Aims. Biofilm formation is intrinsic to prosthetic joint infection (PJI). In the current study, we evaluated the effects of silver-containing hydroxyapatite (Ag-HA) coating and vancomycin (VCM) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation. Methods. Pure titanium discs (Ti discs), Ti discs coated with HA (HA discs), and 3% Ag-HA discs developed using a thermal spraying were inoculated with MRSA suspensions containing a mean in vitro 4.3 (SD 0.8) x 10. 6. or 43.0 (SD 8.4) x 10. 5. colony-forming units (CFUs). Immediately after MRSA inoculation, sterile phosphate-buffered saline or VCM (20 µg/ml) was added, and the discs were incubated for 24 hours at 37°C. Viable cell counting, 3D confocal laser scanning microscopy with Airyscan, and scanning electron microscopy were then performed. HA discs and Ag HA discs were implanted subcutaneously in vivo in the dorsum of rats, and MRSA suspensions containing a mean in vivo 7.2 (SD 0.4) x 10. 6.   or 72.0 (SD 4.2) x 10. 5.   CFUs were inoculated on the discs. VCM was injected subcutaneously daily every 12 hours followed by viable cell counting. Results. Biofilms that formed on HA discs were thicker and larger than those on Ti discs, whereas those on Ag-HA discs were thinner and smaller than those on Ti discs. Viable bacterial counts in vivo revealed that Ag-HA combined with VCM was the most effective treatment. Conclusion. Ag-HA with VCM has a potential synergistic effect in reducing MRSA biofilm formation and can thus be useful for preventing and treating PJI. Cite this article:Bone Joint Res. 2020;9(5):211–218

Introduction: Staphylococcus aureus is a major cause of chronic infections and causes particular problems in relation to implanted prostheses. Biofilm formation on abiotic surfaces affords bacteria innate protection from opsonophagocytosis and antibiotic agents and complicates the eradication of infection from bone and implanted prostheses. Increased concentrations of sodium, the major extracellular cation, have previously been implicated in increased biofilm formation in Staphylococcus aureus. In this study we demonstrate that increased concentrations of potassium, the major intracellular cation, also causes a significant increase in biofilm formation. Furthermore we also show that halide stress also leads to a primary increase in penicillin resistance in Staphylococcus aureus. Methods: Staphylococcus aureus ATCC 9144 was cultured in broth supplemented with variable amounts of potassium chloride and sodium chloride. Biofilm formation was investigated in 96-well microtiter plates using a standard technique. Antibiotic resistance was investigated using graduated E-test strips. Results: There was a positive correlation between bio-film formation and increased concentrations of sodium and potassium. Biofilm formation was noted to be even greater under potassium stress than under sodium stress. Sodium stress also lead to a five-fold increase in penicillin resistance in naïve Staphylococcus aureus cells. Discussion: Cellular injury or insult can lead to cell necrosis and lysis. The intracellular concentration of potassium is 30 times higher than that of the surrounding extracellular fluid. Hence, cell necrosis leads to markedly increased local concentrations of potassium. These experiments show that an increase in potassium concentration leads to an increase in biofilm formation. This suggests that biofilm formation and hence infection of implanted pros-theses may be more likely in areas of major tissue trauma such as large resections and revisions. Furthermore, cellular stress leads to increased antibiotic resistance in naïve cells which may nullify prophylaxis and complicate bacterial eradication in vivo

Aim. To evaluate bacterial adhesion and biofilm formation to metallic cerclage wire versus polymer cerclage system (SuperCable®). Methods. Experimental in vitro study to evaluate quantitative bacterial adherence to different cerclage wire materials. Two types of cerclage wires were compared: a metallic versus a polymer based wire (SuperCable®). A two-centimeter cerclage wire piece of each material was included in 2 mL of tryptic soy broth (TSB) culture media, inoculated with 10 microliters of a 0.5 McFarland of a Staphylococcus epidermidis strain and cultivated at 37°C during 2h for adhesion and 48h for biofilm formation. After this time, the cerclages were washed using a 1% phosphate buffered saline (PBS) and sonicated in new culture medium. After sonication, dilutions of each culture were spread in TSB agar and incubated 37°C during 24h. The number of colonies were counted and the cfu/cm2 was calculated. Results. There were no differences in the number of colonies counted at 2 hours. At 48 hours, the polymer cerclage system showed a clinically and statistically reduction of 95.2% in the biofilm formation of S. epidermidis. The highest bacterial counts were observed in metallic cerclages after 48h. Conclusion. In in vitro conditions, the polymer cerclage system may offer decreased biofilm formation compared with metallic cerclage wires. However, there are many other factors in in vivo conditions that could play a role in bacterial adhesion to cerclage wires. Further research is needed in order to recommend the use of polymer cerclage systems for septic revision surgery

