Biomaterial-associated Infections Research Articles

The influence of antibodies on Staphylococcus epidermidis adherence to polyvinylpyrrolidone-coated silicone elastomer in experimental biomaterial-associated infection in mice

Biomaterial-associated infection (BAI) is a major problem in modern medicine, and is often caused by Staphylococcus epidermidis. We aimed to raise monoclonal antibodies (mAbs) against major surface protein antigens of S. epidermidis, and to assess their possible protective activity in experimental BAI. Mice were vaccinated with a cell wall protein preparation of S. epidermidis. A highly immunodominant antigen was identified as Accumulation-associated protein (Aap). mAbs against Aap and against surface-exposed lipoteichoic acid (LTA) were used for passive immunization of mice in experimental biomaterial-associated infection. Neither anti-Aap nor anti-LTA mAbs showed protection. Either with or without antibodies, tissue surrounding the implants was more often culture positive than the implants themselves, but bacterial adherence to the implants was significantly increased in mice injected with anti-LTA. In vitro, anti-Aap and anti-LTA did show binding to S. epidermidis, but no opsonic activity was observed. We conclude that antibodies against S. epidermidis LTA or Aap showed no opsonic activity and did not protect mice against BAI. Moreover, the increase in binding to implanted biomaterial suggests that passive immunization may increase the risk for BAI.

Microbial biofilm growth vs. tissue integration: “The race for the surface” experimentally studied

Biomaterial-associated infections constitute a major clinical problem. Unfortunately, microorganisms are frequently introduced onto an implant surface during surgery and start the race for the surface before tissue integration can occur. So far, no method has been forwarded to study biofilm formation and tissue integration simultaneously. The aim of this study is to describe an in vitro method to investigate this “race for the surface”. First, a suitable growth medium was prepared that allowed both bacterial and tissue growth in a parallel plate flow chamber. Staphylococci were deposited on the glass bottom plate of the flow chamber in different surface densities, after which U2OS osteosarcoma cells were seeded. U2OS cells did not grow in the absence of flow, possibly due to poisoning by bacterial endotoxins, but under flow both staphylococci and U2OS cells grew. The number of adhering cells and area per spread cell were determined after 48 h in relation to the initial number of bacteria present. Both the number and spread area per cell decreased with increasing density of adhering staphylococci. This demonstrates that the outcome of the race for the surface between bacteria and tissue cells is dependent on the number of bacteria present prior to cell seeding.

Prevention and Treatment of Staphylococcus Biofilms

Staphylococcus growth on medical devices represents a common occurrence that can lead to serious illness and death. Biomaterial-associated infection, mostly caused by Staphylococcus epidermidis and Staphylococcus aureus, is fairly complicated by the organism' development of a biofilm, which provides a microenvironment that protects from attack by the host immune system and antibiotics. In this review we present recent insights regarding S. aureus and S. epidermidis structural and functional factors that are effective in biofilm development and describe the regulation of their expression. On the basis of the knowledge gained, we also present the potential and limits of current biochemical and biophysical strategies aimed at preventing biofilm formation or at the treatment of established mature biofilms.

Tricarboxylic acid cycle-dependent regulation of Staphylococcus epidermidis polysaccharide intercellular adhesin synthesis.

Staphylococcus epidermidis is a major nosocomial pathogen primarily infecting immunocompromised individuals or those with implanted biomaterials (e.g., catheters). Biomaterial-associated infections often involve the formation of a biofilm on the surface of the medical device. In S. epidermidis, polysaccharide intercellular adhesin (PIA) is an important mediator of biofilm formation and pathogenesis. Synthesis of PIA is regulated by at least three DNA binding proteins (IcaR, SarA, and sigma(B)) and several environmental and nutritional conditions. Previously, we observed the environmental conditions that increased PIA synthesis decreased tricarboxylic acid (TCA) cycle activity. In this study, S. epidermidis TCA cycle mutants were constructed, and the function of central metabolism in PIA biosynthesis was examined. TCA cycle inactivation altered the metabolic status of S. epidermidis, resulting in a massive derepression of PIA biosynthetic genes and a redirection of carbon from growth into PIA biosynthesis. These data demonstrate that the bacterial metabolic status is a critical regulatory determinant of PIA synthesis. In addition, these data lead us to propose that the TCA cycle acts as a signal transduction pathway to translate external environmental cues into intracellular metabolic signals that modulate the activity of transcriptional regulators.

