Viscoelasticity of biofilms and their recalcitrance to mechanical and chemical challenges

Abstract

We summarize different studies describing mechanisms through which bacteria in a biofilm mode of growth resist mechanical and chemical challenges. Acknowledging previous microscopic work describing voids and channels in biofilms that govern a biofilms response to such challenges, we advocate a more quantitative approach that builds on the relation between structure and composition of materials with their viscoelastic properties. Biofilms possess features of both viscoelastic solids and liquids, like skin or blood, and stress relaxation of biofilms has been found to be a corollary of their structure and composition, including the EPS matrix and bacterial interactions. Review of the literature on viscoelastic properties of biofilms in ancient and modern environments as well as of infectious biofilms reveals that the viscoelastic properties of a biofilm relate with antimicrobial penetration in a biofilm. In addition, also the removal of biofilm from surfaces appears governed by the viscoelasticity of a biofilm. Herewith, it is established that the viscoelasticity of biofilms, as a corollary of structure and composition, performs a role in their protection against mechanical and chemical challenges. Pathways are discussed to make biofilms more susceptible to antimicrobials by intervening with their viscoelasticity, as a quantifiable expression of their structure and composition.

Keywords: antimicrobial penetration; biofilm; detachment; extracellular polymeric substances (EPS); structure; viscoelasticity.

Recalcitrance of biofilms against mechanical and chemical challenges has been looked at for ages from a microbiological perspective, but an approach based on viscoelastic properties of biofilms yields new insights in this recalcitrance.

Figure 1.

Key-properties of biofilms governing biofilm recalcitrance toward mechanical and chemical challenges. (A) Structure and composition govern biofilm resistance to environmental detachment and deformation forces. Resistance to deformation can be a time-dependent process yielding relaxation to an original shape over time. (B) Structure and composition govern the resistance of biofilms against chemical challenge in combination with altered phenotypes that are intrinsically more resistant to antimicrobials. (C) Composition and structure are jointly reflected in the viscoelasticity of a biofilm. Elasticity is generally presented as a spring with spring constant E, while the viscosity η (or its inverse, the fluidity φ) is shown as a dashpot. Springs and dashpots can be arranged in series (named ‘Maxwell’ element) or in parallel (called ‘Kelvin–Voigt’ element). Springs react immediately to an applied force, while dashpots dampen the speed of reaction. Usually, biological materials like biofilms cannot be represented by a single combination of springs and dashpots.

Figure 2.

2D FIB-SEM cross sections from a 3D image stack of OsO₄-stained, FIB-sectioned S. epidermidis ATCC 35984 biofilms prior to and after exposure to quaternary-ammonium solutions, demonstrating holes in the bacterial cell wall due to the interdigitization of the hydrophobic tail of the quaternary ammonium molecules (unpublished data). (A) Control (exposure to tryptone soya broth), (B) after exposure to 1 x MBC of a quaternary-ammonium solution (Ethoquad C/25) [Cocoalkyl methyl (polyoxyethylene) ammonium chloride], the scale bar denotes 1 μm.

Figure 3.

Viscoelastic measurements of ‘creep’ and ‘stress relaxation’. (A) During creep measurements, a constant stress is induced while the deformation of the biofilm is recorded over time. (B) During stress relaxation measurements, a constant deformation is induced and the stress required to maintain that deformation is recorded over time. (C) In a Maxwell model, response to an induced stress or deformation is mathematically modeled using multiple Maxwell elements in parallel, each with their characteristic time constants representing the viscous part of the response (‘the dashpots’, η_n) and an immediate elastic response component (‘the springs’, E_n). Characteristics time constants of each individual response process follow from τ_n as indicated in the graph. (D) In a Burger's model, this response is mathematically modeled using a Kelvin–Voigt element separated by a spring and dashpot, that together represent a Maxwell element.

Figure 4.

Role of viscoelasticity of biofilms in their survival under mechanical and chemical challenges. (A) Side view of a streamer in a biofilm growing under an applied wall shear stress. The edge of the streamer has been outlined for clarity and the direction of fluid flow is indicated by the thick arrow [(Stoodley et al., 1999a), with permission of the publisher]. (B) Biofilm removal or expansion (negative removal) for different distances between a biofilm and the bristle tips of a powered toothbrush [adapted from Busscher et al., 2010) with permission of the publisher], together with CLSM images (unpublished) of biofilms prior to (a) and after (b) non-contact brushing showing volumetric expansion (scale bar indicates 75 μm). (C) Penetration ratio of chlorhexidine as a generalized function of the relative importance of the three Maxwell elements E₁, E₂ and E₃, denoting the fast, intermediate and slow relaxation components, respectively. Dashed lines represent 95% confidence intervals.