Introduction. Support of appositional bone ingrowth and resistance to bacterial adhesion and biofilm formation are preferred properties for biomaterials used in spinal fusion surgery. Although polyetheretherketone (PEEK) is a widely used interbody spacer material, it exhibits poor osteoconductive and bacteriostatic properties. In contrast, monolithic silicon nitride (Si. 3. N. 4. ) has shown enhanced osteogenic and antimicrobial behavior. Therefore, it was hypothesized that incorporation of Si. 3. N. 4. into a PEEK matrix might improve upon PEEK's inherently poor ability to bond with bone and also impart resistance to biofilm formation. Methods. A PEEK polymer was melted and compounded with three different silicon nitride powders at 15% (by volume, vol.%), including: (i) α-Si. 3. N. 4. ; (ii) a liquid phase sintered (LPS) ß-Si. 3. N. 4. ; and (iii) a melt-derived SiYAlON mixture. These three ceramic powders exhibited different solubilities, polymorphic structures, and/or chemical compositions. Osteoconductivity was assessed by seeding specimens with 5 × 10. 5. /ml of SaOS-2 osteosarcoma cells within an osteogenic media for 7 days. Antibacterial behavior was determined by inoculating samples with 1 × 10. 7. CFU/ml of Staphylococcus epidermidis (S. epi.) in a 1 × 10. 8. /ml brain heart infusion (BHI) agar culture for 24 h. After staining with PureBlu™ Hoechst 33342 or with DAPI and CFDA for SaOS-2 cell adhesion or bacterial presence, respectively, samples were examined with a confocal fluorescence microscope using a 488 nm Krypton/Argon laser source. Images were also acquired using a FEG-SEM in secondary and backscattered modes on gold sputter-coated specimens (∼20–30Å). Hydroxyapatite (HAp) deposition was measured using a laser microscope. Raman spectra were collected for samples in backscattering mode using a triple monochromator using a 532 nm excitation source (Nd:YVO. 4. diode-pumped solid-state laser). Results. PEEK composites with 15 vol.% α-Si. 3. N. 4. , LPS ß-Si. 3. N. 4. , or the SiYAlON mixture showed significantly greater SaOS-2 cell proliferation (>600%, p<0.003, cf., Fig. 1(a)) and HAp deposition (>100%, p<0.003, cf., Fig. 1(b)) relative to monolithic PEEK. The largest increase in cell proliferation was observed with the SiYAlON composite, while the greatest amount of HAp was found on the LPS ß-Si. 3. N. 4. composite. Following exposure to S. epidermidis, the composite containing the LPS β-Si. 3. N. 4. powder showed one order of magnitude reduction in adherent live bacteria (p<0.003, cf., Fig. 1(c)) as compared to the PEEK monolith. It is interesting to note that the composite containing α-Si. 3. N. 4. exhibited the worst bacterial resistance (i.e., ∼100% higher than monolithic PEEK), suggesting that the bacteriostatic effectiveness of Si. 3. N. 4. bioceramics is apparently dependent upon the presence of selective sintering additives, viz. yttria and alumina. Conclusions. The addition of 15 wt.% of specific Si. 3. N. 4. powders to PEEK showed enhanced SaOS-2 cell adhesion, proliferation, and HAp deposition when compared to monolithic PEEK. These same composites also showed resistance to S. epi. adhesion and biofilm formation.. Although improvements in osteoconductivity have been previously observed by compounding or coating PEEK with HAp, titanium, or tantalum, these approaches did not provide anti-microbial properties. Compounding PEEK with Si. 3. N. 4. represents a significant advancement due to its ability to provide both improved bone apposition and resistance to biofilm formation. For any figures or tables, please contact the authors directly

Maggot therapy as an ancient method is succesfully used for treatment of acute and chronic wound infections in traumatology and orthopaedics. In this study, for the first time, the influence of sterile maggot excretions of Lucilia sericata on Pseudomonas aeruginosa (PAO1) biofilm formation on three common used orthopaedic materials was investigated. Sterile maggot excretions were collected according to a standardized method and the protein concentration was measured. The influence of the excretions on PAO1-biofilm formation was tested on comb-like devices, especially designed for these experiments, made from polyethylene, titanium and stainless steel. These combs were made to fit into a flat-bottom 96-wells microtiter plate. In the wells a suspension of PAO1-bacteria, nutrient medium and maggot excretions were pipetted. In the control wells, no excretions were added. Combs were placed in the wells and incubated for 24 hours at 37°C. The formed biofilms were stained in crystal violet and eluted with ethanol. The Optical Density (OD 595 nm) was read to quantify biofilm formation. The experiments were conducted with excretions from young maggots (Instar-1 maggots) and full grown maggots (Instar-3 maggots). All experiments were done in quadruplicate. The following can be concluded: PAO1-biofilm formation is the strongest on polyethylene and the weakest on stainless steel. Sterile maggot excretions are effective at preventing initial biofilm formation (p≤0.013) as well as preventing additional accumulation after its initiation (p≤0.038). The excretions even cause a significant breakdown of an existing biofilm (p≤0.028). Excretions from full grown maggots are more effective than those from young maggots. This study shows for the first time that sterile maggot excretions of Lucilia sericata inhibit biofilm formation, prevent its further grow and break down existing biofilms. While biofilm formation on orthopaedic materials is a severe complication, this experimental study could indicate a new treatment for biofilm formation on infected biomaterials

