Henderina van der Mei

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.

Bond Strengthening in Oral Bacterial Adhesion to Salivary Conditioning Films

ABSTRACT Transition from reversible to irreversible bacterial adhesion is a highly relevant but poorly understood step in initial biofilm formation. We hypothesize that in oral biofilm formation, irreversible adhesion is caused by bond strengthening due to specific bacterial interactions with salivary conditioning films. Here, we compared the initial adhesion of six oral bacterial strains to salivary conditioning films with their adhesion to a bovine serum albumin (BSA) coating and related their adhesion to the strengthening of the binding forces measured with bacteria-coated atomic force microscopy cantilevers. All strains adhered in higher numbers to salivary conditioning films than to BSA coatings, and specific bacterial interactions with salivary conditioning films were accompanied by stronger initial adhesion forces. Bond strengthening occurred on a time scale of several tens of seconds and was slower for actinomyces than for streptococci. Nonspecific interactions between bacteria and BSA coatings strengthened twofold faster than their specific interactions with salivary conditioning films, likely because specific interactions require a closer approach of interacting surfaces with the removal of interfacial water and a more extensive rearrangement of surface structures. After bond strengthening, bacterial adhesion forces with a salivary conditioning film remained stronger than those with BSA coatings.

Simultaneous interaction of bacteria and tissue cells with photocatalytically activated, anodized titanium surfaces

Photocatalytic-activation of anodized TiO2-surfaces has been demonstrated to yield antibacterial and tissue integrating effects, but effects on simultaneous growth of tissue cells and bacteria in co-culture have never been studied. Moreover, it is unknown how human-bone-marrow-mesenchymal-stem (hBMMS) cells, laying the groundwork for integration of titanium implants in bone, respond to photocatalytic activation of anodized TiO2-surfaces. Photocatalytically-activated, anodized titanium and titanium-alloy surfaces achieved 99.99% killing of adhering Staphylococcus epidermidis and Staphylococcus aureus, an effect that lasted for 30 days of storage in air. Surface coverage by osteoblasts was not affected by photocatalytic activation of anodized TiO2-surfaces. Co-cultures of osteoblasts with contaminating S. epidermidis however, enhanced surface coverage on photocatalytically-activated, anodized titanium-alloy surfaces. hBMMS cells grew less on photocatalytically-activated, anodized titanium surfaces, while not at all on photocatalytically-activated, anodized titanium-alloy surfaces and did not survive the presence of contaminating staphylococci. This reduced surface coverage by hBMMS cells disappeared when photocatalytically-activated, anodized titanium-alloy surfaces were exposed to buffer for 60 min, both in absence or presence of contaminating S. aureus. Consequently, it is concluded that photocatalytically-activated, anodized titanium and titanium-alloy surfaces will effectively kill peri-operatively introduced staphylococci contaminating an implant surface and constitute an effective means for antibiotic prophylaxis in cementless fixation of orthopaedic hardware.

Weibull analyses of bacterial interaction forces measured using AFM

Statistically significant conclusions from interaction forces obtained by AFM are difficult to draw because of large data spreads. Weibull analysis, common in macroscopic bond-strength analyses, takes advantage of this spread to derive a Weibull distribution, yielding the probability of occurrence of a force value and the dependability of the data set. Here we propose Weibull distribution as a new way to present nanoscopic bacterial interaction forces obtained using AFM.

Surfactive and antibacterial activity of cetylpyridinium chloride formulations in vitro and in vivo

AIM To compare effects of three cetylpyridinium chloride (CPC) formulations with and without alcohol and Tween80 on physico-chemical properties of salivary pellicles, bacterial detachment in vitro and bacterial killing in vivo. MATERIAL AND METHODS Adsorption of CPC to salivary pellicles in vitro was studied using X-ray photoelectron spectroscopy and water contact angle measurements. Adhesion and detachment of a co-adhering bacterial pair was determined in vitro using a flow chamber. Killing was evaluated after live/dead staining after acute single use in vivo on 24- and 72-h-old plaques after 2-week continuous use. RESULTS The most pronounced effects on pellicle surface chemistry and hydrophobicity were observed after treatment with the alcohol-free formulation, while the pellicle thickness was not affected by any of the formulations. All CPC formulations detached up to 33% of the co-adhering pair from pellicle surfaces. Bacterial aggregate sizes during de novo deposition were enhanced after treatment with the alcohol-free formulation. Immediate and sustained killing in 24 and 72 h plaques after in vivo, acute single use as well as after 2-week continuous use were highest for the alcohol-free formulation. CONCLUSIONS CPC bioavailability in a formulation without alcohol and Tween80 could be demonstrated through measures of pellicle surface properties and bacterial interactions in vitro as well as bacteriocidal actions on oral biofilms in vivo.

