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Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa(P. aeruginosa) is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosabiofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study, we investigate the structural and mechanical impacts of Ca2+supplementation in alginate-dominated biofilms grown in vitro, and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple-particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase or DNase I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in nontrivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

Biofilms are communities of bacteria embedded in a polymeric matrix which are found in infections and in environments outside the body. Breaking down the matrix renders biofilms more susceptible to physical disruption and to treatments such as antibiotics. Different species of bacteria, and different strains within the same species, produce different types of matrix polymers. This suggests that targeting specific polymers for disruption may be more effective than nonspecific approaches to disrupting biofilm matrixes. In this study, we treated Pseudomonas aeruginosabiofilms with enzymes that are specific to different matrix polymers. We measured the resulting alteration in biofilm mechanics using bulk rheology and changes in structure using electron microscopy. We find that, for biofilms grown in vitro, the effect of enzymatic treatment is greatest when the enzyme is specific to a dominant matrix polymer. Specifically matched enzymatic treatment tends to reduce yield strain and yield stress and increase the rate of biofilm drying, due to increased diffusivity as a result of network compromise. Electron micrographs qualitatively suggest that well-matched enzymatic treatments reduce long-range structure and shorten connecting network fibers. Previous work has shown that generic glycoside hydrolases can cause dispersal of bacteria from in vivo and ex vivo biofilms into a free-swimming state, and thereby make antibiotic treatment more effective. For biofilms grown in wounded mice, we find that well-matched treatments that result in the greatest mechanical compromise in vitro induce the least dispersal ex vivo. Moreover, we find that generic glycoside hydrolases have no measurable effect on the mechanics of biofilms grown in vitro, while previous work has shown them to be highly effective at inducing dispersal in vivo and ex vivo. This highlights the possibility that effective approaches to eradicating biofilms may depend strongly on the growth environment.