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 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.

Quantitative Models of Phage-Antibiotics Combination Therapy

“…For example, during chronic infections spatially organized bacterial aggregates of P.a. protect themselves against phage killing by producing exopolysaccharides 30 . Furthermore, modelling efforts have shown that spatial structure affects the therapeutic success of phage therapy 31 and phage-antibiotic combination therapy 32 .…”

Evolutionary dynamics of phage resistance in bacterial biofilms

“…On the basis of simulations and experiments we can also make predictions about the spatial architectures of phage-host interaction that are particular to biofilm environments. Prior work from our groups and others have shown that phages can be immobilized within the biofilm matrix, where they are blocked from gaining access to otherwise susceptible cells (17,18). Our observations here indicate that whenever phage movement is limited within biofilms, it is likely that the extracellular matrix (which is modeled implicitly here as the factor controlling phage diffusion) contains phages which are blocked from host access but not necessarily inert; they remain a threat to susceptible cells after the dispersion of biofilms due to mechanical disturbance or induced by bacteria themselves after depleting local nutrient supplies (53,54).…”

“…This process has been studied for decades, however phage resistance evolution has received little attention in the context of biofilms, in which cells adhere to surfaces and embed themselves in a secreted polymer matrix (14)(15)(16). Biofilm growth is thought to be the most common mode of bacterial life, but we are just beginning to understand the mechanistic and ecological details of phage-bacteria interaction within them (9,(17)(18)(19).…”

“…The extracellular matrix of Pseudomonas aeruginosa, for instance, can block the diffusion of antibiotics such as tobramycin (22,23). Biofilm matrix secreted by Escherichia coli and Pseudomonas aeruginosa can also alter phage movement (17,18). The spatial and temporal complexity of biofilm communities make it difficult to anticipate how they will impact phage-bacteria interaction, including the evolution of physiological resistance to phage attack.…”

“…Optimizing phages for this purpose, including an understanding of phage resistance evolution among host bacteria, requires a thorough look at phagebiofilm interactions (26,27). In particular, biofilm growth may have profound impacts on the relative advantages and disadvantages of phage resistance, which often involves mutations that also carry a growth rate cost, because the spatial structure within biofilms can potentially protect susceptible cells from phage exposure (17,18,28). Furthermore, in some conditions, even susceptible bacterial hosts can outrun a phage infection, for example, if host cell grow sufficiently quickly, or if phages are introduced at low multiplicity of infection (28)(29)(30)(31).…”