Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation

Effect of fibronectin amount and conformation on the strength of endothelial cell adhesion to HEMA/EMA copolymers

The effect of substrate surface hydrophobicity on fibronectin (Fn) adsorption and endothelial cell adhesion strength was studied. Bovine aortic endothelial cells (BAEC) were plated for 2 h with and without preadsorbed Fn on slides coated with homopolymers and copolymers of hydrophilic polyhydroxyethylmethacrylate (polyHEMA) and hydrophobic polyethylmethacrylate (polyEMA). The polarity of the substrate was determined by Wilhelmy plate contact angle. The amount of adsorbed Fn was determined using 125I-labeled Fn. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to detect gross conformational changes of adsorbed Fn on polyHEMA or polyEMA. BAEC were cultured in serum-free medium for 2 h and subjected to a brief exposure of laminar flow in a variable-height flow chamber that provided a range of shear stresses of 15-185 dynes/cm2. The critical shear stress to detach 50% of the cells increased with increasing EMA content to a maximum at 20% HEMA/80% EMA copolymer irrespective of the presence of preadsorbed Fn. However, the critical force increased even though there were similar amounts of Fn adsorbed on all substrates. ATR-FTIR spectroscopy showed only minor changes in beta-sheet structure of Fn adsorbed to polyHEMA and polyEMA. These results show that the force to detach cells did not increase solely with increasing amounts of adsorbed Fn; rather, these results indicate a more complex interplay involving both the amount and conformation of adsorbed Fn

The effect of substrate hydrophobicity on endothelial cell adhesion

The purpose of this study was to investigate the effect of substrate surface hydrophobicity on endothelial cell adhesion strength. Bovine aortic endothelial cells (BAEC) were plated for 2 h with and without preadsorbed fihronectin (Fn) on homopolymer and copolymer coated slides of hydrophilic polyhyd roxyet hylmethacrylate (polyHEMA) and hydrophobic polyethylmethecrylate (polyEMA). The polarity of the substrata was determined from Withelmy piate contact angle measurement Two concentrations of Fn (10 p.g/mL and 1 p.g,\u27mL) were adsorbed onto the substrates for 1 h. The amount of adsorbed Fn was determined using 125]-Iabeled Fn. The cells were subjected to a brief exposure of laminar flow in a variable height flow chamber that provided a range of shear stresses varying from 15 to 146 dynes/cm 2. With and without preadsorbed Fn, the critical shear stress (defined as the shear stress at which 50% of the cells remained attached) increased with increasing EMA content. With the 10 p.g/mL Fn incubation concentration, the amount of adsorbed Fn increased with increasing EMA content. With the 1 p.g/mL Fn incubation concentration, the amount of Fn adsorbed was similar on all substrates. Therefore, BAEC adhesion strength is dependent on more than just the amount of Fn adsorbed (i.e. Fn conformation), and any difference in cell adhesion can ultimately be attributed to the substrate\u27s hydrophobicity

An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal

tolerance; dispersal