Summary Statement. A study to evaluate biofilm development on different coatings of UHMWPE was performed. We observed a species-specific effect, with S. aureus affected mainly by DLC-F and S. epidermidis by DLC. These data correlates with previous adherence studies. Introduction. Prosthetic joint infection is intimately related to bacterial biofilms on implant biomaterials. Recently, diamond-like carbon (DLC) coating has been suggested to improve the antibacterial performance of medical grade GUR1050 ultra high molecular weight polyethylene (UHMWPE) supplied by Orthoplastics bacup, UK versus collection and clinical staphylococcal strains. The aim of this study was to make an approximation towards the actual impact of such coatings in biofilm formation. Material and Methods. Biofilm formation by two collection laboratory strains (S. aureus 15981[4] and S. epidermidis ATCC 35984) was evaluated with raw UHMWPE and two UHMWPEs coated with DLC, and fluorine doped DLC (F-DLC). The coated surfaces were obtained by plasma enhanced chemical vapour deposition, as previously described. All the sterilised surfaces were exposed to ≈10. 8. colony forming units/mL during 48 hours at 35° C, with total medium exchange at 24 hours without shaking. Surfaces were carefully washed with PBS (X 3) and then stained with Backlight. ©. live/dead stain for 15 minutes. Confocal Laser Scanning Microscopy was used for sampling the surfaces and studying biofilm, for which eight random series of photographs (Named SERIES) and four predefined biofilm series (Named BIOFILM) were taken. Biofilm thickness (microns) and covered surface by live/dead bacteria (%) were determined for both SERIES and BIOFILM. Assays were made in triplicates. Photographs were analyzed by ImageJ software, and data, by a Mann-Withney test. Results. Biofilm thickness and bacterial coverage per surface type in SERIES as well as BIOFILM for S. aureus and S.epidermidis, respectively is shown. A diminution of these two variables was observed in the coated surfaces versus raw UHWMPE with statistically significant reductions (p≤0.0001). F-DLC was the most effective coated surface versus S. aureus, with the least biofilm thickness and the highest proportional percentage of dead bacteria, and so DLC was versus S. epidermidis. Of interest, the proportion of dead S. epidermidis was higher in raw UHMWPE. Discussion & Conclusions. Staphylococcal biofilm formation on UHMWPE surfaces is irregular. Both biofilm thicknesses as bacterial coverage were lower in DLC and F-DLC. These preliminary data correlate to our previous bacterial adherence findings and support the better anti-adherence performance of DLC coated UHMWPEs. Fluorine is suggested to exhibit a bacteria-dependant behavior, but at present its role is quite far to be known. Further studies using clinical strains of both species are needed to evaluate the accuracy of these results

Bacterial infection related to prosthetic replacement is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. In order to reduce the incidence of replacement-associated infections, we developed a novel coating technology of Hydroxyapatite (HA) containing silver (Ag). We reported the Ag-HA coating showed high antibacterial activity against E. coli, S. aureus and methicillin-resistant S. aureus (MRSA) under static condition. However, human bodies have a circulating body fluid, which is not a static condition. And the growth and the maturation of biofilm, which is said that a common course of persistent infections at a surgical site, are enhanced by the flow of broth in culture environment. Therefore, we evaluated whether the Ag-HA coating inhibits the biofilm formation on its surface or not by a biofilm-forming test under flow condition in this study. Ag-HA or HA powder was sprayed onto the commercial pure titanium disks using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin sensitive S. aureus (BF-MSSA; Seattle 1945) strain and the BF-MRSA (UOEH6) strain were used. The pre-culture bacterial suspension (about 10. 5. colony forming units; CFU) was inoculated onto the Ag-HA and HA coating disks. After cultivation at 37 °C for 1 h, the disks were rinsed twice with 500 μL sterile PBS (-) to eliminate the non-adherent bacteria. The number of the adherent bacteria on these disks was counted using culture method. After rinsing, the disks were transferred into petri-dish containing Trypto–Soy Broth (TSB) + 0.25% glucose with a stirring bar on the magnetic stirrer and they were cultured at 37°C for 7 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate on the surfaces of them was calculated using the NIH image software. The number of the bacteria on these disks was not so different between Ag-HA and HA coating after rinsing. After biofilm-forming test, the coverage of the biofilm of BF-MSSA was 2.1% and 81.0% on the Ag-HA and HA coatings, respectively. Similarly, in the case of BF-MRSA, it was 7.7% and 72.0% on the Ag-HA and HA coatings, respectively. Though bacteria slightly adhered, biofilm was hardly observed on the Ag-HA coating. The biofilm on the HA coating was extensive and mature. The inhibition effect of biofilm formation on the Ag-HA coating might be ascribed to the antibacterial effect by Ag ions released from the coating. Because Ag ions have a broad spectrum of antibacterial activity against pathogens, including biofilm forming bacteria, they inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria. Even in a flow condition, it was suggested that the AgHA shows the antibacterial activity, though the conditions in this work are different from those in living body

Background and Purpose: The remarkably low wear of metal-on-metal (MOM) bearings involving cobalt-chromium (Co-Cr) alloys has led to a resurgence in its use. However, consequences of these wear particles and the corrosion products are for the most part unclear. Recent research efforts towards the bacteriological influences of the MOM-degradation products suggested that particulate MOM debris promotes planktonic bacterial growth. On the other hand, extremely high concentrations of metal ions, derived from salts, have shown to possess bacteriostatic effects (growth reduction) on planktonic growth and on biofilm formation. The effects of salt-derived metal ions were found to be inhibitory and not bactericidal (lethal to bacteria). However, these two findings were both found under static growth conditions and no studies have investigated these findings under more clinically resembling dynamic growth conditions. In addition, influences of Co-Cr particles on biofilm formation have not yet been studied. Therefore, the aim of this study was to evaluate how Co-Cr particles and Co-Cr ions affect biofilm formation under static and dynamic growth conditions. Methods: A collection of clinically isolated bacterial strains were exposed to Co-Cr particles and Co-Cr ions in concentrations as found in serum and above as found in adjacent tissue. The experiments were conducted as well under static, as under dynamic growth conditions. Biofilm formation in wells, stained with live/dead viability staining and visualized by confocal laser scanning microscopy, was analyzed with COMSTAT, yielding biovolume, biofilm thickness, and live/dead ratio of the bacteria within the biofilm. Results: Co-Cr particle concentrations of 20 g/L reduced biofilm formation significantly. Moreover, these particle concentrations were found to be bactericidal (killed the bacteria). The live/dead ratio decreased when culturing was done under dynamic growth conditions when compared to the static growth condition. Under both growth conditions, biofilm formation was inhibited at concentrations of 10/5 mg/L Co-Cr ions, as reported to occur in synovial fluids. Co-Cr ion concentrations up to 1/0,5 mg/L revealed no consistent influence on biofilm formation. Interpretation: Long-term clinical data on infection rates for Co-Cr MOM-bearings are not yet available, but the current results suggest that Co-Cr ions may yield these prostheses less prone to biofilm formation and subsequent infection

