Abstract
ABSTRACTThe 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.
Highlights
The microbial communities inhabiting chronic infections are often composed of spatially organized micrometer-sized, highly dense aggregates
We show that when grown under static conditions in SCFM2, P. aeruginosa grows as aggregates that readily shed bacteria to colonize new areas and seed new aggregates
As these aggregates have been proposed to be critical for P. aeruginosa tolerance to host factors and antibiotics [13, 19], we first assessed whether P. aeruginosa grows as aggregates in our synthetic sputum (SCFM2)
Summary
The microbial communities inhabiting chronic infections are often composed of spatially organized micrometer-sized, highly dense aggregates. We developed a growth medium that mimics chronic lung infection and promotes natural aggregate formation by the bacterium Pseudomonas aeruginosa. ® mbio.asm.org 1 discovery pipeline, alternative therapies are needed to treat the multidrug-resistant pathogens causing chronic infections One such treatment is the use of the natural viral parasites of bacteria called bacteriophage or phage. Prophylactic or rapid treatment following infection has been shown to be effective in preventing acute lung infection in mice; when treatment was delayed by as little as 2 h following pathogen introduction, efficacy decreased by almost 30% [8] These studies demonstrate that coinoculation of phage with bacterial cells is more effective than the use of phage to treat established infections. The reason for this decreased effect is poorly understood, and a basic question that remains is whether phage therapy should be limited to the early stages of an infection or whether a better understanding of phage-bacterium interactions could be used to more effectively treat established chronic infections
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