title: Phage Inhibit Pathogen Dissemination by Targeting Bacterial Migrants in a Chronic Infection Model.
abstract: The microbial communities inhabiting chronic infections are often composed of spatially organized micrometer-sized, highly dense aggregates. It has recently been hypothesized that aggregates are responsible for the high tolerance of chronic infections to host immune functions and antimicrobial therapies. Little is currently known regarding the mechanisms controlling aggregate formation and antimicrobial tolerance primarily because of the lack of robust, biologically relevant experimental systems that promote natural aggregate formation. Here, we developed anin vitromodel based on chronicPseudomonas aeruginosainfection of the cystic fibrosis (CF) lung. This model utilizes a synthetic sputum medium that readily promotes the formation ofP. aeruginosaaggregates with sizes similar to those observed in human CF lung tissue. Using high-resolution imaging, we exploited this model to elucidate the life history ofP. aeruginosaand the mechanisms that this bacterium utilizes to tolerate antimicrobials, specifically, bacteriophage. In the early stages of growth in synthetic sputum, planktonic cells form aggregates that increase in size over time by expansion. In later growth, migrant cells disperse from aggregates and colonize new areas, seeding new aggregates. When added simultaneously with phage,P. aeruginosawas readily killed and aggregates were unable to form. When added after initial aggregate formation, phage were unable to eliminate all of the aggregates because of exopolysaccharide production; however, seeding of new aggregates by dispersed migrants was inhibited. We propose a model in which aggregates provide a mechanism that allowsP. aeruginosato tolerate phage therapy during chronic infection without the need for genetic mutation.IMPORTANCEBacteria in chronic infections often reside in communities composed of micrometer-sized, highly dense aggregates. A primary challenge for studying aggregates has been the lack of laboratory systems that promote natural aggregate formation in relevant environments. Here, we developed a growth medium that mimics chronic lung infection and promotes natural aggregate formation by the bacteriumPseudomonas aeruginosaHigh-resolution, single-cell microscopy allowed us to characterizeP. aeruginosa's life history-seeding, aggregate formation, and dispersal-in this medium. Our results reveal that this bacterium readily forms aggregates that release migrants to colonize new areas. We also show that aggregates allowP. aeruginosato tolerate therapeutic bacteriophage addition, although this treatment limitsP. aeruginosadissemination by targeting migrants.
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.|Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.|John Ring LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Darch
authors.affiliation: Costerton Biofilm Center, Institute of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kasper N
authors.initials: KN
authors.lastname: Kragh
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.|Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.|John Ring LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Evelyn A
authors.initials: EA
authors.lastname: Abbott
authors.affiliation: Costerton Biofilm Center, Institute of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.|Department of Clinical Microbiology, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bjarnsholt
authors.affiliation: Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.|Department of Integrative Biology, University of Texas at Austin, Austin, Texas, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Bull
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA mwhiteley@austin.utexas.edu.|Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.|John Ring LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Marvin
authors.initials: M
authors.lastname: Whiteley
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001433:Bacteriolysis; D003550:Cystic Fibrosis / Q000150:complications; D024881:Drug Resistance, Bacterial; D006801:Humans; D050296:Microbial Viability; D008962:Models, Theoretical; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000502:physiology* / Q000821:virology*; D013183:Sputum / Q000382:microbiology*
chemical_list: D000900:Anti-Bacterial Agents