Specific Disruption of Established Pseudomonas aeruginosa Biofilms Using Polymer-Attacking Enzymes.

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 aeruginosa biofilms 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.

Assaying How Phagocytic Success Depends on the Elasticity of a Large Target Structure.

Biofilm infections can consist of bacterial aggregates that are an order of magnitude larger than neutrophils, phagocytic immune cells that densely surround aggregates but do not enter them. Because a neutrophil is too small to engulf the entire aggregate, it must be able to detach and engulf a few bacteria at a time if it is to use phagocytosis to clear the infection. Current research techniques do not provide a method for determining how the success of phagocytosis, here defined as the complete engulfment of a piece of foreign material, depends on the mechanical properties of a larger object from which the piece must be removed before being engulfed. This article presents a step toward such a method. By varying polymer concentration or cross-linking density, the elastic moduli of centimeter-sized gels are varied over the range that was previously measured for Pseudomonas aeruginosa biofilms grown from clinical bacterial isolates. Human neutrophils are isolated from blood freshly drawn from healthy adult volunteers, exposed to gel containing embedded beads for 1 h, and removed from the gel. The percentage of collected neutrophils that contain beads that had previously been within the gels is used to measure successful phagocytic engulfment. Both increased polymer concentration in agarose gels and increased cross-linking density in alginate gels are associated with a decreased success of phagocytic engulfment. Upon plotting the percentage of neutrophils showing successful engulfment as a function of the elastic modulus of the gel to which they were applied, it is found that data from both alginate and agarose gels collapse onto the same curve. This suggests that gel mechanics may be impacting the success of phagocytosis and demonstrates that this experiment is a step toward realizing methods for measuring how the mechanics of a large target, or a large structure in which smaller targets are embedded, impact the success of phagocytic engulfment.