Macrophage response to staphylococcal biofilms on crosslinked poly(ethylene) glycol polymer coatings and common biomaterials in vitro.

Biomaterial-associated-infections (BAI) are serious clinical complications that threaten the longevity of implanted devices and lead to high morbidity and mortality. Poly(ethylene)glycol (PEG) coatings have been studied as a strategy to reduce the incidence of BAI by reducing protein deposition that promotes pathogen adhesion and growth on device surfaces. Despite their effectiveness to reduce protein adsorption and a hundred-fold reduction in bacterial adhesion, PEG-based coatings still facilitate weak bacterial adhesion that can form an initial basis for biofilms. Here, we describe a methodology enabling direct, quantitative and detailed qualitative in situ observation of macrophage morphology, migration and phagocytosis of bacteria. In vitro interaction of macrophages with Staphylococcus epidermidis 3399 adhering to commercial, crosslinked PEG-based coatings (OptiChem®) was compared with fluorinated ethylene propylene, silicone rubber and glass. Adhesion, phagocytosis and migration were studied real-time in a parallel-plate-flow-chamber. Macrophages cultured on OptiChem® coatings showed enhanced migration and phagocytosis of bacteria compared to common biomaterials. Bacterial clearance per macrophage on both inert and reactive OptiChem® coatings were about three times higher than on the common biomaterials studied, corresponding with up to 70% reduction in bacterial numbers on OptiChem®, whereas on the biomaterials less than 40% bacterial reduction was obtained. These findings show that bacterial clearance from cross-linked PEG-based coatings by macrophages is more effective than from common biomaterials, possibly resulting from weak adhesion of bacteria on Optichem®. Moreover, macrophages exhibit higher mobility on Optichem® retaining an improved capability to clear bacteria from larger areas than from other common biomaterials, where they appear more immobilized.

Competitive time- and density-dependent adhesion of staphylococci and osteoblasts on crosslinked poly(ethylene glycol)-based polymer coatings in co-culture flow chambers.

Biomaterial-associated infections (BAI) remain a serious clinical complication, often arising from an inability of host tissue-implant integration to out-compete bacterial adhesion and growth. A commercial polymer coating based on polyethylene glycol (PEG), available in both chemically inert and NHS-activated forms (OptiChem(®)), was compared for simultaneous growth of staphylococci and osteoblasts. In the absence of staphylococci, osteoblasts adhered and proliferated well on glass controls and on the NHS-reactive PEG-based coating over 48 h, but not on the inert PEG coating. Staphylococcal growth was low on both PEG-based coatings. When staphylococci were pre-adhered on surfaces for 1.5 h to mimic peri-operative contamination, osteoblast growth and spreading was reduced on glass but virtually absent on both reactive and inert PEG-based coatings. Thus although NHS-reactive, PEG-based coatings stimulated tissue-cell interactions in the absence of contaminating staphylococci, the presence of adhering staphylococci eliminated osteoblast adhesion advantages on the PEG surface. This study demonstrates the importance of using bacterial and cellular co-cultures compared to monocultures when assessing functionalized biomaterials coatings for infectious potential.

The risk of biomaterial-associated infection after revision surgery due to an experimental primary implant infection.

The fate of secondary biomaterial implants was determined by bio-optical imaging and plate counting, after antibiotic treatment of biomaterials-associated-infection (BAI) and surgical removal of an experimentally infected, primary implant. All primary implants and tissue samples from control mice showed bioluminescence and were culture-positive. In an antibiotic treated group, no bioluminescence was detected and only 20% of all primary implants and no tissue samples were culture-positive. After revision surgery, bioluminescence was detected in all control mice. All the implants and 80% of all tissue samples were culture-positive. In contrast, in the antibiotic treated group, 17% of all secondary implants and 33% of all tissue samples were culture-positive, despite antibiotic treatment. The study illustrates that due to the BAI of a primary implant, the infection risk of biomaterial implants is higher in revision surgery than in primary surgery, emphasizing the need for full clearance of the infection, as well as from surrounding tissues prior to implantation of a secondary implant.

