A distinguishable role of eDNA in the viscoelastic relaxation of biofilms

Abstract

Bacteria in the biofilm mode of growth are protected against chemical and mechanical stresses. Biofilms are composed, for the most part, of extracellular polymeric substances (EPSs). The extracellular matrix is composed of different chemical constituents, such as proteins, polysaccharides, and extracellular DNA (eDNA). Here we aimed to identify the roles of different matrix constituents in the viscoelastic response of biofilms. Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Pseudomonas aeruginosa biofilms were grown under different conditions yielding distinct matrix chemistries. Next, biofilms were subjected to mechanical deformation and stress relaxation was monitored over time. A Maxwell model possessing an average of four elements for an individual biofilm was used to fit the data. Maxwell elements were defined by a relaxation time constant and their relative importance. Relaxation time constants varied widely over the 104 biofilms included and were divided into seven ranges (<1, 1 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, and >500 s). Principal-component analysis was carried out to eliminate related time constant ranges, yielding three principal components that could be related to the known matrix chemistries. The fastest relaxation component (<3 s) was due to the presence of water and soluble polysaccharides, combined with the absence of bacteria, i.e., the heaviest masses in a biofilm. An intermediate component (3 to 70 s) was related to other EPSs, while a distinguishable role was assigned to intact eDNA, which possesses a unique principal component with a time constant range (10 to 25 s) between those of EPS constituents. This implies that eDNA modulates its interaction with other matrix constituents to control its contribution to viscoelastic relaxation under mechanical stress.

Importance: The protection offered by biofilms to organisms that inhabit it against chemical and mechanical stresses is due in part to its matrix of extracellular polymeric substances (EPSs) in which biofilm organisms embed themselves. Mechanical stresses lead to deformation and possible detachment of biofilm organisms, and hence, rearrangement processes occur in a biofilm to relieve it from these stresses. Maxwell analysis of stress relaxation allows the determination of characteristic relaxation time constants, but the biofilm components and matrix constituents associated with different stress relaxation processes have never been identified. Here we grew biofilms with different matrix constituents and used principal-component analysis to reveal that the presence of water and soluble polysaccharides, together with the absence of bacteria, is associated with the fastest relaxation, while other EPSs control a second, slower relaxation. Extracellular DNA, as a matrix constituent, had a distinguishable role with its own unique principal component in stress relaxation with a time constant range between those of other EPSs.

FIG 1

Panels a to c represent the measured stress relaxation of a P. aeruginosa SG81 biofilm as a function of time, together with model fits to the data, obtained by using two (panels a and b) or five (panel c) Maxwell elements. Note that panel a extends over 100 s, while panels b and c refer only to the first 5 s of the relaxation process. Panel d represents the quality of the fit, indicated by chi-square values, as a function of the number of Maxwell elements used for the fit.

FIG 2

Relative importance of the individual Maxwell elements of different biofilms as a function of their characteristic relaxation time constants in relation to the different matrix chemistries according to Table 1. Each data point represents one Maxwell element with its time constant plotted against its relative importance. Each individual biofilm possessed an average of four or five Maxwell elements. Similar biofilms were grown and investigated minimally three times with separate initial bacterial cultures. Maxwell elements with 0% relative importance have no accompanying time constant and are not plotted, while characteristic time constants exceeding 7,000 s have been assigned a value of 7,000 s. Vertical lines indicate divisions of relaxation time constant ranges (C_i). Panels: a, P. aeruginosa biofilms; b, S. mutans biofilms; c, S. aureus and S. epidermidis biofilms.

FIG 3

(a) Coefficients (a_ij) of the initial time constant ranges (C_i) for each principal component (PC_j) according to the equation ${{PC}_j={\displaystyle \underset{i=1}{\overset{7}{\mathrm{\Sigma }}}}{\alpha }_{ij}\times {\overline{E}}_i}_{}$ (see also the last equation in Materials and Methods). (b) Assignment of matrix chemistries to the three principal components (PC_j) as distinguished for the different biofilms involved in this study. Principal components are expressed as a function of relaxation time constants based on positive correlations with matrix chemistries defined in Table 1. The matrix chemistries positively associated with PC₁ include water and soluble polysaccharides, while the matrix chemistry for PC₂ includes other EPS polymers, like insoluble polysaccharides (i.e. glucans). PC₃ includes only intact eDNA.