Contact Killing of Gram-Positive and Gram-Negative Bacteria on PDMS Provided with Immobilized Hyperbranched Antibacterial Coatings.

Here we describe in detail the preparation and application of antibacterial coatings on PDMS (poly(dimethylsiloxane)) and the contact-killing properties with 10 bacterial strains. Our aim was to develop a generally applicable coating to prevent biomaterial acquired infections, which is the major mode of failure of biomedical implants. In the first step, the surface was provided with a hydrophobic hyperbranched coating resin that was covalently attached to PDMS, mediated by an appropriate coupling agent. The coupling agent contained a siloxane group that reacts covalently with the silanol groups of air-plasma-treated PDMS and a blocked isocyanate enabling covalent coupling with the amino groups of the hyperbranched coating resins. The coating resins were functionalized with a polyethylenimine and subsequently quaternized with bromohexane and iodomethane. The coatings were highly effective against Gram-positive bacteria (five strains) and sufficiently active against Gram-negative bacteria (five stains). The killing effect on the latter group was strongly enhanced by adding a permeabilizer (EDTA). The biocidal efficacy was not influenced by the presence of (saliva) proteins.

Self-perceived mouthfeel and physico-chemical surface effects after chewing gums containing sorbitol and Magnolia bark extract.

The European Food Safety Authority recognizes the contribution of sugar-free chewing gum to oral health through increased salivation, clearance of food debris, and neutralization of biofilm pH. Magnolia bark extract is a gum additive shown to reduce the prevalence of bad-breath bacteria but its effects on self-perceived mouthfeel are unknown. This paper aims to relate the effects of sorbitol-containing chewing gum, with and without Magnolia bark extract, on tooth-surface hydrophobicity and salivary-film composition with self-perceived mouthfeel. In a crossover clinical trial, volunteers chewed sorbitol-containing gum, with or without Magnolia bark extract added, three times daily during a 4-wk time period. A subset of volunteers also chewed Parafilm as a mastication control. Oral moistness and tooth smoothness were assessed using questionnaires, and intra-oral water-contact angles were measured before, immediately after, and 60 min after, chewing. Simultaneously, saliva samples were collected, placed on glass slides, and the compositions of the adsorbed film were measured using X-ray photoelectron spectroscopy. Chewing of gum, regardless of whether or not it contained Magnolia bark extract, improved self-perceived mouthfeel up to 60 min, concurrent with a more hydrophilic tooth surface and an increased amount of O1s electrons bound at 532.6 eV in salivary films. Chewing of Parafilm affected neither tooth-surface hydrophobicity nor salivary-film composition. Accordingly, adsorption of sorbitol, rather than the presence of Magnolia bark extract or increased salivation, is responsible for improved self-perceived mouthfeel.

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces.

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 µm-3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 µm-3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.

Biofilm formation on stainless steel and gold wires for bonded retainers in vitro and in vivo and their susceptibility to oral antimicrobials.

OBJECTIVE: Bonded retainers are used in orthodontics to maintain treatment result. Retention wires are prone to biofilm formation and cause gingival recession, bleeding on probing and increased pocket depths near bonded retainers. In this study, we compare in vitro and in vivo biofilm formation on different wires used for bonded retainers and the susceptibility of in vitro biofilms to oral antimicrobials. MATERIALS AND METHODS: Orthodontic wires were exposed to saliva, and in vitro biofilm formation was evaluated using plate counting and live/dead staining, together with effects of exposure to toothpaste slurry alone or followed by antimicrobial mouthrinse application. Wires were also placed intra-orally for 72 h in human volunteers and undisturbed biofilm formation was compared by plate counting and live/dead staining, as well as by denaturing gradient gel electrophoresis for compositional differences in biofilms. RESULTS: Single-strand wires attracted only slightly less biofilm in vitro than multi-strand wires. Biofilms on stainless steel single-strand wires however, were much more susceptible to antimicrobials from toothpaste slurries and mouthrinses than on single-strand gold wires and biofilms on multi-strand wires. Also, in vivo significantly less biofilm was found on single-strand than on multi-strand wires. Microbial composition of biofilms was more dependent on the volunteer involved than on wire type. CONCLUSIONS: Biofilms on single-strand stainless steel wires attract less biofilm in vitro and are more susceptible to antimicrobials than on multi-strand wires. Also in vivo, single-strand wires attract less biofilm than multi-strand ones. CLINICAL SIGNIFICANCE: Use of single-strand wires is preferred over multi-strand wires, not because they attract less biofilm, but because biofilms on single-strand wires are not protected against antimicrobials as in crevices and niches as on multi-strand wires.

In vitro oral biofilm formation on triclosan-coated sutures in the absence and presence of additional antiplaque treatment.