Force Analysis of Bacterial Transmission from Contact Lens Cases to Corneas, with the Contact Lens as the Intermediary

PURPOSE To determine the probability of transmission of a Staphylococcus aureus strain from a contact lens case, to the contact lens (CL) surfaces, to the cornea, on the basis of bacterial adhesion forces measured by using atomic force microscopy (AFM). METHODS Adhesion forces between S. aureus strain 835 probes with rigid and soft CLs, storage cases, and porcine corneas were measured with AFM and used to calculate Weibull distributions, from which the transmission probability from one surface to another was derived. Bacterial transmission probabilities from force analyses were compared with experimentally obtained transmission data. RESULTS After bond-strengthening, S. aureus adhered to the surface of a lens case with a median force of 10.8 nN. Adhesion forces were different on the soft and rigid CLs (7.7 and 13.6 nN, respectively). Adhesion forces on porcine corneas amounted to 11.8 nN. Data variations were used to calculate the Weibull distribution, from which the probability of transmission from the lens case to a CL and from the CL to the cornea can be directly read. Final transmission probabilities from lens case to the cornea were slightly higher for the rigid (24%) than for the soft (19%) CL. Bacterial transmission determined experimentally increased with increasing contact times, but were within the range of the probabilities derived from Weibull analyses. CONCLUSIONS Probabilities of bacterial transmission from contaminated lens cases to corneas can be derived from Weibull analyses of measured forces of adhesion to the surfaces involved.

On-demand antimicrobial release from a temperature-sensitive polymer - Comparison with ad libitum release from central venous catheters

Antimicrobial releasing biomaterial coatings have found application for instance in the fixation of orthopedic joint prostheses and central venous catheters. Most frequently, the release kinetics is such that antimicrobially-effective concentrations are only reached within the first days to weeks after implantation, leaving no local antimicrobial release available when a biomaterial-associated infection occurs later. Here we compare the ad libitum release of chlorhexidine and silver-sulfadiazine from a central venous catheter with their release from a new, on-demand release coating consisting of a temperature-sensitive copolymer of styrene and n-butyl (meth)acrylate. The copolymer can be loaded with an antimicrobial, which is released when the temperature is raised above its glass transition temperature. Ad libitum release of chlorhexidine and silver-sulfadiazine from a commercially-purchased catheter and associated antimicrobial efficacy against Staphylococcus aureus was limited to 16days. Consecutive temperature-triggers of our on-demand coating yielded little or no antimicrobial efficacy of silver-acetate release, but antimicrobially-effective chlorhexidine concentrations were observed over a time period of 60-80days. This attests to the clear advantage of on-demand coatings above ad libitum releasing coatings, that may have released their antimicrobial content before it is actually needed. Importantly, glass transition temperature of chlorhexidine loaded copolymers was lower (48°C) than of silver loaded ones (61°C), facilitating their clinical use.

Measurements of softness of microbial cell surfaces.

Publisher Summary This chapter compares the dependence of the electrophoretic mobility on the ionic strength of a fibrillated and a nonfibrillated oral streptococcal strain. It reports that a soft, ion-penetrable strain was fibrillated, while an electrophoretically harder strain was devoid of a proteinaceous fibrillar surface layer, demonstrable by electron microscopy after negative staining. The bald streptococcal strain had a diffusion coefficient, as measured by dynamic light scattering that was independent of ionic strength and composition of the suspending fluid. The fibrillar strain shows an almost twofold decrease in diffusion coefficient on increasing the suspension pH from 2 to 7. Direct probing of the softness of microbial cell surfaces has been described using atomic force microscopy and suggested that the mechanical softness of microbial cell surfaces related with the electrophoretic one and the dynamic nature of microbial cell surfaces upon changes of their ionic environment. Thus, particulate microelectrophoresis, dynamic light scattering, and atomic force microscopy have become valuable, partly complementary methods to assess the softness and related ion penetrability of microbial cell surfaces. Considering the implications of these dynamic properties of microbial cell surfaces for their adhesion to surfaces, the chapter describes the application of these three methods as geared toward the analysis of microbial cell surfaces.