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Phage Inhibit Pathogen Dissemination by Targeting Bacterial Migrants in a Chronic Infection Model
Sophie E Darch1,2,3, Kasper N Kragh4, Evelyn A Abbott1,2,3
1Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.
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 an in vitro model based on chronic Pseudomonas aeruginosa infection of the cystic fibrosis (CF) lung. This model utilizes a synthetic sputum medium that readily promotes the formation of P. aeruginosa aggregates with sizes similar to those observed in human CF lung tissue. Using high-resolution imaging, we exploited this model to elucidate the life history of P. aeruginosa and 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. aeruginosa was 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 allows P. aeruginosa to tolerate phage therapy during chronic infection without the need for genetic mutation.IMPORTANCE Bacteria 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 bacterium Pseudomonas aeruginosa High-resolution, single-cell microscopy allowed us to characterize P. 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 allow P. aeruginosa to tolerate therapeutic bacteriophage addition, although this treatment limits P. aeruginosa dissemination by targeting migrants.
Insights
Microbial aggregates in chronic infections like cystic fibrosis lung infections help bacteria like Pseudomonas aeruginosa survive antimicrobial treatments. This study developed a model to show how aggregates form and protect bacteria from phage therapy.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacterial Pathogenesis
Background:
- Chronic infections harbor dense bacterial aggregates, hypothesized to confer tolerance to immune responses and antimicrobials.
- Limited understanding of aggregate formation mechanisms and tolerance due to lack of relevant experimental models.
- Pseudomonas aeruginosa in cystic fibrosis (CF) lungs forms aggregates, contributing to treatment resistance.
Purpose of the Study:
- To develop a biologically relevant in vitro model for studying Pseudomonas aeruginosa aggregate formation in a chronic infection context.
- To elucidate the life history and antimicrobial tolerance mechanisms of P. aeruginosa aggregates, particularly against bacteriophage therapy.
- To investigate the role of bacterial aggregates in P. aeruginosa tolerance to bacteriophage treatment in a synthetic sputum medium.
Main Methods:
- Development of a synthetic sputum medium to promote natural P. aeruginosa aggregate formation.
- Utilized high-resolution imaging to observe bacterial life history, including aggregate seeding, expansion, and dispersal.
- Assessed the impact of bacteriophage addition at different stages of aggregate formation on bacterial survival and dissemination.
Main Results:
- The synthetic sputum medium successfully promoted P. aeruginosa aggregate formation, mimicking sizes observed in CF lung tissue.
- P. aeruginosa exhibits a life cycle involving aggregate expansion and dispersal of migrant cells to seed new aggregates.
- Simultaneous addition of bacteriophage prevented aggregate formation; post-formation addition inhibited migrant dispersal but not aggregate survival due to exopolysaccharide production.
Conclusions:
- Bacterial aggregates in P. aeruginosa chronic infections provide a mechanism for tolerance to bacteriophage therapy without requiring genetic mutation.
- Aggregate formation and expansion are key to P. aeruginosa survival and persistence during chronic infections.
- Targeting migrant dispersal may be a strategy to limit P. aeruginosa dissemination and enhance bacteriophage therapy efficacy in chronic infections.