Predicting biofilm viscoelasticity

Kristin Kovach and Megan Davis-Fields

Phase space and connections examined in this study (colored areas). Our goal is to map single-cell microscopy onto biofilm structure and rheology.  Single-cell microscopy is easier, quicker, and cheaper to do and has much higher potential for automation than does rheology.

Our research involves studying the viscoelastic properties of Pseudomonas aeruginosa biofilms. When P. aeruginosa forms a biofilm, the bacteria create a matrix of extracellular polymeric substances that allow these biofilms to attach and persist on surfaces. In the case of P. aeruginosa, the persistence of these biofilms is a source of major health problems in immunocompromised patients, such as patients with cystic fibrosis. The main contributors to biofilm persistence are extracellular polysaccharides Pel, Psl, and alginate, and extracellular DNA.

The mechanics of single cells can be investigated by microscopically tracking the motion of cells on a surface. Atomic force microscopy can also be used with these single cell populations to study their surface adhesion. The mechanics of the biofilm as a whole can be probed by using a rheometer’a device which can apply strain at controlled amounts to a material in order to measure that material’s response, and thereby its viscoelastic properties’and also by atomic force microscopy, to examine the response of the biofilm at different locations within the biofilm. Using these sets of data, we will create the predictive metrics for biofilm behavior of P. aeruginosa which we hope to later expand to other bacterial biofilms.

Expression of specific EPS elements results in distinct traits of single PAO1 cells on surfaces. (a)  The times that cells remain on the surface, normalized to the average doubling time for each strain.  Wild type (WT) and {delta}pel (solid red and dashed blue lines) remain on the surface.  WT produces both Pel and Psl; {delta}pel produces Psl but not Pel.  {delta}psl (dotted yellow line) have approximately equal probability of detaching from the surface or remaining attached.  {delta}psl produces Pel but not Psl.  {delta}pel{delta}psl (dash-dot green line) almost always detach from the surface before an individual cell’s life-cycle is 25% complete.  {delta}pel{delta}psl produces neither Pel nor Psl.  (b and c)  The tracked projected aspect ratio gives a proxy measurement for cells tilting up off the surface.  WT lies flat on the horizontal surface.  Similarly, {delta}psl is most likely to lie flat, with a smaller probability of standing upright on-end.  However, Pel-knockout strains spend at least an order of magnitude more time tilted at a non-90° angle from the surface than do Pel-expressing strains.  Data, analysis, and methodology published in Cooley et al, 2013 Soft Matter.

Genetic knockouts that do not make specific EPS materials produce softer biofilms. Knocking out Psl production lowers the elastic modulus to about 66% of the WT value.  Knocking out Pel lowers the elastic modulus to about 50% of the WT value.  The {delta}pelpsl double knockout modulus is comparable to the {delta}pel knockout modulus and is not shown for clarity.  Also not shown for clarity are the viscous moduli, which are about an order of magnitude less than the elastic modulus until the biofilm fails, when the two moduli become comparable.