Transmission of Monospecies and Dual-Species Biofilms from Smooth to Nanopillared Surfaces.

The transmission of bacteria in biofilms from donor to receiver surfaces precedes the formation of biofilms in many applications. Biofilm transmission is different from bacterial adhesion, because it involves biofilm compression in between two surfaces, followed by a separation force leading to the detachment of the biofilm from the donor surface and subsequent adhesion to the receiver surface. Therewith, the transmission depends on a balance between donor and receiver surface properties and the cohesiveness of the biofilm itself. Here, we compare bacterial transmission from biofilms of an extracellular-polymeric-substance (EPS)-producing and a non-EPS-producing staphylococcal strain and a dual-species oral biofilm from smooth silicon (Si) donor surfaces to smooth and nanopillared Si receiver surfaces. Biofilms were fully covering the donor surface before transmission. However, after transmission, the biofilms only partly covered the donor and receiver surfaces regardless of nanopillaring, indicating bacterial transmission through adhesive failure at the interface between biofilms and donor surfaces as well as through cohesive failure in the biofilms. The numbers of bacteria per unit volume in EPS-producing staphylococcal biofilms before transmission were 2-fold smaller than in biofilms of the non-EPS-producing strain and of dual species. This difference increased after transmission in the biofilm left behind on the donor surfaces due to an increased bacterial density for the non-EPS-producing strain and a dual-species biofilm. This suggests that biofilms of the non-EPS-producing strain and dual species remained compressed after transmission, while biofilms of the EPS-producing strain were induced to produce more EPS during transmission and relaxed toward their initial state after transmission due to the viscoelasticity conferred to the biofilm by its EPS.IMPORTANCE Bacterial transmission from biofilm-covered surfaces to surfaces is mechanistically different from bacterial adhesion to surfaces and involves detachment from the donor and adhesion to the receiver surfaces under pressure. Bacterial transmission occurs, for instance, in food processing or packaging, in household situations, or between surfaces in hospitals. Patients admitted to a hospital room previously occupied by a patient with antibiotic-resistant pathogens are at elevated infection risk by the same pathogens through transmission. Nanopillared receiver surfaces did not collect less biofilm from a smooth donor than a smooth receiver, likely because the pressure applied during transmission negated the smaller contact area between bacteria and nanopillared surfaces, generally held responsible for reduced adhesion. Biofilm left behind on smooth donor surfaces of a non-extracellular-polymeric-substance (EPS)-producing strain and dual species had undergone different structural changes than an EPS-producing strain, which is important for their possible further treatment by antimicrobials or disinfectants.

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.

Osteoblast integration of dental implant materials after challenge by sub-gingival pathogens: a co-culture study in vitro.

Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium (Ti), titanium-zirconium (TiZr) alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.

Soft tissue integration versus early biofilm formation on different dental implant materials.

OBJECTIVE: Dental implants anchor in bone through a tight fit and osseo-integratable properties of the implant surfaces, while a protective soft tissue seal around the implants neck is needed to prevent bacterial destruction of the bone-implant interface. This tissue seal needs to form in the unsterile, oral environment. We aim to identify surface properties of dental implant materials (titanium, titanium-zirconium alloy and zirconium-oxides) that determine the outcome of this "race-for-the-surface" between human-gingival-fibroblasts and different supra-gingival bacterial strains. METHODS: Biofilms of three streptococcal species or a Staphylococcus aureus strain were grown in mono-cultures on the different implant materials in a parallel-plate-flow-chamber and their biovolume evaluated using confocal-scanning-laser-microscopy. Similarly, adhesion, spreading and growth of human-gingival-fibroblasts were evaluated. Co-culture experiments with bacteria and human-gingival-fibroblasts were carried out to evaluate tissue interaction with bacterially contaminated implant surfaces. Implant surfaces were characterized by their hydrophobicity, roughness and elemental composition. RESULTS: Biofilm formation occurred on all implant materials, and neither roughness nor hydrophobicity had a decisive influence on biofilm formation. Zirconium-oxide attracted most biofilm. All implant materials were covered by human-gingival-fibroblasts for 80-90% of their surface areas. Human-gingival-fibroblasts lost the race-for-the-surface against all bacterial strains on nearly all implant materials, except on the smoothest titanium variants. SIGNIFICANCE: Smooth titanium implant surfaces provide the best opportunities for a soft tissue seal to form on bacterially contaminated implant surfaces. This conclusion could only be reached in co-culture studies and coincides with the results from the few clinical studies carried out to this end.

Generalized relationship between numbers of bacteria and their viability in biofilms.

Bacterial biofilms are confined communities that are encapsulated in protective layers of extracellular polymeric substances. Microscopic evaluation of biofilms of diverse bacterial strains on various substrata reveals that, in general, the percentage of viable bacteria decreases with the total number of bacteria in a biofilm.

Oral biofilm models for mechanical plaque removal.

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24-40%), while the manual brush as a control (5-11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro.

Bond strengthening in oral bacterial adhesion to salivary conditioning films.

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.

Adhesion and viability of waterborne pathogens on p-DADMAC coatings.

The attachment of waterborne pathogens onto surfaces can be increased by coating the surfaces with positive charge-enhancing polymers. In this paper, the increased efficacy of polydiallyldimethylammonium chloride (p-DADMAC) coatings on glass was evaluated in a parallel plate flow chamber with the use of waterborne pathogens (Raoultella terrigena, Escherichia coli, and Brevundimonas diminuta). p-DADMAC coatings strongly compensated the highly negative charges on the glass surface and even yielded a positively charged surface when applied from a 500 ppm solution. Whereas none of the strains adhered from water to glass due to electrostatic repulsion, R. terrigena and E. coli readily adhered in high numbers to p-DADMAC coated glass slides applied from 1, 100, or 500 ppm aqueous solutions. B. diminuta only adhered to a positively charged p-DADMAC coating applied from a 500 ppm solution. In addition, all p-DADMAC coatings indicated strong contact killing with the bacterial species used in this study by live/dead staining techniques. In summary, this paper demonstrates the potential of p-DADMAC coatings to strongly enhance bacterial adhesion. Moreover, once adhered, bacterial viability can be reduced by the positively charged ammonium groups in the coating.