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

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. 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. 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. 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.

Assessing infection risk in implanted tissue-engineered devices

Peri-operative contamination is the major cause of biomaterial-associated infections, highly complicating surgical patient outcomes. While this risk in traditional implanted biomaterials is well-recognised, newer cell-seeded, biologically conducive tissue-engineered (TE) constructs now targeted for human use have not been assessed for this possibility. We investigated infection incidence of implanted, degradable polyester TE scaffold biomaterials in rabbit knee osteochondral defects. Sterile, polyester copolymer scaffolds of different compositions and cell-accessible pore volumes were surgically inserted into rabbit osteochondral defects for periods of 3 weeks up to 9 months, either with or without initial seeding with autologous or allogeneic chondrocytes. Infection assessment included observation of pus or abscesses in or near the knee joint and post-mortem histological evaluation. Of 228 implanted TE scaffolds, 10 appeared to be infected: 6 scaffolds without cell seeding (3.6%) and 4 cell-seeded scaffolds (6.3%). These infections were evident across all scaffold types, independent of polymer composition or available pore volume, and up to 9 months. We conclude that infections in TE implants pose a serious problem with incidences similar to current biomaterials-associated infections. Infection control measures should be developed in tissue engineering to avoid further complications when TE devices emerge clinically.

Treatment and Prevention of Staphylococcus epidermidis Experimental Biomaterial-Associated Infection by Bactericidal Peptide 2

Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices and are predominantly caused by staphylococci, especially Staphylococcus epidermidis. We investigated the in vitro microbicidal activity of the synthetic antimicrobial peptide bactericidal peptide 2 (BP2) and its efficacy in a murine model of S. epidermidis BAI. BP2 showed potent microbicidal activity at micromolar concentrations against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The staphylocidal activity of BP2 was not affected by physiological salt concentrations and was only slightly affected by the presence of human plasma. In the BAI model, injection of BP2 (5 mg/kg of body weight) 1 h after challenge with S. epidermidis resulted in an 80% reduction in the number of culture-positive implants and a 100-fold reduction in survival of S. epidermidis in peri-implant tissue at 24 h postchallenge. When BP2 was injected along implants 3 h prior to bacterial challenge, the median numbers of CFU cultured from biomaterial implants and peri-implant tissue were reduced by 85% and 90%, respectively. In conclusion, BP2 has potent, broad-spectrum in vitro microbicidal activity and showed potent in vivo activity in a murine model of S. epidermidis biomaterial-associated infection.

Fibronectin binding by Propionibacterium acnes.

Strains of Propionibacterium acnes, isolated from different kinds of orthopaedic and biomaterial-associated infections and from skin flora were shown to express binding of soluble as well as immobilized fibronectin. Among these 7 strains isolated from orthopaedic infections, 2 from breast prostheses, and 9 skin isolates, 2, 2, and 5 strains respectively bound immobilized fibronectin. The fibronectin binding was sensitive to protease and heat treatment, and was inhibited by a cell surface extract from one of the binding strains. In SDS-PAGE and autoradiography of cell surface extracts, a band corresponding to a MW of about 80 kD reacted with fibronectin and the 150 kD fragment of fibronectin. Binding to fibronectin and the 150 kD fragment of fibronectin could be inhibited with heparin. We thus present a first Fn binding protein of P. acnes, a surface exposed protein of 80 kD. None of the strains bound soluble collagen, and only one strain expressed weak binding of vitronectin and bone sialoprotein II.

