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.

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

“…pilA transcription in various P. aeruginosa strains was measured in 96-well plate liquid cultures similar to the method described in ref. 55. Briefly, bacterial strains carrying the pMS402+ ppilA plasmid were grown for 16 h at 37°C in 5 mL LB-Kan, 150 μg/mL Cultures were then diluted 1:20 in fresh media and grown to a standardized OD 600 = 0.15, and the OD 600 and relative luminescence of each culture were measured at 15-min intervals using a Synergy 4 microtiter plate reader (BioTek) for 5h.…”

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…The cause of such increased tolerances seen in biofilms has been investigated in a number of different studies, focusing on the extracellular matrix, the involvement of quorum sensing (QS), and the physiological factors observed within the biofilm [19,[26][27][28][29]. The extracellular matrix itself has been shown to obstruct the diffusion of some antimicrobial agents through both chemical and physiological means [27,30,31]. The matrix however is not selective against antimicrobial agents, and therefore the diffusion of many substances, such as oxygen, metabolites and waste products, can also be altered.…”

Genome‐wide transcriptome analysis of fluoroquinolone resistance in clinical isolates of Escherichia coli

“…[2][3][4][5][6] In E. coli cells, FQN can be extruded from the outer or inner membrane through the action of various efflux pumps. Previous studies have reported that acrAB, mdfA and norE are involved in FQN resistance.…”

“…The mechanisms of FQN resistance in various bacteria include biofilm formation, overexpression of multidrug efflux pump related genes and chromosomal mutations of DNA gyrase and topoisomerase IV. [2][3][4][5][6] Phage shock protein (psp) response and pilus operons play an important role in acquiring pathogenesis of UTI and biofilm formation in E. coli. [7][8][9] Many investigators are making every effort to overcome FQN resistance by investigating novel mechanisms.…”