Introduction: One of the most important mechanisms S. epidermidis uses to establish infection on biomaterials is biofilm formation, in which adhesion and the production of polysaccharide intercellular adhesin (PIA) are key factors. Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to inhibit S. epidermidis biofilm formation and may be useful in prevention or treatment of implant infections (. 1. ,. 2. ). The potential of these drugs was evaluated by determining the effects of the NSAIDs on bacterial growth, adhesion to bare and conditioned polymethyl-methacrylate (PMMA), on biofilm development, and on established biofilms. Methods: A PIA-deficient mutant and wild type strain (gift of Prof. D. Mack, Hamburg) and 3 clinical isolates of S. epidermidis were used. The NSAIDs were salicylic acid, acetylsalicylic acid, ibuprofen and phenylbutazone. Their effects on bacterial growth rate and viability were assessed. For adhesion assay, bacteria were exposed to a 1mM concentration of each drug and allowed to adhere for 1h to bare or human plasma – conditioned PMMA before being sonicated and quantified by chemiluminescence and culture. For biofilm assays, bacteria were grown on silicone discs in the presence of various drug concentrations for 4 days before being sonicated and quantified as above. Mature (4 day) biofilms were also exposed to the drugs for a further 4 days and quantified similarly, to assess the effect on established biofilms. Results: All NSAIDs tested significantly (P< 0.05) reduced the growth rate and viability of each strain, in a concentration – dependent manner. Reduction of adhesion was observed on bare PMMA suggesting interference with either vitronectin – binding protein or charge / hydrophobic interactions. This was independent of the effect on growth. However, adhesion to plasma – conditioned PMMA, presumably mediated by MSCRAMMs, was not significantly affected. Reduction of biofilm formation was observed for all strains and was concentration – dependent, suggesting that inhibition of PIA synthesis was not responsible. There was a significant effect on established biofilms, this was also concentration dependent. Conclusions:. All four NSAIDs reduced S. epidermidis growth rate and viability, but at concentrations above those achievable therapeutically. The effect on adherence was confined to unconditioned PMMA. The effect on biofilm formation and on established biofilms appeared to be related to that on growth and viability. On these grounds, NSAIDs appear to have a limited prospect for use in prevention or treatment of S. epidermidis biomaterial-related infection. However, catheter coating, NSAID-antibiotic combinations, and potential for other types of infection may have greater prospects

Problems. Biofilm infections are increasingly associated with orthopedic implants. Bacteria form biofilms on the surfaces of orthopedic devices. The biofilm is considered to be a common cause of persistent infections at a surgical site. The growth and the maturation of biofilm are enhanced by the flow of broth in culture environment. In order to reduce the incidence of implant-associated infections, we developed a novel coating technology of hydroxyapatite (HA) containing silver (Ag). We previously reported that the Ag-HA coating inhibits biofilm formation under flow condition of Trypto Soy Broth + 0.25% glucose for 7 days. In this study, we evaluated whether the Ag-HA coating continuously inhibits the biofilm formation on its surface under flow condition of fetal bovine serum, which contains many in vivo substrates such as proteins. Materials and Method. The commercial pure titanium disks were used as substrates. Ag-HA or HA powder was sprayed onto the substrates using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin resistant Staphylococcus aureus (BF-MRSA; UOEH6) strain was used. The bacterial suspension (about 10. 5. colony forming units) was inoculated into 24-well sterile polystyrene tissue culture plates. The Ag-HA and HA coating disks were aseptically placed in the wells. After cultivation at 37°C for 1 hour, the disks were rinsed twice with 500 μL sterile PBS (−) to eliminate the non-adherent bacteria. After rinsing, the disks were transferred into petri-dish containing heat-inactivated FBS with a stirring bar on the magnetic stirrer and they were cultured at 37 °C for 24 hours, 7 and 14 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate (BCR) on the surfaces of them was calculated using the NIH image software. Results. Biofilm was hardly observed on the Ag-HA coating. However, the biofilm on the HA coating was extensive and mature (Fig. 1). At 24h after cultivation, BCRs of BF-MRSA were 2.1% and 19.8% on the Ag-HA and HA coatings, respectively. Similarly, they were 6.3% and 12.4% on the Ag-HA and HA coatings at 7 days. At 14 days they were 20.6% and 39.4% on the Ag-HA and HA coatings, respectively. These results demonstrate that BCRs on the Ag-HA coating were significantly lower than those on the HA coating (Fig. 2). Discussion. The Ag-HA coating continuously showed the inhibiting ability for biofilm formation under flow condition for 14 days. Ag ions inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria in the vicinity of the surface, although the release rate of Ag ions was high until 24h after immersion and decreased thereafter. The Ag-HA coating would be expected to contribute to reduction of implant-related biofilm infection

