Rene J. B. Dijkstra

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.

The potential for bio-optical imaging of biomaterial-associated infection in vivo

This review presents the current state of Bioluminescence and Fluorescent Imaging technologies (BLI and FLI) as applied to Biomaterial-Associated Infections (BAI). BLI offers the opportunity to observe the in vivo course of BAI in small animals without the need to sacrifice animals at different time points after the onset of infection. BLI is highly dependent on the bacterial cell metabolism which makes BLI a strong reporter of viable bacterial presence. Fluorescent sources are generally more stable than bioluminescent ones and specifically targeted, which renders the combination of BLI and FLI a promising tool for imaging BAI. The sensitivity and spatial resolution of both imaging tools are, however, dependent on the imaging system used and the tissue characteristics, which makes the interpretation of images, in terms of the location and shape of the illuminating source, difficult. Tomographic reconstruction of the luminescent source is possible in the most modern instruments, enabling exact localization of a colonized implant material, spreading of infecting organisms in surrounding tissue and immunological tissue reactions. BLI studies on BAI have successfully distinguished between different biomaterials with respect to the development and clearance of BAI in vivo, simultaneously reducing animal use and experimental variation. It is anticipated that bio-optical imaging will become an indispensable technology for the in vivo evaluation of antimicrobial coatings.

Influence of a chitosan on oral bacterial adhesion and growth in vitro

Generally, mechanical plaque control without chemical support is insufficient to prevent oral diseases, and an ongoing quest exists for new antimicrobials for use in oral healthcare. Chitosans are polycationic, naturally occurring antimicrobials that are rapidly finding their way into oral healthcare. In this study we determined the effects of pellicle treatment with chitosan on bacterial adhesion and growth. Chitosan caused a reduction in bacterial adhesion and was responsible for bacterial death upon contact compared with a buffer control. Exposure of adhering bacteria to a chitosan solution or a buffer control did not cause detachment, but the chitosan solution left a small proportion of the adhering bacteria alive. Growth after exposure to chitosan was similar to (Streptococcus mutans ATCC700610, Streptococcus oralis HM1, Streptococcus sobrinus HG1025) or less than (S. mutans NS, Actinomyces naeslundii HM1) that of the control, while biofilm viability after chitosan treatment was lower than that of the control, except for S. oralis HM1. Therefore, chitosan is a promising antimicrobial for use in oral healthcare.

Intermolecular Forces and Enthalpies in the Adhesion of Streptococcus mutans and an Antigen I/II-Deficient Mutant to Laminin Films

The antigen I/II family of surface proteins is expressed by most oral streptococci, including Streptococcus mutans, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient S. mutans isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 x 10(6) and 1.1 x 10(6) cells cm(-2) at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 x 10(6) and 26.1 x 10(6) cells cm(-2)). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to -5.0 and -4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to -1.5 and -2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on S. mutans LT11 is on the order of 6 x 10(4) sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.

Antibacterial efficacy of a new gentamicin-coating for cementless prostheses compared to gentamicin-loaded bone cement

Cementless prostheses are increasingly popular but require alternative prophylactic measures than the use of antibiotic‐loaded bone cements. Here, we determine the 24‐h growth inhibition of gentamicin‐releasing coatings from grit‐blasted and porous‐coated titanium alloys, and compare their antibacterial efficacies and gentamicin release‐profiles to those of a commercially available gentamicin‐loaded bone cement. Antibacterial efficacy increased with increasing doses of gentamicin in the coating and loading with 1.0 and 0.1 mg gentamicin/cm2 on both grit‐blasted and porous‐coated samples yielded comparable efficacy to gentamicin‐loaded bone cement. The coating had a higher burst release than bone cement, and also inhibited growth of gentamicin‐resistant strains. Antibacterial efficacy of the gentamicin coatings disappeared after 4 days, while gentamicin‐loaded bone cement exhibited efficacy over at least 7 days. Shut‐down after 4 days of gentamicin‐release from coatings is advantageous over the low‐dosage tail‐release from bone cements, as it minimizing risk of inducing antibiotic‐resistant strains. Both gentamicin‐loaded cement discs and gentamicin‐coated titanium coupons were able to kill gentamicin‐sensitive and ‐resistant bacteria in a simulated prothesis‐related interfacial gap. In conclusion, the gentamicin coating provided similar antibacterial properties to those seen by gentamicin‐loaded bone cement, implying protection of a prosthesis from being colonized by peri‐operatively introduced bacteria in cementless total joint arthroplasty.