Influence of day and night wear on surface properties of silicone hydrogel contact lenses and bacterial adhesion.

PURPOSE: The aim of this study was to determine the effect of continuous wear on physicochemical surface properties of silicone hydrogel (S-H) lenses and their susceptibility to bacterial adhesion. METHODS: In this study, volunteers wore 2 pairs of either "lotrafilcon A" or "balafilcon A" S-H contact lenses. The first pair was worn continuously for a week and the second pair for 4 weeks. One lens of each pair was used for surface characterization and the other one for bacterial adhesion experiments. Lens surfaces were characterized by examination of their wettability, roughness, elemental composition, and proteins attached to their surfaces. Adhesion of Staphylococcus aureus 835 and Pseudomonas aeruginosa #3 to a lens was studied using a parallel plate flow chamber. RESULTS: Before use, the lotrafilcon A lens was rougher than the balafilcon A lens and had a lower water contact angle and a higher affinity for S. aureus 835. After wear, both lens types had similar water contact angles, whereas the differences in elemental surface composition decreased as well. S. aureus 835 adhered in higher numbers to worn balafilcon A lenses, whereas the opposite was seen for P. aeruginosa #3. The initial deposition rates of both bacterial strains to lotrafilcon A lenses decreased by wearing and were found to correlate significant (P < 0.001) with the surface roughness of worn lenses. CONCLUSIONS: In this study, the differences in surface properties between 2 types of S-H lenses were found to change after 1 week of continuous wear. Generally, bacteria adhered in lower numbers and less tenaciously to worn lenses, except S. aureus 835, adhering in higher numbers to worn balafilcon A lenses.

Resistance to a polyquaternium-1 lens care solution and isoelectric points of Pseudomonas aeruginosa strains.

OBJECTIVES: The aim of this study was to correlate the cell surface hydrophobicity and charge of various strains of Pseudomonas aeruginosa with their resistance to a polyquaternium-1 lens care solution. METHODS: The 11 P. aeruginosa strains included were isolated from eyes, contact lenses, lens cases and lens care solutions. Cell surface hydrophobicities were determined from water contact angle measurements and surface charges were measured as a function of pH using particulate micro-electrophoresis. RESULTS: Strains resistant to polyquaternium-1 had an isoelectric point (IEP; pH where the bacterial zeta potential is zero) ranging from 4.0 to 5.5, whereas susceptible strains were more negatively charged than resistant strains and had an IEP between 1.3 and 1.9. Water contact angles ranged from hydrophilic (34 degrees) to hydrophobic (124 degrees), without showing a relation with antimicrobial resistance. CONCLUSIONS: Results suggest that electrostatic repulsion between cationic molecules on the cell surface and quaternary ammonium compounds impedes the antimicrobial entering the cell.

Influence of wear and overwear on surface properties of etafilcon A contact lenses and adhesion of Pseudomonas aeruginosa.

PURPOSE: To determine changes in physicochemical surface properties of contact lenses (CLs) during daily wear and effects of lens wear on adhesion of a Pseudomonas aeruginosa strain from a patient with CL-related keratitis. METHODS: Ten new CL wearers used ionic, etafilcon A lenses with 58% water on both eyes for approximately 10 hours each day during 10 and 50 days. All lenses were treated daily with an appropriate lens care solution. After the CLs were worn for 10 days (first pair of lenses) and 50 days (second pair, representing overwear), hydrophobicity by water contact angles, surface roughness by atomic force microscope, elemental surface composition by x-ray photoelectron spectroscopy (XPS), and adsorbed proteins by SDS-PAGE were determined on one lens. The lens from the contralateral eye was placed in a parallel plate flow chamber for bacterial adhesion after each time interval. RESULTS: Water contact angles on lenses changed from 45 degrees on unused lenses to 61 degrees +/- 25 degrees after 10 days of wear and changed significantly (P < 0.05) to 27 degrees +/- 14 degrees after 50 days of wear. Surface roughness increased significantly (P < 0.05) from 4 +/- 2 nm (unused) to 10 +/- 7 nm after 50 days of wear. These changes were accompanied by adsorption of proteinaceous material, as evidenced by XPS and SDS-PAGE, demonstrating adsorption of lysozyme, tear lipocalin, and a 30-kDa protein. Initial bacterial adhesion to worn CLs was lower than to unworn CLs. Furthermore, detachment of adhering bacteria from worn lenses was easier than from unworn lenses. The changes observed in the physicochemical surface properties of the lenses after the CLs were worn for 50 days were accompanied by reports of discomfort by 6 of the 10 new CL wearers. Multiple regression analysis revealed that the most predictive variables for an effect on initial deposition after 10 days of wear were hydrophobicity, roughness, the presence of nitrogen-rich material, including the presence of a 30-kDa protein, and the presence of oxygen-rich material-that is, the type of oxygen adsorbed (O equal or parallel C or Ocjs0807;C). After 50 days of wear, roughness and the presence of tear lipocalin were most predictive. CONCLUSIONS: This study demonstrates that the physicochemical surface properties changed after wear and overwear, whereas overwear of the lenses decreased initial adhesion of P. aeruginosa #3 under the present experimental conditions.