Introduction: Staphylococcus aureus is a major cause of chronic infections and causes particular problems in relation to implanted prostheses. Biofilm formation on abiotic surfaces affords bacteria innate protection from opsonophagocytosis and antibiotic agents and complicates the eradication of infection from bone and implanted prostheses. Increased concentrations of sodium, the major extracellular cation, have previously been implicated in increased biofilm formation in Staphylococcus aureus. In this study we demonstrate that increased concentrations of potassium, the major intracellular cation, also causes a significant increase in biofilm formation. Furthermore we also show that halide stress also leads to a primary increase in penicillin resistance in Staphylococcus aureus. Finally we demonstrate that pbp4, a key gene in cell wall synthesis, is down-regulated under sodium and potassium stress. Methods: Staphylococcus aureus ATCC 9144 was cultured in broth supplemented with variable amounts of potassium chloride and sodium chloride. Biofilm formation was investigated in 96-well microtiter plates using a standard technique. Antibiotic resistance was investigated using graduated E-test strips. Gene transcription was assessed using RT-PCR. Results: There was a positive correlation between biofilm formation and increased concentrations of sodium and potassium. Biofilm formation was noted to be even greater under potassium stress than under sodium stress. Sodium stress also lead to a five-fold increase in penicillin resistance in naïve Staphylococcus aureus cells. A key gene involved in cell wall production (pbp4) was down-regulated under sodium (p = 0.03) and potassium (p = 0.03) stress. Discussion: Cellular injury or insult can lead to cell necrosis and lysis. The intracellular concentration of potassium is 30 times higher than that of the surrounding extracellular fluid. Hence, cell necrosis leads to markedly increased local concentrations of potassium. These experiments show that an increase in potassium concentration leads to an increase in biofilm formation. This suggests that biofilm formation and hence infection of implanted prostheses may be more likely in areas of major tissue trauma such as large resections and revisions. Furthermore, cellular stress leads to increased resistance to penicillin, a cell wall active antibiotic, in naïve cells which may nullify prophylaxis and complicate bacterial eradication in vivo. Finally we postulate a link between the experimental rise in penicillin resistance and the down-regulation of pbp4 demonstrated by RT-PCR under the same halide stresses

Introduction. The use of antibiotic-loaded polymethylmethacrylate bone-cement spacers during two-stage exchange procedures is the standard in the treatment of patients with delayed prosthetic joint infection. The real antimicrobial activity of these spacers is unclear because the adherence of bacteria to cement might result in clinical recurrence of infection. The purpose of the study is to evaluate the in vitro formation of Pseudomonas Aeruginosa (PA) and Staphylococcus spp. biofilm on antibiotic-loaded bone cement. Materials and methods. Cement disks (diameter = 6 mm) impregnated with gentamicin and colistin were submerged in bacterial suspensions of Methicillin-resistant Staphylococcus Aureus(MRSA), Staphylococcus epidermidis (SE), and PA. Negative controls (specimen disks without antibiotic) were similarly prepared. Biofilm formation was visualized by confocal scanning laser microscopy (CSLM), after staining the discs with the live/dead BacLight viability stain containing SYTO 9 dye and propidium iodide. Images from five randomly selected areas were acquired for each disc. Sequential optical sections of 2 µm were collected in sequence along the z-axis over the complete thickness of the sample. The resulting stacks of images were analyzed, quantified and rendered into three-dimensional (3D). The biofilm thickness on antibiotic bone cement compared with the controls was automatically evaluated. Results. CSLM showed living bacteria and bacterial biofilm on the surface of all cement disks, either antibiotic-loaded or controls. Mean biofilm thickness on the controls was 29.6 µm for MRSA, 32.3 µm for SE, and 59.7 µm for PA. The 3D rendering showed decrease in the biofilm thickness for all bacterial strains on gentamicin- and colistin-impragnated cement disks as compared with the controls. The incorporation of gentamicin into cement resulted in a 54%, 74%, and 45% reduction in the bacterial biofilm thickness for MRSA, PA and SE, respectively. The use of colistin leaded to a 51 % reduction in the PA biofilm thickness. Conclusion. The bacterial viability and biofilm formation are reduced by adding antibiotics to bone cement but antibiotic-loaded bone cement does not completely inhibit the formation of an infectious biofilm in vitro

Aim. To investigate the local intra-operative concentration of gentamicin needed to prevent biofilm formation in a porcine model of implant-associated osteomyelitis. Method. In total 24 pigs were allocated to six groups. Group A (n=6) was inoculated with saline. Groups B (n=6), C (n=3), D (n=3), E (n=3) and F (n=4) were inoculated with 10 μL saline containing 10. 4. CFU of Staphylococcus aureus, however, different minimal inhibitory concentrations (MIC) of gentamicin were added to the inoculum of Groups C(160xMIC), D(1600xMIC), E(16000xMIC) and F(160000xMIC). The inoculums were injected into a pre-drilled implant cavity proximally in the right tibial bone. Following inoculation, a steel implant (2 × 15 mm) was placed in the cavity. The pigs were euthanized after five days. The implants were sonicated and swabs were taken from the implant cavity for microbiological evaluation. The peri-implant tissue was analyzed by histopathology including estimation of neutrophil infiltration. Results. The microbiological samples from Group A pigs were sterile. All implants and implant cavities of pigs inoculated with bacteria and bacteria + 160 or 1.600xMIC were positive for S. aureus. In each of the Groups E (16000xMIC) and F (160000xMIC) only one animal was found positive and 1/3 and 3/4 of the implants were sterile after sonication, respectively. All positive swabs were confirmed to be same spa-type as used for inoculation. By adding Groups C + D (<10000xMIC) and Groups E + F (>10000xMIC) a strong significant decrease (one-way ANOVA, P value = 0.001) of implant attached bacteria was only seen between the high MIC values and Group B (bacteria only). The histological examination demonstrated that 1600, 16000 and 160000 × MIC resulted in a peri-implant tissue reaction, including neutrophil estimation, comparable to saline inoculated animals. Patho-morphologically, it was not possible to distinguish between pigs inoculated with bacteria and bacteria + 160xMIC as both groups had a strong inflammatory response and an equal estimation of neutrophils. Discussion. The antibiotic susceptibility for prevention of an in vivo biofilm infection is influenced by body fluids, host immune response, extracellular host proteins like fibrin, tissue necrosis and development of an anaerobic environment. With the present in-vivo setup, we have demonstrated that local intra-operative gentamicin might be given in concentrations of more than 10000 times the MIC value in order to prevent biofilm formation by planktonic bacteria. Our study supports that biofilm susceptibility testing performed in-vitro is yet still unreliable for prediction of prophylactic and therapeutic success

