(462) Pseudomonas aeruginosa Biofilm Viscoelasticity is Length Scale-Dependent: Therapeutic Consequences

Background: Biofilm viscoelasticity is correlated with susceptibility to mechanical and chemical stress, and the cystic fibrosis (CF) airway facilitates robust biofilm formation. Hyperconcentrated and viscoelastic mucus promotes chronic inflammation and infection, resulting in increased mucin and DNA concentrations. Muco-modulating therapies decrease viscoelasticity via cleaving or disrupting interactions between the polymeric components (mucins and DNA). Similar to mucus, biofilm viscoelasticity is regulated by polymers including polysaccharides and DNA and is dependent upon the length scale. Macrorheology characterizes bulk properties relevant to structural rigidity, microrheology describes biofilm heterogeneity and the polymer network, and nanorheology quantifies nanoparticle diffusion through the biofilm. We hypothesize that biofilm viscoelasticity will vary with length scale, and that macro-, micro-, and nanorheology are essential to understanding the host-pathogen relationship in chronic CF respiratory infections. Together these data provide a better understanding of biofilm mechanics and inform the design of improved antibiofilm therapies.

Methods: Pseudomonas aeruginosa biofilms were grown in human airway mucus and evaluated with cone and plate rheometry (C&P), particle tracking microrheology (PTM), and a novel 1-dimensional migration assay (1-DMA). The diameter and surface chemistry of the fluorescent tracer particles used in PTM and 1-DMA were varied to evaluate a range of sizes and probe-biofilm interactions. Biofilms were also treated with DNase or dithiothreitol (DTT) to compromise structural integrity and then evaluated with all three assays. Susceptibility to tobramycin was evaluated via disruption and colony counting.

Results: Bulk measurements of P. aeruginosa biofilms with C&P were in agreement with previously published work and differ from bulk mucus rheology. Viscoelasticity measured via PTM varied with probe size and surface chemistry, though confocal microscopy suggested similar distribution of probes throughout the biofilm. The 1-DMA indicated that neutrally charged dextran probes had minimal interactions with the biofilm while poly-lysine (cationic) caused entanglement. Indeed, treating biofilms with poly-lysine increased biofilm macrorheology. Probes smaller than 20 nm in diameter diffused through the biofilm with water. Cleavage of polymeric components with DNase or DTT decreased microscopic moduli and universally improved probe diffusion through the biofilm. Further, decreased viscoelastic moduli correlated with increased susceptibility to tobramycin.

Conclusions: Biofilm viscoelasticity is associated with increased resistance to mechanical and chemical challenge. Thus, characterizing the length scale-dependent biophysical properties of biofilms will lead to improved treatment design. The ideal antibiofilm agent would thoroughly penetrate the biofilm, decrease viscoelastic moduli, and improve antibiotic efficacy. Targeting the polymeric components in the biofilm with micro- or nano-scale treatments that do not interact with the mesh will improve biofilm degradation and subsequent clearance. Nanoscale agents that are biofilm-inert diffused with water, suggesting that small, non-interacting agents may efficiently penetrate and degrade the polymeric mesh and ultimately improve antibiotic action.

Acknowledgements: This work was funded by the Cystic Fibrosis Foundation (BOUCHE19R0, HILL19G0, HILL20y2-OUT) and the NIH (T32HL007106-45)

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