Inhibition of implant-associated infections via nitric oxide release

The in vivo antibacterial activity of nitric oxide (NO)-releasing xerogel coatings was evaluated against an aggressive subcutaneous Staphylococcus aureus infection in a rat model. The NO-releasing implants were created by coating a medical-grade silicone elastomer with a sol–gel-derived (xerogel) film capable of storing NO. Four of the bare or xerogel-coated silicone materials were subcutaneously implanted into male rats. Ten rats were administered 10 μl of a 10 8 cfu ml −1 S. aureus colony directly into the subcutaneous pocket with the implant prior to wound closure. Infection was quantitatively and qualitatively evaluated after 8 d of implantation with microbiological and histological methods, respectively. A 82% reduction in the number of infected implants was achieved with the NO-releasing coating. Histology revealed that the capsule formation around infected bare silicone rubber controls was immunoactive and that a biofilm may have formed. Capsule formation in response to NO-releasing implants had greater vascularity in comparison with uninoculated or untreated controls. These results suggest that NO-releasing coatings may dramatically reduce the incidence of biomaterial-associated infection.

Search for the insertion element IS256 within the ica locus of Staphylococcus epidermidis clinical isolates collected from biomaterial-associated infections

Staphylococcus epidermidis biofilm-forming strains produce a polysaccharide intercellular adhesin (PIA), which mediates bacterial cell aggregation and favours the colonisation on prosthetic implants. PIA synthesis is regulated by the icaADBC locus. In vitro, by repeated subcultures of a biofilm-producing strain, the loss of the ability to produce biofilm appears associated with the insertion of the IS256 element into the ica locus. This study was aimed (i) to investigate if the five genes of ica locus are always all present in different strains of S. epidermidis, and (ii) to search if IS256 insertion naturally occurs in ica locus without making recourse to the experimental procedure of repeated subcultures of strains. 120 S. epidermidis clinical isolates from peri-prosthesis infections were investigated both by an original multiplex PCR analysis of the ica genes and by PCR amplification of the IS256 element. Also two reference strains (the biofilm-negative S. epidermidis ATCC 12228 and the biofilm-forming ATCC 35984 [RP62A]) and two biofilm-negative RP62A-derived acriflavin mutants (D9 and HAM892) were analysed. D9 e HAM892 were for the first time shown to contain in ica locus, at the base 3319, a 1300-bp insertion with a DNA sequence corresponding to IS256. Among the 120 clinical isolates, 51 (43%) turned out completely ica-positive, 69 completely ica-negative (57%). The genes of the ica locus appear, in all cases of the present collection, strictly linked each other, so they are either all present or all absent. In this collection, IS256 was present in eight out of the 69 ica-negative strains and in 34 out of the 51 ica-positive strains. IS256, also when present in bacterial genomic DNA, was never found inside the ica locus, thus suggesting that insertion/excision of this element is not a natural occurring mechanism for off/on switching of biofilm production.

Electric-current-induced detachment of Staphylococcus epidermidis strains from surgical stainless steel.

Infection of percutaneous biomaterials implants, such as fixation frames used for the repair of complicated fractures in orthopedics, is a major complication that almost inevitably leads to replacement of the implant. As antibiotic therapy usually has little impact on biomaterial-associated infections, it is the aim of this article to examine whether implant-associated Staphylococcus epidermidis and Staphylococcus aureus strains could be stimulated to detach from a surgical stainless steel anode during application of an electric current. First, bacteria were allowed to adhere from a flowing suspension of physiological ionic strength in a parallel plate flow chamber to a stainless-steel surface, after which the suspension was replaced by a bacterium-free solution with a specified ionic strength (0.5-150-mM potassium phosphate). DC currents ranging from 15 to 125 microA were applied to induce bacterial detachment. Initial detachment decreased with increasing ionic strength at 100 microA. The percentage detachment achieved by application of an electric current after 2.5 h was highest (95%) in 1-mM potassium phosphate and decreased to 15% when the ionic strength exceeded 40 mM. The electric current did not significantly affect the percentage detachment, but initial detachment rates increased with increasing current from 1000 cm(-2) s(-1) at 15 microA to 7000 cm(-2) s(-1) at 125 microA. Although different isolates of S. epidermidis and S. aureus showed different patterns of current-induced detachment, all strains could be stimulated to detach. The results of this study define ionic-strength conditions and electric currents yielding staphylococcal detachment from surgical stainless steel and therewith point to a pathway for the treatment and prevention of percutaneous metal-implant infection.

