Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions.

The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-ß) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola (as mono culture or selected mixed-culture). IFN-ß expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-ß expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.

In Vitro Antibacterial Effectiveness of a Naturopathic Oral Care Product on Oral Pathogens.

PURPOSE: Currently, the prevention of periodontal diseases focuses on mechanical removal of pathogenic biofilms combined with oral antiseptics as supportive chemical antibacterial control. Due to the risk of resistance development and side effects of existing antiseptics, the interest in alternative medicine with naturopathic treatment modalities is growing in dentistry. In the present study, the antibacterial effect of the naturopathic oral care product Repha OS and some of its derivatives, based on medicinal plant extracts and essential oils, with a specific focus on added sweeteners, was investigated on periodontal pathogenic and halitosis-associated bacteria. MATERIALS AND METHODS: The antibacterial efficacy was investigated by agar dilution assay. The minimum inhibitory concentration (MIC) for the bacterial species Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei was determined. RESULTS: A concentration-dependent antibacterial effect on oral bacterial species by Repha OS and its derivatives was demonstrated. For the original product, the maximum MIC was 10% of the calculated test solution concentration in agar for all examined bacterial species. The removal of essential oils reduced the antibacterial efficacy, whereas the displacement or replacement of sweeteners had almost no effect. CONCLUSION: In addition to other individual effects of the ingredients, the results of this study show that an antibacterial effect of the naturopathic oral care product on the tested oral bacterial species was achieved in vitro. In vivo, the combination of this antibacterial effect with other properties of the various ingredients may be interesting for a holistic approach in preventive dentistry.

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the "slippery liquid-infused porous surfaces" (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

Development and characterization of an oral multispecies biofilm implant flow chamber model.

Peri-implant infections are the most common cause of implant failure in modern dental implantology. These are caused by the formation of biofilms on the implant surface and consist of oral commensal and pathogenic bacteria, which harm adjacent soft and hard tissues and may ultimately lead to implant loss. In order to improve the clinical situation, there has to be a better understanding of biofilm formation on abiotic surfaces. Therefore, we successfully developed a system to cultivate an oral multispecies biofilm model in a flow chamber system, optimized for the evaluation of biofilm formation on solid materials by direct microscopic investigation. The model contains four relevant oral bacterial species: Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis in ratios similar to the native situation. The reliability of the developed "Hanoverian Oral Multispecies Biofilm Implant Flow Chamber" (HOBIC) model was verified. Biofilm volume and live/dead distribution within biofilms were determined by fluorescence staining and confocal laser scanning microcopy (CLSM). The individual species distribution was analyzed using quantitative real time PCR with propidium monoazide pretreatment (PMA-qRT-PCR) and by urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH). This in vitro model may be used to analyze biofilm formation on dental implants in more detail and to develop future implant systems with improved material properties.

A single-step transconjugation system for gene deletion in Aggregatibacter actinomycetemcomitans.

Aggregatibacter (A.) actinomycetemcomitans is a periodontopathogenic bacterium causing aggressive periodontitis. Here we describe a single-step transconjugation system as novel and easily applicable protocol for site-specific genetic manipulation of A. actinomycetemcomitans. Deletion of PgaC, which is involved in the synthesis of biofilm matrix, led to a reduced biofilm formation.

Biofilm formation by the oral pioneer colonizer Streptococcus gordonii: an experimental and numerical study.

For decades, extensive research efforts have been conducted to improve the functionality and stability of implants. Especially in dentistry, implant treatment has become a standard medical practice. The treatment restores full dental functionality, helping patients to maintain high quality of life. However, about 10% of the patients suffer from early and late device failure due to peri-implantitis, an inflammatory disease of the tissues surrounding the implant. Peri-implantitis is caused by progressive microbial colonization of the device surface and the formation of microbial communities, so-called biofilms. This infection can ultimately lead to implant failure. The causative agents for the inflammatory disease, periodontal pathogenic biofilms, have already been extensively studied, but are still not completely understood. As numerical simulations will have the potential to predict oral biofilm formation precisely in the future, for the first time, this study aimed to analyze Streptococcus gordonii biofilms by combining experimental studies and numerical simulation. The study demonstrated that numerical simulation was able to precisely model the influence of different nutrient concentration and spatial distribution of active and inactive biomass of the biofilm in comparison with the experimental data. This model may provide a less time-consuming method for the future investigation of any bacterial biofilm.

Development of Laser-Structured Liquid-Infused Titanium with Strong Biofilm-Repellent Properties.

Medical implants are commonly used in modern medicine but still harbor the risk of microbial infections caused by bacterial biofilms. As their retrospective treatment is difficult, there is a need for biomedical materials that inhibit bacterial colonization from the start without using antibacterial agents, as these can promote resistance development. The promising concept of slippery liquid-infused porous surfaces (SLIPS) possesses enormous potential for this purpose. In the present study, this principle was applied to titanium, a common material in implantology, and its biofilm-repellent properties were demonstrated. To simplify prospective approval of the medical device and to avoid chemical contamination, surface structuring was performed by ultrashort pulsed laser ablation. Four different structures (hierarchical micro- and nanosized spikes, microsized grooves, nanosized ripples, and unstructured surfaces) and five infusing perfluoropolyethers of different viscosities were screened; the best results were obtained with the biomimetic, hierarchical spike structure combined with lubricants of medium viscosities (20-60 cSt at 37 °C, 143 AZ, and GPL 104). The surfaces exhibited extremely low contact angle hysteresis, as is typical for liquid-infused materials and a reliable 100-fold reduction of human oral pathogen Streptococcus oralis biofilms. This characteristic was maintained after exposure to shear forces and gravity. The titanium SLIPS also inhibited adherence of human fibroblasts and osteoblasts. Toxicity tests supported the explanation that solely the surface's repellent properties are responsible for the vigorous prevention of the adhesion of bacteria and cells. This use of physically structured and liquid-infused titanium to avoid bioadhesion should support the prevention of bacterial implant-associated infections without the use of antibacterial agents.

Quantifying implant-associated biofilms: Comparison of microscopic, microbiologic and biochemical methods.

Biofilm-associated infections pose severe problems in modern implant medicine. Screening for new implant materials with antibacterial properties requires reliable quantification of colonizing bacteria. There are many different methods to quantify biofilms on solid surfaces in vitro, employing different (bio-)chemical/microbiological reference parameters. It is therefore difficult to compare studies with different quantification techniques. Here, we have evaluated commonly used microscopic, microbiologic and biochemical methods to quantify bacterial biofilms, in order to clarify their comparability and applicability. Two bacterial species frequently involved in biofilm-associated infections, Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, were used as model organisms; their initial adhesion and biofilm formation on titanium and on antibacterial copper were analyzed using the following methods: LIVE/DEAD fluorescence staining and confocal laser-scanning microscopy, ultrasonic or a newly developed enzymatic detachment followed by standard plate counting (CFU method), a resazurin-based assay, the BacTiter-Glo™ assay and crystal violet staining. The methods differed greatly in complexity, reliability and the applicability to initial adhesion and biofilm formation. To screen biofilm formation on a multitude of surfaces, the resazurin-based and the BacTiterGlo™ assay are well suited. LIVE/DEAD staining and confocal laser-scanning microscopy can be applied for a more detailed analysis of both, initial adhesion and biofilm formation. When using the CFU method for screening purposes, the introduced enzymatic detachment procedure is to be favored over ultrasonic detachment. There is not one single method, which is suitable for all purposes. The appropriate biofilm quantification method has to be chosen on the basis of the specific scientific question.