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

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      PLoS Pathogens

      Public Library of Science

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          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.

          Author Summary

          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.

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          Most cited references 65

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          Physiological heterogeneity in biofilms.

          Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.
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            Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.

            The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.
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              Persister cells and tolerance to antimicrobials.

              Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics. Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology. Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters. The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase. Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin). This shows that production of persisters depends on growth stage. Maintaining a culture of E. coli at early exponential phase by reinoculation eliminated persisters. This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics. Our data indicate that persisters are specialized survivor cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                November 2008
                November 2008
                21 November 2008
                : 4
                : 11
                Affiliations
                Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
                Schepens Eye Research Institute, United States of America
                Author notes

                Conceived and designed the experiments: HM SL. Performed the experiments: HM LCM SL. Analyzed the data: HM LCM SL. Wrote the paper: HM SL.

                Article
                08-PLPA-RA-0820R2
                10.1371/journal.ppat.1000213
                2581603
                19023416
                Mulcahy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 12
                Categories
                Research Article
                Infectious Diseases/Antimicrobials and Drug Resistance
                Infectious Diseases/Bacterial Infections
                Microbiology
                Microbiology/Cellular Microbiology and Pathogenesis
                Microbiology/Environmental Microbiology
                Microbiology/Innate Immunity
                Microbiology/Medical Microbiology

                Infectious disease & Microbiology

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