Detection of biomaterial-associated infections in orthopaedic joint implants.

Biomaterial-associated infection of orthopaedic joint replacements is the second most common cause of implant failure. Yet, the microbiologic detection rate of infection is relatively low, probably because routine hospital cultures are made only of swabs or small pieces of excised tissue and not of the surfaces of potentially infected implants. Joint replacements from patients in whom infection was suspected, after clinical, radiologic, and biochemical examinations, were used in this study. The aim of the current study was to compare the detection rate of infection in total joint replacements based on cultures of the excised tissue and scrapings from the biomaterial surface. Joint prostheses were retrieved from 22 patients requiring orthopaedic revision surgery because of suspected infection of their prostheses. Routine hospital culturing of tissue only showed bacterial growth in nine patients (41%). However, after prolonged culturing, bacterial growth was observed in 14 patients (64%), whereas extensive culturing of scrapings from the biomaterial surface indicated bacterial growth in 19 of the 22 patients (86%). In addition, confocal laser scanning microscopy enabled observation of biofilm bacteria on the surfaces of the explanted prostheses. Diagnosis in orthopaedic revision surgery should consider using a microbial or microscopic analysis of the surface of an explanted prosthesis, where the biofilm mode of growth firmly anchors and protects the infecting organisms. Improved detection of infection by analysis of the implant surface is expected to yield ameliorated therapy and a reduced need for revision surgery.

Antibacterial poly(D,L-lactic acid) coating of medical implants using a biodegradable drug delivery technology.

Biomaterial-associated bacterial infections present common and challenging complications with medical implants. The purpose of this study was to determine the antibacterial properties of a low molecular weight biodegradable poly(D,L-lactic acid) coating with integrated antibiotics gentamicin and teicoplanin. Coating of Kirschner-wires was carried out by a solvent casting technique under aseptic conditions with and without incorporated antibiotics. Release kinetics of gentamicin and teicoplanin were studied in phosphate-buffered saline. Initial bacterial adhesion of Staphylococcus epidermidis on coated and bare implants was determined by radiolabelling and counts of detached viable organisms. The incorporated antibiotics showed a continuous release over a period of at least 96 h with an initial peak of release in the first 6 h. Attachment of non-viable microorganisms, detected by radiolabelled bacteria, was increased significantly by the polymer coatings (P < 0.05). In contrast, the number of viable bacteria was reduced by the pure polymer (P < 0.01) and further by the polymer-antibiotic combinations (P < 0.05). Poly(D,L-lactic acid) coating of implants could offer new perspectives in preventing biomaterial-associated infections. Combinations with other drugs to formulate custom-tailored implant surfaces are feasible.

Granulocyte colony-stimulating factor for the treatment of biomaterial-associated staphylococcal infections in-vitro.