In vitro and in vivo comparisons of staphylococcal biofilm formation on a cross-linked poly(ethylene glycol)-based polymer coating.

Poly(ethylene glycol) (PEG) coatings are known to reduce microbial adhesion in terms of numbers and binding strength. However, bacterial adhesion remains of the order of 10(4)cm(-2). It is unknown whether this density of bacteria will eventually grow into a biofilm. This study investigates the kinetics of staphylococcal biofilm formation on a commercially produced, robust, cross-linked PEG-based polymer coating (OptiChem) in vitro and in vivo. OptiChem inhibits biofilm formation in vitro, and although adsorption of plasma proteins encourages biofilm formation, microbial growth kinetics are still strongly delayed compared to uncoated glass. In vivo, OptiChem-coated and bare silicone rubber samples were inserted into an infected murine subcutaneous pocket model. In contrast to bare silicone rubber, OptiChem samples did not become colonized upon reimplantation despite the fact that surrounding tissues were always culture-positive. We conclude that the commercial OptiChem coating considerably slows down bacterial biofilm formation both in vitro and in vivo, making it an attractive candidate for biomaterials implant coating.

Bacterial colonization of polymer brush-coated and pristine silicone rubber implanted in infected pockets in mice.

OBJECTIVES: Curing biomaterial-associated infection (BAI) frequently includes antibiotic treatment, implant removal and re-implantation. However, revision implants are at a greater risk of infection as they may attract bacteria from their infected surroundings. Polymer brush-coatings attract low numbers of bacteria, but the virtue of polymer brush-coatings in vivo has seldom been investigated. Here, we determine the possible benefits of polymer brush-coated versus pristine silicone rubber in revision surgery, using a murine model. METHODS: BAI was induced in 26 mice by subcutaneous implantation of silicone rubber discs with a biofilm of Staphylococcus aureus Xen29. During the development of BAI, half of the mice received rifampicin/vancomycin treatment. After 5 days, the infected discs were removed from all mice, and either a polymer brush-coated or pristine silicone rubber disc was re-implanted. Revision discs were explanted after 5 days, and the number of cfu cultured from the discs and the surrounding tissue was determined. RESULTS: None of the polymer brush-coated discs after antibiotic treatment appeared colonized by staphylococci, whereas 83% of the pristine silicone rubber discs were re-infected. Polymer brush-coated discs also showed reduced colonization rates in the absence of antibiotic treatment when compared with pristine silicone rubber discs. Tissue surrounding the discs was culture-positive in all cases. CONCLUSIONS: Polymer brush-coatings are less prone to re-infection than pristine silicone rubber when used in revision surgery, i.e. when implanted in a subcutaneous pocket infected by a staphylococcal BAI. Antibiotic pre-treatment during the development of BAI hardly had any effect in preventing the colonization of pristine silicone rubber.

The inhibition of the adhesion of clinically isolated bacterial strains on multi-component cross-linked poly(ethylene glycol)-based polymer coatings.

This study examined bacterial adhesion to a new multi-component cross-linked poly(ethylene glycol)-based polymer coating that can be applied by spin-coating or spraying onto diverse biomaterials. Adhesion of five clinically isolated bacterial strains involved in biomaterial-centered infections were studied in a parallel-plate flow chamber at different shear rates and after exposure of the coating to different physiological fluids. The new chemistry inhibits non-specific biomolecular and cell binding interactions. Relative to glass, the coating reduced adhesion of all strains used in this study by more than 80%, with the exception of Escherichia coli O2K2. Reductions in adhesion of Staphylococcus epidermidis 3,399 persisted beyond 168h exposure of the coatings to phosphate buffered saline or urine, but not after exposure to protein-rich fluids as saliva and blood plasma, despite evidence from X-ray photoelectron spectroscopy that the coating integrity was not affected by exposure to these fluids. We conclude that this new coating chemistry provides beneficial properties to prevent or hinder bacterial adhesion and colonization in applications where low protein-conditions prevail.