A gentamicin-releasing coating for cementless hip prostheses - Longitudinal evaluation of efficacy using in vitro bio-optical imaging and its wide-spectrum antibacterial efficacy

Cementless prostheses are increasingly popular in total hip arthroplasties. Therewith, common prophylactic measures to reduce the risk of postoperative infection like the use of antibiotic-loaded bone cements, will no longer be available. Alternative prophylactic measures may include the use of antibiotic-releasing coatings. Previously, we developed a gentamicin-releasing coating for cementless titanium hip prostheses and derived an appropriate dosing of this coating by adjusting the amount of gentamicin in the coating to match the antibacterial efficacy of clinically employed gentamicin-loaded bone cement. In this manuscript, we investigated two important issues regarding the prophylactic use of this 1 mg cm(-2) bioactive gentamicin-releasing coating in cementless total hip arthroplasty: (1) its ability to prevent bacterial growth in a geometrically relevant set-up and (2) its antibacterial spectrum. A geometrically relevant set-up was developed in which miniature titanium stems were surrounded by agar, contaminated with bioluminescent Staphylococcus aureus. Novel, bio-optical imaging was performed allowing noninvasive, longitudinal monitoring of staphylococcal growth around miniature stems with and without the gentamicin-releasing coating. Furthermore, the antibacterial efficacy of the gentamicin-releasing coating was determined against a wide variety of clinical isolates, including bioluminescent Staphylococcus aureus strains, using traditional zone of inhibition measurements. The gentamicin-releasing coating demonstrated a wide-spectrum of antibacterial efficacy and successfully prevented growth of bioluminescent staphylococci around a miniature stem mounted in bacterially contaminated agar for at least 60 h. This implies that the gentamicin-releasing coating has potential to contribute to the improvement of infection prophylaxis in cementless total hip arthroplasty.

Persistence of a bioluminescent Staphylococcus aureus strain on and around degradable and non-degradable surgical meshes in a murine model

Biomaterials are increasingly used for the restoration of human function, but can become infected as a result of peri- or early post-operative bacterial contamination, although biomaterial-associated infections (BAIs) can also initiate at any time from hematogenous spreading of bacteria from an infection elsewhere in the body. Infecting bacteria in BAIs not only seek shelter in their own protective biofilm matrix, but also hide in surrounding tissue. This study compares staphylococcal persistence on and around a degradable and non-degradable surgical mesh through the use of longitudinal bioluminescence imaging in a murine model, including histological evaluation of surrounding tissue after sacrifice. Surgical meshes were first contaminated with bioluminescent Staphylococcus aureus Xen29 and subsequently subcutaneously implanted in mice. Bioluminescent staphylococci persisted on and around non-degradable meshes during the 28-day course of the study, whereas bioluminescence returned to control levels and bacteria disappeared from surrounding tissues once a degradable mesh had fully dissolved. Thus the application of degradable biomaterials yields major advantages with respect to the prevention of BAIs, as dissolution of the implant not only is associated with elimination of the protective biofilm mode of growth of the infecting organisms, but also allows the immune system to clear the surrounding tissue from infecting organisms.

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.

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.

Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus

Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in Etests demonstrated increased bioluminescence at sub-MICs of different antibiotics. This study aimed to further evaluate the influence of antibiotic pressure on bioluminescence in S. aureus Xen29. Bioluminescence of S. aureus Xen29, grown planktonically in tryptone soy broth, was quantified in the absence and presence of different concentrations of vancomycin, ciprofloxacin, erythromycin or chloramphenicol and was related to expression of the luxA gene under antibiotic pressure measured using real-time PCR. In the absence of antibiotics, staphylococcal bioluminescence increased over time until a maximum after ca. 6h of growth, and subsequently decreased to the detection threshold after 24h of growth owing to reduced bacterial metabolic activity. Up to MICs of the antibiotics, bioluminescence increased according to a similar pattern up to 6h of growth, but after 24h bioluminescence was higher than in the absence of antibiotics. Contrary to expectations, bioluminescence per organism (CFU) after different growth periods in the absence and at MICs of different antibiotics decreased with increasing expression of luxA. Summarising, antibiotic pressure impacts the relation between CFU and bioluminescence. Under antibiotic pressure, bioluminescence is not controlled by luxA expression but by co-factors impacting the bacterial metabolic activity. This conclusion is of utmost importance when evaluating antibiotic efficacy in live animals using bioluminescent bacterial strains.