Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth. Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated. Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.

Bacteria form multicellular and resistant structures named biofilms. Biofilm formation starts with the attachment phase, and the molecular actors involved in this phase, except adhesins, are poorly characterized. There is growing evidence that phospholipids are more than simple structural bricks. They are involved in bacterial adaptive physiology, but little is known about their role in biofilm formation. Here, we report a mass spectrometry analysis of the phospholipid (PL) profile of several strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients. The aim of our study was to evaluate a possible link between the PL profile of a strain and its attachment phenotype. Our results showed that PL profile is strongly strain-dependent. The PL profile of P. aeruginosa PAO1, a collection strain, was different from those of 10 clinical isolates characterized either by a very low or a very high attachment capacity. We observed also that the clinical strain’s PL profiles varied even more importantly between isolates. By comparing groups of strains having similar attachment capacities, we identified one PL, PE 18:1-18:1, as a potential molecular actor involved in attachment, the first step in biofilm formation. This PL represents a possible target in the fight against biofilms.

Biofilms are the source of numerous issues in the food, pharmaceutical, and production industries, making their control a major component of economic and public health. Among anti-biofilm strategies, enzyme-based products that target the biofilm matrix have proven effectiveness against multiple bacterial species. We tested the efficacy of a commercial product, Baso Bionil SL40^® (SL40; Stockmeier France, Saint-Jacques de la lande, France), against biofilms of Pseudomonas aeruginosa under various conditions of temperature, concentration, pH, and incubation time. SL40 contains two enzymes: a subtilisin protease and an α-amylase glycosidase. Our results showed that SL40 removed up to 85% of the biofilm biomass compared to tris solutions. SL40’s efficacy was strongly influenced by the presence of the enzymes and both temperature and concentration. Enzymatic activity was maintained from 20 °C to 60 °C and at pH values ranging from 5 to 9, with effective concentrations corresponding to SL40 dilutions from 3/200 to 1/200 in 50 mM tris solutions. Additionally, we observed that the P. aeruginosa biofilm biomass after pH 9 tris solution treatment was twice compared to a H₂O washing. Our results confirm the potential of enzymes against biofilms, highlight the need to define optimal application conditions, and support their integration into combined strategies for complete biofilm removal.