Introduction:. Periprosthetic infections that accompany the use of total joint replacement devices cause unwanted and catastrophic outcomes for patients and clinicians. These infections become particularly problematic in the event that bacterial biofilms form on an implant surface. Previous reports have suggested that the addition of Vitamin E to ultra-high-molecular-weight polyethylene (UHMWPE) may prevent the adhesion of bacteria to its surface and thus reduce the risk of biofilm formation and subsequent infection. 1–3. In this study, Vitamin E was blended with two types of UHMWPE material. It was hypothesized that the Vitamin E blended UHMWPE would resist the adhesion and formation of clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) biofilms. Methods and Materials:. Five sample types were manufactured, machined and sterilized (Table 1). To determine if MRSA biofilms would be reduced or prevented on the surface of the Vitamin E (VE) loaded samples (HXL VE 150 kGy and HXL VE 75 kGy) in comparison to the other three clinically relevant material types, each was tested for biofilm formation using a flow cell system. 4. Direct Bacterial Quantification – An n = 7 samples of each material type were placed individually into a chamber of the flow cell. A solution of 10% modified brain heart infusion (BHI) broth containing 10. 5. MRSA cells/mL was flowed through each chamber. Using previously established protocols,. 4–7. after 48 hours of growth, each sample was removed, and the number of colony forming units (CFU) determined using a 10-fold dilution series. SEM Imaging – Using the same protocol as above, after the 48-hour incubation period, an n = 7 of each material type were fixed in 2.5% glutaraldehyde, dehydrated in ascending concentrations of ethanol, coated with carbon and imaged using scanning electron microscopy (SEM). Results:. Results indicated that the Vitamin E blended materials did not resist the attachment/formation of MRSA biofilms to any greater degree than the other three material types. All materials had greater than 10. 7. CFU/cm. 2. (Figure 1). SEM images corroborated with the quantification data (Figure 2). Discussion:. In contrast to previously published results,. 1–3. these data indicated that Vitamin E blended UHMWPE may not have the ability to prevent biofilm formation of a clinical MRSA isolate from occurring

Copal bone cement loaded with gentamicin and clindamicin was developed recently as a response to the emerging occurrence of gentamicin-resistant strains in periprothetic infections. The objective of this study was to compare the in vitro antibiotic release and antimicrobial efficacy of gentamicin/clindamicin-loaded Copal bone cement and gentamicin-loaded Palacos R-G bone cement, as well as biofilm formation on these cements. In order to determine antibiotic release, cement blocks were placed in phosphate buffer and aliquots were taken at designated times for measurement of antibiotic release. In addition, the bone cement discs were pressed on agar to study the effects of antibiotic release on bacterial growth. Biofilm formation on the different bone cements was also investigated after 1 and 7 days using plate counting and confocal laser scanning microscopy (CLSM). Experiments were done with a gentamicin-sensitive S. aureus and a gentamicin-resistant CNS. Antibiotic release after 672 h from Copal bone cement was more extensive (65% of the clindamycin and 41% of the gentamicin incorporated) than from Palacos R-G (4% of the gentamicin incorporated). The higher antibiotic release from Copal resulted in a stronger and more prolonged inhibition of bacterial growth on agar. Plate counting and CLSM of biofilms grown on the bone cements showed that antibiotic release reduced bacterial viability, most notably close to the cement surface. Moreover, the gentamicin-sensitive S. aureus formed gentamicin-resistant small colony variants on Palacos R-G, and therefore, Copal was much more effective in decreasing biofilm formation than Palacos R-G. Biofilm formation on bone cement could be more effectively reduced by incorporation of a second antibiotic, next to gentamicin. Antibiotic release from the cements had a stronger effect on bacteria close to the cement than on bacteria at the outer surface of the bio-film. Clinically, bone cement with two antibiotics may be more effective than cement loaded with only gentamicin. The clinical efficacy of antibiotic loaded bone cements in combination with systemic antibiotics can be explained because antibiotics released from cements kill predominantly the bacteria in the bottom of the biofilm, whereas systemic antibiotics can only deal with bacteria at the outer surface of the biofilm

