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      Complete Genome Sequence of the Cystic Fibrosis Pathogen Achromobacter xylosoxidans NH44784-1996 Complies with Important Pathogenic Phenotypes

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          Abstract

          Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-β-1,6- N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen.

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          Most cited references70

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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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            Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili.

            We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient-dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One-half of these mutations was found to perturb normal flagellar function. Using defined fli, flh, mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose-specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.
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              Mechanisms of biofilm resistance to antimicrobial agents.

              Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The physical and/or chemical structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                22 July 2013
                : 8
                : 7
                : e68484
                Affiliations
                [1 ]Costerton Biofilm Center, Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark
                [2 ]Department of Biology, University of Copenhagen, Copenhagen, Denmark
                [3 ]Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
                [4 ]Department of Pediatrics, Rigshospitalet, Copenhagen, Denmark
                [5 ]Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark
                [6 ]Department of Biomedical Sciences, CFIM, University of Copenhagen, Copenhagen, Denmark
                [7 ]Department of Biology, University of Copenhagen, Helsingør, Denmark
                [8 ]Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
                Ghent University, Belgium
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: THJ MAH PØJ LH LR AC MK WR CRH SE MH PK MA KQ MB CM MK OC MG SJS NH TB. Performed the experiments: THJ MAH PØJ LR AC WR MK SE MH PK MA KQ OC TB. Analyzed the data: THJ MAH PØJ LH LR AC WR MK CRH SE MH PK MA KQ MB CM MK OC MG SJS NH TB. Contributed reagents/materials/analysis tools: MAH PØJ KQ MK OC MG SJS TB. Wrote the manuscript: THJ MAH PØJ LH LR AC WR MK CRH SE MH PK MA KQ MB CM MK OC MG SJS NH TB.

                Article
                PONE-D-13-04233
                10.1371/journal.pone.0068484
                3718787
                23894309
                b31e5cb9-49b2-4335-8c21-fd3063f09d64
                Copyright @ 2013

                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.

                History
                : 29 January 2013
                : 29 May 2013
                Funding
                This work was supported by a grant from the Lundbeck Foundation to TB and by a grant from the Danish Strategic Research Council to MG. CFIM (Core Facility for Integrated Microscopy, The Faculty of Health, University of Copenhagen) is acknowledged for access to SEM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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