Stress relaxation analysis facilitates a quantitative approach towards antimicrobial penetration into biofilms

Abstract

Biofilm-related infections can develop everywhere in the human body and are rarely cleared by the host immune system. Moreover, biofilms are often tolerant to antimicrobials, due to a combination of inherent properties of bacteria in their adhering, biofilm mode of growth and poor physical penetration of antimicrobials through biofilms. Current understanding of biofilm recalcitrance toward antimicrobial penetration is based on qualitative descriptions of biofilms. Here we hypothesize that stress relaxation of biofilms will relate with antimicrobial penetration. Stress relaxation analysis of single-species oral biofilms grown in vitro identified a fast, intermediate and slow response to an induced deformation, corresponding with outflow of water and extracellular polymeric substances, and bacterial re-arrangement, respectively. Penetration of chlorhexidine into these biofilms increased with increasing relative importance of the slow and decreasing importance of the fast relaxation element. Involvement of slow relaxation elements suggests that biofilm structures allowing extensive bacterial re-arrangement after deformation are more open, allowing better antimicrobial penetration. Involvement of fast relaxation elements suggests that water dilutes the antimicrobial upon penetration to an ineffective concentration in deeper layers of the biofilm. Next, we collected biofilms formed in intra-oral collection devices bonded to the buccal surfaces of the maxillary first molars of human volunteers. Ex situ chlorhexidine penetration into two weeks old in vivo formed biofilms followed a similar dependence on the importance of the fast and slow relaxation elements as observed for in vitro formed biofilms. This study demonstrates that biofilm properties can be derived that quantitatively explain antimicrobial penetration into a biofilm.

Figure 1. Measurement and Maxwell model of the viscoelasticity of biofilms.

(A) Stress versus time diagram for relaxation of a compressed biofilm. (B) Schematic of a three element Maxwell model: E_i represent the spring constants and τ_i the relaxation time constants, which are equal to η_i/E_i.

Figure 2. Penetration of chlorhexidine and stress relaxation of differently grown biofilms in vitro.

(A) The schematics of parallel plate flow chamber and constant depth film fermenter. (B) Penetration ratio of chlorhexidine as a function of relaxation of different biofilms for 10%, 20% and 50% induced deformation. Dashed lines indicate 95% confidence intervals.

Figure 3. Chlorhexidine penetration and Maxwell analyses of in vitro grown biofilms.

Penetration ratio as a function of the relative importance of the three Maxwell elements E₁, E₂ and E₃, denoting the fast, intermediate and slow relaxation components, respectively for different biofilms after 10%, 20% and 50% induced deformation. All data points refer to single experiments, while symbols are explained in Fig. 2. Dashed lines represent 95% confidence intervals.

Figure 4. Stress relaxation properties of intra-orally grown oral biofilms.

The in vivo biofilms were obtained in five volunteers as indicated by different colors in comparison with the average relaxation properties of different single-species biofilms formed in a PPFC and CDFF, falling within the black rectangles.

Figure 5. Chlorhexidine penetration and Maxwell analyses of intra-orally grown biofilms.

Penetration ratio of chlorhexidine as a function of the relative importance of the fast, intermediate and slow Maxwell elements E₁, E₂ and E₃ for in vivo biofilms formed in different volunteers after 10%, 20% and 50% induced deformation. All data points refer to single experiments in one volunteer. Different volunteers are indicated by the same color codes as used in Fig. 4. Dashed lines represent 95% confidence intervals.