Enterococcus faecalis is a rare but recognized cause of prosthetic joint infection. It is notorious for formation of biofilm on prosthetic surfaces. We hypothesized that a ‘serum factor’ was responsible for transformation of E. faecalis from its planktonic form to a biofilm existence upon making contact with prostheses. Using a novel ‘proteomic approach’, we studied the protein expression profiles of this bacterium when grown on an artificial surface in a serum environment against a control. E.faecalis 628 transconjugant formed by conjugation clinical strain (E55) and laboratory strain (JH2-2) was used to inoculate each of rabbit serum (RS) and Brain Heart Infusion (BHI) agar as a control and grown for 24 hours. Proteins were harvested for analysis in fractions including cell surface, membrane and cytosolic proteins. Recovered proteins were separated using 2-dimentional polyacrylamide gel electrophoresis (2D PAGE). Gels were stained and spots of interest harvested. These were analyzed using MALDI mass spectrometry followed by peptide mass fingerprinting using online database searches. Two surface exclusion proteins Sea1 and PrgA were only expressed from the serum culture. These proteins are both encoded by genes very close to the gene for enterococcal aggregation substance PrgB, which plays an integral role in biofilm formation. PrgA and PrgB are both encoded by the prgQ operon and hence expressed simultaneously upon activation of the operon. This tendency for serum only protein expression suggests the possibility of a pheromone-like activator in serum that could be a potential therapeutic target for management of biofilm associated E. faecalis prosthetic infections

Background: Bacteria form a biofilm on the surface of orthopaedic devices, causing persistent and infection. Little is known about biofilms formation on bone grafts and bone substitutes. We analyzed various representative materials regarding their propensity for biofilm formation caused by Staphylococcus aureus. Methods: As bone graft beta-tricalciumphosphate (b-TCP, CyclOsTM) and as bone substitute a tantalum metal mesh (trabecular metalTM) and PMMA (Pala-cosTM) were investigated. As test organism S. aureus (strain ATCC 29213) was used. Test materials were incubated with bacterial solution of 105 colony-forming units (cfu)/ml at 37°C for 24 h without shaking. After 24 h, the test materials were removed and washed 3 times in normal saline, followed by sonication in 50 ml Ringer solution at 40 kHz for 5 minutes. The resulting sonication fluid was plated in aliquots of 0.1 ml onto aerobe blood agar with 5% sheep blood and incubated at 37°C with 5% CO2 for 24 h. Then, bacterial counts were enumerated and expressed as cfu/ml. All experiments were performed in triplicate to calculate the mean ± standard deviation. The Wilcoxon test was used for statistical calculations. Results: The three investigated materials show a differing specific surface with b-TCB> trabecular metal> PMMA per mm2. S. aureus formed biofilm on all test materials as confirmed by quantitative culture after washing and sonication. The bacterial counts in sonication fluid (in cfu/ml) were higher in b-TCP (5.1 x 106 ± 0.6 x 106) and trabecular metal (3.7 x 106 ± 0.6 x 106) than in PMMA (3.9 x 104 ± 1.8 x 104), p< 0.05. Conclusion: Our results demonstrate that about 100-times more bacteria adhere on b-TCP and trabecular metal than on PMMA, reflecting the larger surface of b-TCP and trabecuar metal compared to the one of PMMA. This in-vitro data indicates that bone grafts are susceptible to infection. Further studies are needed to evaluate efficient approaches to prevent and treat infections associated with bone grafts and substitutes, including modification of the surface or antibacterial coating

Background: Bacteria form biofilms on the surface of orthopaedic devices, causing persistent infections. Monitoring biofilm formation on bone grafts and bone substitutes is challenging due to heterogeneous surface characteristics. We analyzed various bone grafts and bone substitutes regarding their propensity for in-vitro biofilm formation caused by S. aureus and S. epidermidis. Methods: Beta-tricalciumphosphate (β-TCP, Chro-nOsTM), processed human spongiosa (TutoplastTM) and PMMA (EndobonTM) were investigated. PE was added as a growth control. As test strains S. aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) were used. Test materials were incubated with defined bacterial solution (105 colony-forming units (cfu)/ml) at 37°C for 24 h without shaking. After 24 h, the test materials were removed and washed 3 times in PBS, followed by a standardised sonication protocol (Trampuz et al. 2007, NEJM). The resulting sonication fluid was plated in aliquots of 100μl onto aerobe blood agar with 5% sheep blood and incubated at 37°C with 5% CO2 for 24 h. Bacterial counts were enumerated and expressed as cfu/ml. Sonicated samples were transferred to a microcalorimeter (TA Instrument) and heat flow at 37°C was continuously monitored over a 24h period with a precision of 0.0001°C and a sensitiviy of 200μW. All experiments were performed in triplicates to calculate the mean ± standard deviation. ANOVA analysis was used for statistical calculations. Results: For S. aureus bacterial counts (log10 cfu/ sample) were significantly higher (p< 0.001) for the porous (β-TCP 7.67 ± 0.17, Tutoplast 7.65 ± 0.15) than for the solid samples (PMMA 6.12 ± 0.18, PE 5.17 ± 0.22). Bacterial density (log10 cfu/surface) was 10^1–10^2 times higher for the S. epidermidis than for the S. aureus. In calorimetry the shape of the heat flow curves was characteristic for the individual strain and was not influenced by the test materials. The time to detection (TTD) was shortest for β-TCP for both strains and TTD was always shorter for S. aureus than S. epidermidis with corresponding material. Cfu/sample calculated from the calorimetric data was concordant with the standard culturing method. Conclusion: Our results demonstrate biofilm formation with both strains on all tested materials. The calorimetry in all cases was able to detect quantitatively the amount of biofilm. Further studies are needed to see whether calorimetry is a suitable tool also to monitor approaches to prevent and treat infections associated with bone grafts and bone substitutes