Staphylococcal infections are a common and severe complication after the implantation of a prosthesis. We developed an in-vitro model for biomaterial-associated infections and studied the effects of human recombinant granulocyte colony-stimulating factor (rhuG-CSF; filgrastime) on the eradication of bacteria from the surface of biomaterial. Latex beads (25 micro m) were incubated with 10(7) colony forming units of either a slime producing (DSM 3269) or non-slime producing strain (ATCC 14990) of Staphylococcus epidermidis. Infected particles were consecutively confronted with effector cells, derived from heparinized whole blood samples taken from healthy volunteers, after stimulation with rhuG-CSF (5,000 IU/ml, 10,000 IU/ml). Control blood specimens were not stimulated or conditioned with normal saline. The results indicate that stimulation with rhuG-CSF induced an increased rate of phagocytosis and lead to a more rapid reduction of adhering bacteria from the surface of the beads. Therefore, the in-vitro data suggest that patients with prosthesis infection may profit from an additional treatment with rhuG-CSF.

Detection of slime production by means of an optimised Congo red agar plate test based on a colourimetric scale in Staphylococcus epidermidis clinical isolates genotyped for ica locus

This investigation was conduced on a collection of 113 S. epidermidis strains isolated from biomaterial-associated infections. All strains were examined both for the presence of icaA and icaD genes responsible for slime synthesis by a PCR method and for the in vitro slime production ability by the Congo red agar (CRA) plate test. In the present study, the original CRA test was optimised adopting a six-colour reference scale for a fine classification of colonies colours. The six-colour tones of the scale were as follows: very black (vb), black (b), almost black (ab), which were considered as positive results, and bordeaux (brd), red (r), and very red (vr), interpreted as negative. 57.5% of all the strains were found to be icaA icaD-positive as well as slime-forming onto CRA, exhibiting the following colonies colours: vb (35.4%); b (15.9%); ab (6.2%). The percentage of icaA icaD-negative strains was 42.5% and all of them were negative onto CRA: brd (19.5%), r (14.2%), vr (8.8%). The comparison of colour classification with the information on ica genes confirmed the validity of the scale adopted, providing support to the criteria used for a correct interpretation of the colonies colour during the execution of the CRA test. Overall these results indicate a fine consistency between these two experimental methods and a good reliability of CRA plate test, especially when this is supported by a colourimetric scale.

In vitro adherence and accumulation of Staphylococcus epidermidis RP 62 A and Staphylococcus epidermidis M7 on four different bone cements.

Bacterial resistance of Staphylococcus epidermidis, a serious pathogen of implant-related infections, to antibiotics is related to the production of a glycocalyx slime that impairs antibiotic access and the killing by host defense mechanisms. In vitro studies of different bone cements containing antibiotics, developed for the prevention of biomaterial-associated infection, could not always demonstrate complete eradication of biomaterial-adherent bacteria. We have investigated four different bone cements in regard to bacterial accumulation of a slime-producing strain RP 62 A and its isogenic mutant M7 lacking the ability to produce exopolysaccharide slime using a bacterial adhesion assay and modified Kirby-Bauer technique. A significant effect of exopolysaccharide production for the accumulation on bone cement could be demonstrated. The gentamicin/clindamycin bone cement was the only tested biomaterial that produced a large zone of bacterial inhibition in the inoculated area adjacent to the biomaterial. The bacterial adhesion was not reduced significantly and there was no correlation between zones of inhibition on blood agar plates and the quantitative adhesion assay. The clinical efficacy of the gentamicin/clindamycin bone cement must be proven in vivo.

Biomaterial-associated infection of gentamicin-loaded PMMA beads in orthopaedic revision surgery.

In two-stage orthopaedic revision surgery, high local levels of antibiotics are achieved after removal of an infected prosthesis through temporary implantation of gentamicin-loaded beads. However, despite their antibiotic release, these beads act as a biomaterial surface to which bacteria preferentially adhere, grow and potentially develop antibiotic resistance. Gentamicin-loaded beads were retrieved from 20 patients with prosthesis-related infections. Excised tissue samples were taken for routine culture, while beads were analysed in an extensive laboratory procedure. Extensive culture procedures indicated the presence of bacteria on gentamicin-loaded beads in 18 of the 20 patients involved, while 12 of these 18 patients were considered free of infection by routine culture. Nineteen of 28 bacterial strains isolated were gentamicin resistant and cultures from three patients yielded highly gentamicin-resistant sub-populations. It is concluded that routine culture of excised tissues in orthopaedic revision surgery is inadequate to ascertain full eradication of infection, especially as infecting, antibiotic-resistant bacteria preferentially adhere to and grow on gentamicin-loaded beads. Extensive examination of the bead surfaces is proposed as a more reliable indication that infection has been eradicated.