PURPOSE: This study evaluated the in vitro plaque inhibitory effect of triclosan-coated polyglactin 910 sutures in the absence and presence of an additional antiplaque agent commonly used after oral surgery. MATERIALS AND METHODS: Triclosan-coated sutures were incubated for 4 hours in freshly collected human saliva and, when appropriate, subsequently treated with an antiplaque rinse containing chlorhexidine-cetyl pyridinium as active components. Sutures without a triclosan-coating served as a control. RESULTS: Triclosan-coated sutures harbored similar amounts of plaque as did uncoated sutures. Exposure to the antiplaque rinse caused significant decreases in viable organisms for uncoated and triclosan-coated sutures. However, after application of the antiplaque rinse, more micro-organisms were found on triclosan-coated than on uncoated sutures. CONCLUSION: Sutures coated with triclosan do not provide a sufficient antimicrobial effect to prevent in vitro colonization by oral bacteria, whereas use in combination with a chlorhexidine-cetyl pyridinium-containing antiplaque rinse appears to be counterproductive.

Comparison of methods to evaluate bacterial contact-killing materials.

Cationic surfaces with alkylated quaternary-ammonium groups kill adhering bacteria upon contact by membrane disruption and are considered increasingly promising as a non-antibiotic based way to eradicate bacteria adhering to surfaces. However, reliable in vitro evaluation methods for bacterial contact-killing surfaces do not yet exist. More importantly, results of different evaluation methods are often conflicting. Therefore, we compared five methods to evaluate contact-killing surfaces. To this end, we have copolymerized quaternary-ammonium groups into diurethane dimethacrylate/glycerol dimethacrylate (UDMA/GDMA) and determined contact-killing efficacies against five different Gram-positive and Gram-negative strains. Spray-coating bacteria from an aerosol onto contact-killing surfaces followed by air-drying as well as ASTM E2149-13a (American Society for Testing and Materials) were found unsuitable, while the Petrifilm® system and JIS Z 2801 (Japanese Industrial Standards) were found to be excellent methods to evaluate bacterial contact-killing surfaces. It is recommended however, that these methods be used in combination with a zone of inhibition on agar assay to exclude that leakage of antimicrobials from the material interferes with the contact-killing ability of the surface. STATEMENT OF SIGNIFICANCE: Bacterial adhesion to surfaces of biomaterials implants can be life-threatening. Antimicrobials to treat biomaterial-associated infections often fail due to the bacterial biofilm-mode-of-growth or are ineffective due to antibiotic-resistance of causative organisms. Positively-charged, quaternized surfaces can kill bacteria upon contact and are promising as a non-antibiotic-based treatment of biomaterial-associated infections. Reliable methods to determine efficacies of contact-killing surfaces are lacking, however. Here, we show that three out of five methods compared, including an established ASTM, are unsuitable. Methods found suitable should be used in combination with a zone-of-inhibition-assay to establish absence of antimicrobial leaching, potentially interfering with contact-killing. Identification of suitable assays for evaluating bacterial contact-killing will greatly assist this emerging field as an alternative for antibiotic-based treatment of biomaterial-associated-infections.

Self-defensive antibiotic-loaded layer-by-layer coatings: Imaging of localized bacterial acidification and pH-triggering of antibiotic release.

Self-defensive antibiotic-loaded coatings have shown promise in inhibiting growth of pathogenic bacteria adhering to biomaterial implants and devices, but direct proof that their antibacterial release is triggered by bacterially-induced acidification of the immediate environment under buffered conditions remained elusive. Here, we demonstrate that Staphylococcus aureus and Escherichia coli adhering to such coatings generate highly localized acidification, even in buffered conditions, to activate pH-triggered, self-defensive antibiotic release. To this end, we utilized chemically crosslinked layer-by-layer hydrogel coatings of poly(methacrylic acid) with a covalently attached pH-sensitive SNARF-1 fluorescent label for imaging, and unlabeled-antibiotic (gentamicin or polymyxin B) loaded coatings for antibacterial studies. Local acidification of the coatings induced by S. aureus and E. coli adhering to the coatings was demonstrated by confocal-laser-scanning-microscopy via wavelength-resolved imaging. pH-triggered antibiotic release under static, small volume conditions yielded high bacterial killing efficiencies for S. aureus and E. coli. Gentamicin-loaded films retained their antibacterial activity against S. aureus under fluid flow in buffered conditions. Antibacterial activity increased with the number of polymer layers in the films. Altogether, pH-triggered, self-defensive antibiotic-loaded coatings become activated by highly localized acidification in the immediate environment of an adhering bacterium, offering potential for clinical application with minimized side-effects. STATEMENT OF SIGNIFICANCE: Polymeric coatings were created that are able to uptake and selectively release antibiotics upon stimulus by adhering bacteria in order to understand the fundamental mechanisms behind pH-triggered antibiotic release as a potential way to prevent biomaterial-associated infections. Through fluorescent imaging studies, this work importantly shows that adhering bacteria produce highly localized pH changes even in buffer. Accordingly such coatings only demonstrate antibacterial activity by antibiotic release in the presence of adhering bacteria. This is clinically important, because ad libitum releasing antibiotic coatings usually show a burst release and have often lost their antibiotic content when bacteria adhere.

Voice prosthetic biofilm formation and Candida morphogenic conversions in absence and presence of different bacterial strains and species on silicone-rubber.

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.