In siturheology of Staphylococcus epidermidis bacterial biofilms

Abstract

We developed a method to grow Staphylococcus epidermidis bacterial biofilms and characterize their rheological properties in situ in a continuously fed bioreactor incorporated into a parallel plate rheometer. The temperature and shear rates of growth modeled bloodstream conditions, a common site of S. epidermidis infection. We measured the linear elastic (G′) and viscous moduli (G″) of the material using small-amplitude oscillatory rheology and the yield stress using non-linear creep rheology. We found that the elastic and viscous moduli of the S. epidermidis biofilm were 11 ± 3 Pa and 1.9 ± 0.5 Pa at a frequency of 1 Hz (6.283 rad per s) and that the yield stress was approximately 20 Pa. We modeled the linear creep response of the biofilm using a Jeffreys model and found that S. epidermidis has a characteristic relaxation time of approximately 750 seconds and a linear creep viscosity of 3000 Pa s. The effects on the linear viscoelastic moduli of environmental stressors, such as NaCl concentration and extremes of temperature, were also studied. We found a non-monotonic relationship between moduli and NaCl concentrations, with the stiffest material properties found at human physiological concentrations (135 mM). Temperature dependent rheology showed hysteresis in the moduli when heated and cooled between 5 °C and 60 °C. Through these experiments, we demonstrated that biofilms are rheologically complex materials that can be characterized by a combination of low modulus (~10 Pa), long relaxation time (~103 seconds), and a finite yield stress (20 Pa). This suggests that biofilms should be viewed as soft viscoelastic solids whose properties are determined in part by local environmental conditions. The in situ growth method introduced here can be adapted to a wide range of biofilm systems and applied over a broad spectrum of rheological and environmental conditions because the technique minimizes the risk of irreversible, non-linear deformation of the microbial specimen before analysis.

Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms

“…The majority of recent studies focuses on the analysis of the mechanical properties of wild-type biofilms (25-27). These studies comprise work on biofilm elasticity (16,17,28,29), biofilm erosion stability (30), and adhesion properties (31-33), as well as theoretical investigations of mechanical biofilm characteristics (34,35). Biofilm formation by the bacterium Bacillus subtilis has been studied intensively (36)(37)(38)(39)(40)(41)(42)(43), but information about the mechanical properties of these biofilm-forming bacteria is just emerging (24,30,44).…”

Biomechanical Analysis of Infectious Biofilms

“…), with the motion of one controlled to impose a predefined strain or stress schedule. Biofilms can be grown ex situ and transplanted to the rheometer, either intact on plates [41,66] or by a destructive process [55,104], or the rheometer modified to permit growth in situ [69,70]. Macrorheometry can extract bulk properties [68,82] but is not well suited to studying heterogeneities.…”

A microfluidic method and custom model for continuous, non-intrusive biofilm viscosity measurements under different nutrient conditions

“…19 Reported mechanical responses to different ions have also been varied. 20,23,36 Moreover, living biofilms demonstrate intriguing time-dependent behaviour. For example, temporary increases to shear stress can be elastically absorbed by biofilms, but under long-term application, viscous flow becomes dominant as a mechanism to dissipate internal stresses.…”

“…41,48,53 Often elasticity is the focus of rheological studies of biofilms, with viscosity being produced as a by-product. For example, parallel plate rheometers 36 or analysis of stress-strain curves produced using time-limited changes to shear forces by cycling nutrient flow rates in growth cells. 11,19,45,51,52 However, changes to imposed shear can induce measurement biases in viscosity for non-Newtonian biofilms.…”