly longer periods of time-therefore opening the way toward long-term biofilm experiments in microfluidic devices. Our approach is easily generalizable to existing devices at low cost and may, thus, become a standard in biofilm experiments in PDMS microfluidics.

Seaweed Extracts as an Effective Gateway in the Search for Novel Antibiofilm Agents against Staphylococcus aureus.

Treatment of biofilm-associated infections has become a major challenge in biomedical and clinical fields due to the failure of conventional treatments in controlling this highly complex and tolerant structure. Therefore, the search for novel antibiofilm agents with increased efficacy as those provided by natural products, presents an urgent need. The aim of this study was to explore extracts derived from three algae (green Ulva lactuca, brown Stypocaulon scoparium, red Pterocladiella capillacea) for their potential antibiofilm activity against Staphylococcus aureus, bacterium responsible for several acute and chronic infections. Seaweed extracts were prepared by successive maceration in various solvents (cyclohexane (CH), dichloromethane (DCM), ethyl acetate (EA), and methanol (MeOH)). The ability of the different extracts to inhibit S. aureus biofilm formation was assessed using colony-forming unit (CFU) counts method supported by epifluorescence microscopic analysis. Effects of active extracts on the biofilm growth cycle, as well as on S. aureus surface hydrophobicity were evaluated. Results revealed the ability of four extracts to significantly inhibit S. aureus biofilm formation. These findings were supported by microscopy analyses. The gradual increase in the number of adherent bacteria when the selected extracts were added at various times (t0, t2h, t4h, t6h, and t24h) revealed their potential effect on the initial adhesion and proliferation stages of S. aureus biofilm development. Interestingly, a significant reduction in the surface hydrophobicity of S. aureus treated with dichloromethane (DCM) extract derived from U. lactuca was demonstrated. These findings present new insights into the exploration of seaweeds as a valuable source of antibiofilm agents with preventive effect by inhibiting and/or delaying biofilm formation.

Investigation of Direct and Retro Chromone-2-Carboxamides Based Analogs of Pseudomonas aeruginosa Quorum Sensing Signal as New Anti-Biofilm Agents.

Biofilm formation is considered a major cause of therapeutic failure because bacteria in biofilms have higher protection against antimicrobials. Thus, biofilm-related infections are extremely challenging to treat and pose major concerns for public health, along with huge economic impacts. Pseudomonas aeruginosa, in particular, is a "critical priority" pathogen, responsible for severe infections, especially in cystic fibrosis patients because of its capacity to form resistant biofilms. Therefore, new therapeutic approaches are needed to complete the pipeline of molecules offering new targets and modes of action. Biofilm formation is mainly controlled by Quorum Sensing (QS), a communication system based on signaling molecules. In the present study, we employed a molecular docking approach (Autodock Vina) to assess two series of chromones-based compounds as possible ligands for PqsR, a LuxR-type receptor. Most compounds showed good predicted affinities for PqsR, higher than the PQS native ligand. Encouraged by these docking results, we synthesized a library of 34 direct and 25 retro chromone carboxamides using two optimized routes from 2-chromone carboxylic acid as starting material for both series. We evaluated the synthesized carboxamides for their ability to inhibit the biofilm formation of P. aeruginosa in vitro. Overall, results showed several chromone 2-carboxamides of the retro series are potent inhibitors of the formation of P. aeruginosa biofilms (16/25 compound with % inhibition ≥ 50% at 50 µM), without cytotoxicity on Vero cells (IC50 > 1.0 mM). The 2,4-dinitro-N-(4-oxo-4H-chromen-2-yl) benzamide (6n) was the most promising antibiofilm compound, with potential for hit to lead optimization.

Seaweed Extracts: A Promising Source of Antibiofilm Agents with Distinct Mechanisms of Action against Pseudomonas aeruginosa.

The organization of bacteria in biofilms is one of the adaptive resistance mechanisms providing increased protection against conventional treatments. Thus, the search for new antibiofilm agents for medical purposes, especially of natural origin, is currently the object of much attention. The objective of the study presented here was to explore the potential of extracts derived from three seaweeds: the green Ulva lactuca, the brown Stypocaulon scoparium, and the red Pterocladiella capillacea, in terms of their antibiofilm activity against P. aeruginosa. After preparation of extracts by successive maceration in various solvents, their antibiofilm activity was evaluated on biofilm formation and on mature biofilms. Their inhibition and eradication abilities were determined using two complementary methods: crystal violet staining and quantification of adherent bacteria. The effect of active extracts on biofilm morphology was also investigated by epifluorescence microscopy. Results revealed a promising antibiofilm activity of two extracts (cyclohexane and ethyl acetate) derived from the green alga by exhibiting a distinct mechanism of action, which was supported by microscopic analyses. The ethyl acetate extract was further explored for its interaction with tobramycin and colistin. Interestingly, this extract showed a promising synergistic effect with tobramycin. First analyses of the chemical composition of extracts by GC-MS allowed for the identification of several molecules. Their implication in the interesting antibiofilm activity is discussed. These findings suggest the ability of the green alga U. lactuca to offer a promising source of bioactive candidates that could have both a preventive and a curative effect in the treatment of biofilms.