Extracellular Polymeric Matrix Production and Relaxation under Fluid Shear and Mechanical Pressure in Staphylococcus aureus Biofilms

The viscoelasticity of a biofilm's EPS (extracellular polymeric substance) matrix conveys protection against mechanical challenges, but adaptive responses of biofilm inhabitants to produce EPS are not well known. Here, we compare the responses of a biofilm of an EPS-producing (ATCC 12600) and a non-EPS producing (5298) Staphylococcus aureus strain to fluid shear and mechanical challenge. Confocal laser scanning microscopy confirmed absence of calcofluor-white-stainable EPS in biofilms of S. aureus 5298. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with tribometry indicated that polysaccharide production per bacterium in the initial adhering layer was higher during growth at high shear than at low shear and that this increased EPS production extended to entire biofilms, as indicated by tribometrically measured coefficients of friction (CoF). CoF of biofilms grown under high fluid shear were higher than those when grown under low shear, likely due to wash-off polysaccharides. Measurement of a biofilm's CoF implies application of mechanical pressure that yielded an immediate increase in the polysaccharide band area of S. aureus ATCC 12600 biofilms due to their compression. Compression decreased after relief of pressure to the level observed prior to mechanical pressure. For biofilms grown under high shear, this coincided with a higher percent whiteness in optical coherence tomography-images indicative of water outflow, returning back into the biofilm during stress relaxation. Biofilms grown under low shear, however, were stimulated during tribometry to produce EPS, also after relief of stress. Knowledge of factors that govern EPS production and water flow in biofilms will allow better control of biofilms under mechanical challenge and better understanding of the barrier properties of biofilms against antimicrobial penetration. IMPORTANCE Adaptive responses of biofilm inhabitants in nature to environmental challenges such as fluid shear and mechanical pressure often involve EPS production with the aim of protecting biofilm inhabitants. EPS can assist biofilm bacteria in remaining attached or can impede antimicrobial penetration. The TriboChemist is a recently introduced instrument, allowing the study of initially adhering bacteria to a germanium crystal using ATR-FTIR spectroscopy, while simultaneously allowing measurement of the coefficient of friction of a biofilm, which serves as an indicator of the EPS content of a biofilm. EPS production can be stimulated by both fluid shear during growth and mechanical pressure, while increased EPS production can continue after pressure relaxation of the biofilm. Since EPS is pivotal in the protection of biofilm inhabitants against mechanical and chemical challenges, knowledge of the factors that make biofilm inhabitants decide to produce EPS, as provided in this study, is important for the development of biofilm control measures

Load- and velocity-dependent friction behavior of cow milk fat

Milk fat can influence the friction between oral surfaces and simultaneously contribute to the sensory perception of food products. The aim of this study was to gain insight into the friction mechanism of low- and high-fat cow milk. Here, we have measured the friction of milk (0.3% and 3.5% fat) between sliding hydrophobic (soft) surfaces, at different loads and velocity decay rates, using a pin-on-disk instrument. Results show that friction coefficient of 0.3% low-fat milk and 3.5% high-fat milk was lower than that of water, and the difference was two orders of magnitude in some cases. Low- and high-fat milk show a shear thinning effect; that is, the friction coefficient increases as the sliding velocity is decreased. The friction coefficient of 0.3% fat milk was lower than that of 3.5% fat milk at 1.5 N load, and this behavior reversed as the load was increased to 6.5 N. We hypothesize that this switch is due to a complex interaction between fat molecules and casein in the adsorbed surface layers formed after shear thinning. More adsorbed casein from low-fat milk increases the binding of casein with water molecules (through the hydrophilic tail) to reduce the friction at low load, whereas more adsorbed fat molecules in the case of high-fat milk reduce the friction at high load. Furthermore, as the velocity decay rate is increased from 0.003 to 0.005, low-fat milk still maintains the low friction compared with high-fat milk. This is attributed to casein’s (the hydrophilic tail) ability to quickly recover and restore water molecules at the interface. Overall, the synergistic effect of casein and fat molecules, depending on the fat content in milk, is vital to the friction mechanism. Hopefully, this study will be useful in dairy food product research

Role of salivary film structure in biolubrication

Conditioning film is a mixture of proteins that facilitate the articulation of the body parts through lubrication. Compared to other body parts, oral cavity is a unique system where the lubrication is changed not only because of the diseases but also due to perturbation. By using the oral cavity as the model system we examine the changes in the lubrication due to chemical perturbation, for example, after treatment with tooth paste with sodium fluoride - sodium lauryl sulfate combination and only sodium lauryl sulfate. In general, salivary protein films after chemical perturbation is revived with a different structural configuration that is comprised of a rigid basal and a soft over layer, as shown by using quartz crystal microbalance with dissipation monitoring and water contact angle measurements. Perturbed salivary protein films has better wear resistant property compared with unperturbed salivary protein films (no chemical treatment). And, the stable coefficient of friction values for perturbed salivary protein films is 2 times lower than unperturbed salivary protein film at physiologically relevant contact pressures (72 to 250 kPa), as determined by the nanotribometer. In vivo smoothness scores also indicated a significant improvement in the smoothness feeling immediately after perturbation by toothpastes. Smooth feeling after using the toothpastes as a routine to manage oral hygeine is attributed to the re-structuring of interfacial salivary protein film into rigid and hydrophilic layers that provides sustained lubrication and simultaneously triggering the smooth experience by the tongue in the oral cavity.</p

