title: Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System ExhibitsIn Vivo-Like Characteristics.
abstract: Alginate beads represent a simple and highly reproduciblein vitromodel system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated withPseudomonas aeruginosaand followed for up to 72 h. Confocal microscopy revealed thatP. aeruginosaformed dense clusters similar in size toin vivoaggregates observedex vivoin cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3-as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulatedP. aeruginosaconfirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulatedP. aeruginosaexhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth ofP. aeruginosain alginate beads represent a simple and flexiblein vivo-like biofilm model system, wherein bacterial growth exhibits central features ofin vivobiofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3-as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosahas been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.
authors.affiliation: Costerton Biofilm Centre, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Majken
authors.initials: M
authors.lastname: Sønderholm
authors.affiliation: Costerton Biofilm Centre, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kasper Nørskov
authors.initials: KN
authors.lastname: Kragh
authors.affiliation: Marine Biology Section, Department of Biology, University of Copenhagen, Elsinore, Denmark.
authors.forename: Klaus
authors.initials: K
authors.lastname: Koren
authors.affiliation: Costerton Biofilm Centre, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.|Department of Clinical Microbiology, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Tim Holm
authors.initials: TH
authors.lastname: Jakobsen
authors.affiliation: College of Natural Sciences, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Darch
authors.affiliation: Costerton Biofilm Centre, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Maria
authors.initials: M
authors.lastname: Alhede
authors.affiliation: Department of Clinical Microbiology, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Peter Østrup
authors.initials: PØ
authors.lastname: Jensen
authors.affiliation: College of Natural Sciences, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Marvin
authors.initials: M
authors.lastname: Whiteley
authors.affiliation: Marine Biology Section, Department of Biology, University of Copenhagen, Elsinore, Denmark.|Climate Change Cluster, University of Technology Sydney, Ultimo, New South Wales, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Kühl
authors.affiliation: Costerton Biofilm Centre, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark tbjarnsholt@sund.ku.dk.|Department of Clinical Microbiology, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bjarnsholt
mesh_terms: D000332:Aerobiosis; D000464:Alginates; D001422:Bacterial Adhesion; D001672:Biocompatible Materials; D004579:Electron Transport; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008863:Microspheres; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000502:physiology*
chemical_list: D000464:Alginates; D001672:Biocompatible Materials; D006603:Hexuronic Acids; D009566:Nitrates; D020723:Glucuronic Acid; D010100:Oxygen
keywords: Pseudomonas aeruginosa; antibiotics; biofilm; chronic infection; growth; model system; respiration; spatial structure