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

Abstract

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. Pseudomonas aeruginosa is an opportunistic pathogen, which causes a variety of serious infections in immunocompromised patients and cystic fibrosis (CF) sufferers. The biofilm-forming ability of P. aeruginosa is thought to contribute to chronic P. aeruginosa infection of the CF lung. Biofilms are dense communities of bacteria, encased in an extracellular matrix, that are practically impossible to eradicate using available antimicrobial therapies. Understanding the mechanisms by which biofilm bacteria develop resistance to antibiotics is paramount to expanding the treatment options available to patients with chronic biofilm infections. In this study we have identified a novel mechanism of biofilm-specific antibiotic resistance. Extracellular DNA, a known component of biofilms, was found to induce antibiotic resistance. This previously unidentified function of DNA was due to its ability to bind and sequester cations, including magnesium, from the surrounding environment. This environmental cue was then detected by P. aeruginosa leading to induction of genes involved in modification of the cell surface component, lipopolysaccharide (LPS), resulting in physical alterations in the bacterial outer membrane (OM). These results demonstrate a novel function for DNA in biofilms and identify cation chelation by DNA as a previously unrecognized mechanism, which can explain the increased resistance of biofilms to antimicrobial agents.