Joop de Vries

Comparison of Atomic Force Microscopy Interaction Forces between Bacteria and Silicon Nitride Substrata for Three Commonly Used Immobilization Methods

ABSTRACT Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.

Dynamic Cell Surface Hydrophobicity of Lactobacillus Strains with and without Surface Layer Proteins

Variations in surface hydrophobicity of six Lactobacillus strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.

Staphylococcus aureus-fibronectin interactions with and without fibronectin-binding proteins and their role in adhesion and desorption

ABSTRACT Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.

Survival of Adhering Staphylococci during Exposure to a Quaternary Ammonium Compound Evaluated by Using Atomic Force Microscopy Imaging

ABSTRACT Effects of a quaternary ammonium compound (QAC) on the survival of adhering staphylococci on a surface were investigated using atomic force microscopy (AFM). Four strains with different minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) for the QAC were exposed to three different concentrations of the QAC in potassium phosphate buffer (0.5×, 1×, and 2× MBC) while adhering to glass. Adhering staphylococci were repeatedly imaged with AFM in the contact mode, and the cell surface was found to wrinkle upon progressive exposure to the QAC until bacteria disappeared from the substratum. Higher concentrations of QAC yielded faster wrinkling and the disappearance of bacteria during imaging. Two slime-producing staphylococcal strains survived longer on the surface than two non-slime-producing strains despite similar MICs and MBCs. All staphylococci adhering in unscanned areas remained adhering during exposure to QAC. Since MICs and MBCs did not relate to bacterial cell surface hydrophobicities and zeta potentials, survival on the surface is probably not determined by the direct interaction of QAC molecules with the cell surface. Instead, it is suggested that the pressure of the AFM tip assists the incorporation of QAC molecules in the membrane and enhances their bactericidal efficacy. In addition, the prolonged survival under pressure from slime-producing strains on a surface may point to a new protective role of slime as a stress absorber, impeding the incorporation of QAC molecules. The addition of Ca2+ ions to a QAC solution yielded longer survival of intact, adhering staphylococci, suggesting that Ca2+ ions can impede the exchange of membrane Ca2+ ions required for QAC incorporation.

Detachment and successive re-attachment of multiple, reversibly-binding tethers result in irreversible bacterial adhesion to surfaces

Bacterial adhesion to surfaces occurs ubiquitously and is initially reversible, though becoming more irreversible within minutes after first contact with a surface. We here demonstrate for eight bacterial strains comprising four species, that bacteria adhere irreversibly to surfaces through multiple, reversibly-binding tethers that detach and successively re-attach, but not collectively detach to cause detachment of an entire bacterium. Arguments build on combining analyses of confined Brownian-motion of bacteria adhering to glass and their AFM force-distance curves and include the following observations: (1) force-distance curves showed detachment events indicative of multiple binding tethers, (2) vibration amplitudes of adhering bacteria parallel to a surface decreased with increasing adhesion-forces acting perpendicular to the surface, (3) nanoscopic displacements of bacteria with relatively long autocorrelation times up to several seconds, in absence of microscopic displacement, (4) increases in Mean-Squared-Displacement over prolonged time periods according to tα with 0 < α ≪ 1, indicative of confined displacement. Analysis of simulated position-maps of adhering particles using a new, in silico model confirmed that adhesion to surfaces is irreversible through detachment and successive re-attachment of reversibly-binding tethers. This makes bacterial adhesion mechanistically comparable with the irreversible adsorption of high-molecular-weight proteins to surfaces, mediated by multiple, reversibly-binding molecular segments.

Influence of biofilm lubricity on shear-induced transmission of staphylococcal biofilms from stainless steel to silicone rubber

In real‐life situations, bacteria are often transmitted from biofilms growing on donor surfaces to receiver ones. Bacterial transmission is more complex than adhesion, involving bacterial detachment from donor and subsequent adhesion to receiver surfaces. Here, we describe a new device to study shear‐induced bacterial transmission from a (stainless steel) pipe to a (silicone rubber) tube and compare transmission of EPS‐producing and non‐EPS‐producing staphylococci. Transmission of an entire biofilm from the donor to the receiver tube did not occur, indicative of cohesive failure in the biofilm rather than of adhesive failure at the donor‐biofilm interface. Biofilm was gradually transmitted over an increasing length of receiver tube, occurring mostly to the first 50 cm of the receiver tube. Under high‐shearing velocity, transmission of non‐EPS‐producing bacteria to the second half decreased non‐linearly, likely due to rapid thinning of the lowly lubricious biofilm. Oppositely, transmission of EPS‐producing strains to the second tube half was not affected by higher shearing velocity due to the high lubricity and stress relaxation of the EPS‐rich biofilms, ensuring continued contact with the receiver. The non‐linear decrease of ongoing bacterial transmission under high‐shearing velocity is new and of relevance in for instance, high‐speed food slicers and food packaging.

A Trifunctional, Modular Biomaterial Coating: Nonadhesive to Bacteria, Chlorhexidine-Releasing and Tissue-Integrating

Various potential anti-infection strategies can be thought of for biomaterial implants and devices. Permanent, tissue-integrated implants such as artificial joint prostheses require a different anti-infection strategy than, for instance, removable urinary catheters. The different requirements set to biomaterials implants and devices in different clinical applications call for tailor-made strategies. Here, a modular coating-concept for biomaterials is reported, which in its full, trifunctional form comprises nonadhesiveness to bacteria and antimicrobial release, combined with enhanced tissue integration characteristics. Nonadhesiveness to proteins and bacteria is accomplished by a hydrophilic brush coating (Vitrostealth). The antimicrobial release module is constituted by a chlorhexidine releasing poly(ethylene glycol) diacrylamide based-coating that continues to release its antimicrobial content also when underneath the nonadhesive top-coating. The third module, enhancing tissue integration, is realized by the incorporation of the penta-peptide Glycine-Arginine-Glycine-Aspartic acid-Serine (GRGDS) within the nonadhesive top-coating. Modules function in concert or independently of each other. Specifically, tissue integration by the GRGDS-module does not affect the nonadhesiveness of the Vitrostealth-module toward bovine serum albumin and Staphylococcus aureus, while the antimicrobial release module does not affect tissue-integration by the GRGDS-module. Uniquely, using this modular system, tailor-made anti-infection strategies can thus readily be made for biomaterials in different clinical applications.

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.