Role of salivary film structure in biolubrication

Conditioning film is a mixture of proteins that facilitate the articulation of the body parts through lubrication. Compared to other body parts, oral cavity is a unique system where the lubrication is changed not only because of the diseases but also due to perturbation. By using the oral cavity as the model system we examine the changes in the lubrication due to chemical perturbation, for example, after treatment with tooth paste with sodium fluoride - sodium lauryl sulfate combination and only sodium lauryl sulfate. In general, salivary protein films after chemical perturbation is revived with a different structural configuration that is comprised of a rigid basal and a soft over layer, as shown by using quartz crystal microbalance with dissipation monitoring and water contact angle measurements. Perturbed salivary protein films has better wear resistant property compared with unperturbed salivary protein films (no chemical treatment). And, the stable coefficient of friction values for perturbed salivary protein films is 2 times lower than unperturbed salivary protein film at physiologically relevant contact pressures (72 to 250 kPa), as determined by the nanotribometer. In vivo smoothness scores also indicated a significant improvement in the smoothness feeling immediately after perturbation by toothpastes. Smooth feeling after using the toothpastes as a routine to manage oral hygeine is attributed to the re-structuring of interfacial salivary protein film into rigid and hydrophilic layers that provides sustained lubrication and simultaneously triggering the smooth experience by the tongue in the oral cavity.</p

Influence of fluoride-detergent combinations on the visco-elasticity of adsorbed salivary protein films

The visco-elasticity of salivary-protein films is related to mouthfeel, lubrication, biofilm formation, and protection against erosion and is influenced by the adsorption of toothpaste components. The thickness and the visco-elasticity of hydrated films (determined using a quartz crystal microbalance) of 2-h-old in vitro-adsorbed salivary-protein films were 43.5 nm and 9.4 MHz, respectively, whereas the dehydrated thickness, measured using X-ray photoelectron spectroscopy, was 2.4 nm. Treatment with toothpaste slurries decreased the thickness of the film, depending on the fluoride-detergent combination involved. Secondary exposure to saliva resulted in a regained thickness of the film to a level similar to its original thickness; however, no association was found between the thickness of hydrated and dehydrated films, indicating differences in film structure. Treatment with stannous fluoride/sodium lauryl sulphate (SnF(2)/SLS)-containing toothpaste slurries yielded a strong, immediate two-fold increase in characteristic film frequency (f(c)) with respect to untreated films, indicating cross-linking in adsorbed salivary-protein films by Sn2+ that was absent when SLS was replaced with sodium hexametaphosphate (NaHMP). Secondary exposure to saliva of films treated with SnF(2) caused a strong, six-fold increase in f(c) compared with primary salivary-protein films, regardless of whether SLS or NaHMP was the detergent. This suggests that ionized stannous is not directly available for cross-linking in combination with highly negatively charged NaHMP, but becomes slowly available after initial treatment to cause cross-linking during secondary exposure to saliva

Structured free-water clusters near lubricating surfaces are essential in water-based lubrication

Water-based lubrication provides cheap and environmentally friendly lubrication and, although hydrophilic surfaces are preferred in water-based lubrication, often lubricating surfaces do not retain water molecules during shear. We show here that hydrophilic (42° water contact angle) quartz surfaces facilitate water-based lubrication to the same extent as more hydrophobic Si crystal surfaces (61°), while lubrication by hydrophilic Ge crystal surfaces (44°) is best. Thus surface hydrophilicity is not sufficient for water-based lubrication. Surface-thermodynamic analyses demonstrated that all surfaces, regardless of their water-based lubrication, were predominantly electron donating, implying water binding with their hydrogen groups. X-ray photoelectron spectroscopy showed that Ge crystal surfaces providing optimal lubrication consisted of a mixture of -O and =O functionalities, while Si crystal and quartz surfaces solely possessed -O functionalities. Comparison of infrared absorption bands of the crystals in water indicated fewer bound-water layers on hydrophilic Ge than on hydrophobic Si crystal surfaces, while absorption bands for free water on the Ge crystal surface indicated a much more pronounced presence of structured, free-water clusters near the Ge crystal than near Si crystal surfaces. Accordingly, we conclude that the presence of structured, free-water clusters is essential for water-based lubrication. The prevalence of structured water clusters can be regulated by adjusting the ratio between surface electron-donating and electron-accepting groups and between -O and =O functionalities