Abstract
Particle tracking microrheology was used to investigate the viscoelasticity of Staphylococcus aureus biofilms grown in microfluidic cells at various flow rates and when subjected to biofilm-degrading enzymes. Biofilm viscoelasticity was found to harden as a function of shear rate but soften with increasing height away from the attachment surface in good agreement with previous bulk results. Ripley’s K-function was used to quantify the spatial distribution of the bacteria within the biofilm. For all conditions, biofilms would cluster as a function of height during growth. The effects of proteinase K and DNase-1 on the viscoelasticity of biofilms were also investigated. Proteinase K caused an order of magnitude change in the compliances, softening the biofilms. However, DNase-1 was found to have no significant effects over the first 6 h of development, indicating that DNA is less important in biofilm maintenance during the initial stages of growth. Our results demonstrate that during the preliminary stages of Staphylococcus aureus biofilm development, column-like structures with a vertical gradient of viscoelasticity are established and modulated by the hydrodynamic shear caused by fluid flow in the surrounding environment. An understanding of these mechanical properties will provide more accurate insights for removal strategies of early-stage biofilms.
Highlights
Following attachment to a boundary, such as the glass interface in a flow chamber or a catheter surface in vivo, many bacteria species produce a complex extracellular matrix of polymeric material collectively called a biofilm.[1]
Previous authors have examined the mechanical response of microscale biofilms to differing hydrodynamic shear stresses using magnetic force modulation atomic force microscopy,[11] but in general the microscale effects of shear have been relatively little studied with biofilms and never with S. aureus or using particle tracking microrheology
A detailed statistical analysis of images from bright-field microscopy using a fast CMOS camera shows that the spatial heterogeneity of the bacteria within biofilms decreases over time using an analysis based on Ripley’s K-function
Summary
Following attachment to a boundary, such as the glass interface in a flow chamber or a catheter surface in vivo, many bacteria species produce a complex extracellular matrix of polymeric material collectively called a biofilm.[1]. A detailed statistical analysis of images from bright-field microscopy using a fast CMOS camera shows that the spatial heterogeneity of the bacteria within biofilms decreases over time using an analysis based on Ripley’s K-function (not to our knowledge previously used with biofilms) This indicates that biofilms grow in tapered columns, which could be a precursor to the filamentary structure exploited by biofilms to increase dispersal efficiency or as a response to local nutrient concentrations. An order of magnitude change in viscoelasticity was observed using proteinase K (the biofilms soften by this amount), whereas DNase-1 had a negligible effect on the biofilms Overall, these data suggest that for S. aureus initial biofilm growth away from the surface occurs in narrowing columns, rather than homogeneously, that there is a response to higher shear stresses to produce more rigid early-stage biofilms and that in the early-stages extracellular DNA plays a minor role in the structural integrity of the biofilm
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