Introduction. According to the Australian registry 2014, periprosthetic joint infection (PJI) is the fourth important reason for revision of a primary total hip arthroplasty (THA). PJI is frequently caused by commensal strains of the skin such as Staphylococcus aureus or Staphylococcus epidermis. Deep infection is depending on many factors, such as implant surface chemical and physical behaviour, device design, host site, surgery and host response. Nevertheless, a lack of knowledge is seen concerning the specific effects of different surfaces on the biological response of different biomaterials. In addition, it is difficult to discriminate the material chemico-physical properties by the topological features, such as surface roughness. Indeed, it has been widely demonstrated that surface composition, electric charge, wettability and roughness of implant surfaces have a strong influence on their interactions with biological fluids and tissues. Therefore, also bearing surface properties can influence the incidence of PJI, just shown recently. Objectives. To verify the capability of ceramic bearings to reduce bacteria biofilm adhesion by means of their surface chemico-physical properties. Methods. The surface chemico-physical properties of the most common materials in THA as monolithic alumina, zirconia platelet toughened alumina (ZPTA), zirconia (TZP), titanium alloy (Ti6Al4V), stainless-steel and cobalt alloy (Co28Cr6Mo) were compared. All materials were characterized using x-ray photoelectron spectroscopy (XPS), fourier transform spectroscopy (FTIR), x-ray diffraction (XRD) and zeta-potential. Additionaly wettability by contact angle measurement with various media as simulated body fluid (SBF), bacterial broth, cell culture media and fetal bovine serum (FBS) was determined. Furthermore, the surface protein adsorption amount was evaluated by bicinchoninic acid (BCA) assay analysis using FBS as protein source. Selective protein adsorption was also evaluated by electroforetic technique. The specimens' surface anti-bacterial adhesion activity was evaluated by Staphylococcus aureus biofilm formation after 24h by colonies forming units count. Cytocompatibility was assessed using human primary osteoblasts cell culture and MTT assay. Results. The surface of all tested materials was found to be electronegative at physiological pH by means of zeta-potential measurement. Nevertheless, monolithic alumina and ZPTA have the isolectric point at lower pHs and adsorbed a larger amount of proteins (albumin and fibronectin) in comparison with metal surfaces. Such feature might be correlated with bacteria biofilm growth, since the ceramic surfaces were also less colonized by Staphylococcus aureus in comparison to metal surfaces (p<0.005) while they maintained the ability to promote osteoblasts adhesion and proliferation. The above results were confirmed by XPS technique where the ceramic surfaces had less hydroxyl groups and consequently were less prone to adhere with biological species as the bacteria. No correlation was observed using the FTIR and XRD surface characterization techniques. Conclusions. The ceramic bearing surfaces were found to reduce the bacteria biofilm adhesion, because of their surface chemico-physical properties

Background. The main reasons for hip prosthesis failure are aseptic loosening and periprosthetic joint infection (PJI). The real frequency of PJI is probably largely underestimated because of non-standardized definition criteria, diagnostic procedure, treatment algorithm and other confounders. Therefore, data from joint registries are not reflecting the frequency of PJI and can be misleading; particularly low-grade PJI can be frequently misdiagnosed as aseptic failure. Therefore, prospective clinical studies with standardized protocol, comprehensive diagnostic procedure and sufficient follow-up should be performed. Sonication of explanted prosthesis is highly sensitive for detection of biofilms on prosthetic surface and allows quantitative analysis of biofilm formation. We hypothesize that by using sonication, ceramic components (BIOLOX®delta, BIOLOX®forte) will show higher resistance against biofilm adhesion compared to polyethylene (PE) and metal (CoCrMo). Methods. In this prospective multicentre study (level of evidence: Ia), we included all consecutive adults ≥18 years of age, who underwent explantation of the hip prosthesis for infection or aseptic reason. Excluded were patients in whom part of the prosthetic components were retained. A standardized and comprehensive diagnostic algorithm was applied, including sonication of all removed prosthetic components for qualitative and quantitative microbiological analysis (ultrasound bath 40 kHz, 1 W/cm2, 1 min). Individual components (metal, PE, ceramic) were separately placed in sterile boxes for investigation. All patients were simultaneously included in the European Prosthetic joint infection cohort (EPJIC, . www.epjic.org. ) to ensure long-term follow-up. Results. Up to date, 79 patients were included, of whom 47 (60%) were diagnosed with aseptic failure and 32 (40%) with PJI. Mean age was 73 years (27–87 years), 32 (41%) were males. Table 1 summarizes the demographic characteristics. In 32 patients with PJI, most frequently isolated organisms were coagulase-negative staphylococci (n=12, 38%), Staphylococcus aureus (n=7, 22%) and Propionibacterium acnes (n=4, 13%), followed by enterococci (n=2; 6%) and gram-negative bacilli (n=2; 6%); 2 infections (6%) were polymicrobial and 3 were culture-negative (9%). Table 2 summarizes the microbiological results from sonication of removed components. Causative microorganism could be detected in sonication fluid from polyethylene in 100%, from metal in 92% and from ceramic in 69%. Significantly lower bacterial counts expressed as colony-forming units (CFU) were detected in sonication fluid from ceramic components (230 CFU/ml) than from PE (6’250 CFU/ml) and metal components (5’870 CFU / ml) (p < 0.01). Conclusions. These first results support the hypothesis that significantly less biofilm biomass is formed on ceramic surface, compared to PE and metal surfaces, potentially indicating higher ceramic “resistance” against bacterial adhesion. These findings should be confirmed with non-microbiological investigation such as imaging (fluorescent in situ hybridization, confocal laser scanning or electron microscopy). Furthermore, in 6 of 32 patients (19%) with PJI, an aseptic loosening was preoperatively suspected. Infection was found only by systematic application of an optimized diagnostic method, particularly sonication of the removed implant. Final study results are expected to be available by the end of 2016