Bart Gottenbos

Streptococcus mutans biofilm transient viscoelastic fluid behaviour during high-velocity microsprays

Using high-speed imaging we assessed Streptococcus mutans biofilm-fluid interactions during exposure to a 60-ms microspray burst with a maximum exit velocity of 51m/s. S. mutans UA159 biofilms were grown for 72h on 10mm-length glass slides pre-conditioned with porcine gastric mucin. Biofilm stiffness was measured by performing uniaxial-compression tests. We developed an in-vitro interproximal model which allowed the parallel insertion of two biofilm-colonized slides separated by a distance of 1mm and enabled high-speed imaging of the removal process at the surface. S. mutans biofilms were exposed to either a water microspray or an air-only microburst. High-speed videos provided further insight into the mechanical behaviour of biofilms as complex liquids and into high-shear fluid-biofilm interaction. We documented biofilms extremely transient fluid behaviour when exposed to the high-velocity microsprays. The presence of time-dependent recoil and residual deformation confirmed the pivotal role of viscoelasticity in biofilm removal. The air-only microburst was effective enough to remove some of the biofilm but created a smaller clearance zone underlying the importance of water and the air-water interface of drops moving over the solid surface in the removal process. Confocal and COMSTAT analysis showed the high-velocity water microspray caused up to a 99.9% reduction in biofilm thickness, biomass and area coverage, within the impact area.

Removal of biofilms by impinging water droplets

The process of impinging water droplets on Streptococcus mutans biofilms was studied experimentally and numerically. Droplets were experimentally produced by natural breakup of a cylindrical liquid jet. Droplet diameter and velocity were varied between 20 and 200 μm and between 20 and 100 m/s, respectively. The resulting erosion process of the biofilm was determined experimentally with high-speed recording techniques and a quantitative relationship between the removal rate, droplet size, and velocity was determined. The shear stress and the pressure on the surface during droplet impact were determined by numerical simulations, and a qualitative agreement between the experiment and the simulation was obtained. Furthermore, it was shown that the stresses on the surface are strongly reduced when a water film is present.

High-Velocity Microsprays Enhance Antimicrobial Activity in Streptococcus mutans Biofilms

Streptococcus mutans in dental plaque biofilms play a role in caries development. The biofilm’s complex structure enhances the resistance to antimicrobial agents by limiting the transport of active agents inside the biofilm. The authors assessed the ability of high-velocity water microsprays to enhance delivery of antimicrobials into 3-d-old S. mutans biofilms. Biofilms were exposed to a 90° or 30° impact, first using a 1-µm tracer bead solution (109 beads/mL) and, second, a 0.2% chlorhexidine (CHX) or 0.085% cetylpyridinium chloride (CPC) solution. For comparison, a 30-s diffusive transport and simulated mouthwash were also performed. Confocal microscopy was used to determine number and relative bead penetration depth into the biofilm. Assessment of antimicrobial penetration was determined by calculating the killing depth detected by live/dead viability staining. The authors first demonstrated that the microspray was able to deliver significantly more microbeads deeper in the biofilm compared with diffusion and mouthwashing exposures. Next, these experiments revealed that the microspray yielded better antimicrobial penetration evidenced by deeper killing inside the biofilm and a wider killing zone around the zone of clearance than diffusion alone. Interestingly the 30° impact in the distal position delivered approximately 16 times more microbeads and yielded approximately 20% more bacteria killing (for both CHX and CPC) than the 90° impact. These data suggest that high-velocity water microsprays can be used as an effective mechanism to deliver microparticles and antimicrobials inside S. mutans biofilms. High shear stresses generated at the biofilm-burst interface might have enhanced bead and antimicrobial delivery inside the remaining biofilm by combining forced advection into the biofilm matrix and physical restructuring of the biofilm itself. Further, the impact angle has potential to be optimized both for biofilm removal and active agents’ delivery inside biofilm in those protected areas where some biofilm might remain.

Influence of Weight on Removal of Co-Adhering Bacteria from Salivary Pellicles by Different Modes of Brushing

This study compared removal of pairs of co-adhering and non-co-adhering oral actinomyces and streptococci from salivary pellicles by manual, rotating/oscillating electric and sonic toothbrushes, applying weights up to 240 g. First, actinomyces were allowed to adhere to a pellicle in a parallel plate flow chamber, after which streptococci suspended in saliva were perfused through the chamber at 33°C. On average, 34–39% of the adhering bacteria were adhering as single organisms. For co-adhering and non-co-adhering pairs, 33 and 10% of the adhering bacteria were involved, respectively, in aggregates comprising more than 10 organisms. Brushing by hand removed 82% at low weight (40 g), which was less than by electric (93%) or sonic (92%) brushing, while for all modes of brushing bacterial removal increased with increasing weight to 95–99%. For a non-co-adhering pair, subsequent exposure of brushed pellicles to a streptococcal suspension yielded only 2–16% of bacteria involved in large aggregates, regardless of the mode of brushing. For the co-adhering pair, however, de novo streptococcal adhesion to hand-brushed pellicles yielded 34–57% of bacteria involved in large aggregates, while electric and sonic brushing left 22–35% of the bacteria involved in large aggregates. De novo streptococcal adhesion for the co-adhering pair increased with increasing weight for the electric and sonic brush in contrast to the manual brush. Since a strong influence of co-adhesion is evident in de novo streptococcal adhesion, despite nearly complete removal of all actinomyces, these observations suggest that the three modes of brushing leave footprints to which streptococci preferentially adhere.

Fluid-driven Interfacial instabilities and turbulence in bacterial biofilms

Biofilms are thin layers of bacteria embedded within a slime matrix that live on surfaces. They are ubiquitous in nature and responsible for many medical and dental infections, industrial fouling and are also evident in ancient fossils. A biofilm structure is shaped by growth, detachment and response to mechanical forces acting on them. The main contribution to biofilm versatility in response to physical forces is the matrix that provides a platform for the bacteria to grow. The interaction between biofilm structure and hydrodynamics remains a fundamental question concerning biofilm dynamics. Here, we document the appearance of ripples and wrinkles in biofilms grown from three species of bacteria when subjected to high-velocity fluid flows. Linear stability analysis suggested that the ripples were Kelvin-Helmholtz Instabilities. The analysis also predicted a strong dependence of the instability formation on biofilm viscosity explaining the different surface corrugations observed. Turbulence through Kelvin-Helmholtz instabilities occurring at the interface demonstrated that the biofilm flows like a viscous liquid under high flow velocities applied within milliseconds. Biofilm fluid-like behavior may have important implications for our understanding of how fluid flow influences biofilm biology since turbulence will likely disrupt metabolite and signal gradients as well as community stratification.