Interleukin-1 receptor type I gene-deficient mice are less susceptible to Staphylococcus epidermidis biomaterial-associated infection than are wild-type mice.

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected with Staphylococcus epidermidis. To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R(-/-)) and wild-type mice received subcutaneous implants of silicon elastomer (SE) or polyvinylpyrrolidone-grafted SE (SEpvp), combined with an injection of 10(6) CFU of S. epidermidis or sterile saline. Neither mouse strain was susceptible to BAI around SE. IL-1R(-/-) mice with SEpvp implants had a no abscess formation and a reduced susceptibility to persistent S. epidermidis infection. The normal foreign body response, characterized by giant-cell formation and encapsulation, was delayed around SEpvp in wild-type mice but not in IL-1R(-/-) mice. This coincided with enhanced local IL-4 production in IL-1R(-/-) mice. These data suggest that inhibition of local IL-1 activity may be beneficial for the outcome of BAI.

Toward new biomaterials.

Polymers are widely used for a large range of medical devices used as biomaterials on a temporary, intermittent, and long-term basis. It is now well accepted that the initial rapid adsorption of proteins to polymeric surfaces affects the performance of these biomaterials. However, protein adsorption to a polymer surface can be modulated by an appropriate design of the interface. Extensive study has shown that these interactions can be minimized by coating with a highly hydrated layer (hydrogel), by grafting on the surface different biomolecules, or by creating domains with chemical functions (charges, hydrophilic groups). Our laboratory has investigated the latter approach over the past 2 decades, in particular the synthesis and the biological activities of polymers to improve the biocompatibility of blood-contacting devices. These soluble and insoluble polymers were obtained by chemical substitution of macromolecular chains with suitable groups able to develop specific interactions with biological components. Applied to compatibility with the blood and the immune systems, this concept has been extended to interactions of polymeric biomaterials with eukaryotic and prokaryotic cells. The design of new biomaterials with low bacterial attachment is thus under intensive study. After a brief overview of current trends in the surface modifications of biocompatible materials, we will describe how biospecific polymers can be obtained and review our recent results on the inhibition of bacterial adhesion using one type of functionalized polymer obtained by random substitution. This strategy, applied to existing or new materials, seems promising for the limitation of biomaterial-associated infections.

Biomaterial-associated persistence of Staphylococcus epidermidis in pericatheter macrophages.

Biomaterial surfaces may be modified to reduce bacterial adhesion. The susceptibility in mice to Staphylococcus epidermidis infection in tissue surrounding the commonly used catheter materials-silicon elastomer (SE), polyamide (PA), and their surface-modified polyvinylpyrrolidone (PVP)-grafted derivatives, SE-PVP and PA-PVP, respectively-was assessed. Abscesses developed around SE-PVP. Around SE, PA, and PA-PVP catheters, no signs of infection were observed, although mice carrying PA-PVP developed septicemia after 14-21 days. S. epidermidis was cultured from the tissue surrounding PA-PVP segments. Cells around PA-PVP segments containing large numbers of bacteria were identified as macrophages by use of immunohistochemistry and electron microscopy. This persistence of intracellular bacteria was also observed around SE-PVP, SE, and PA catheters, although to a lesser extent. The cytokine profiles around the 4 materials were different. Implanted biomaterial induces an inflammatory response favorable to the persistence of S. epidermidis. Intracellular persistence of bacteria inside macrophages may be a pivotal process in the pathogenesis of biomaterial-associated infection.