Aims. Biofilm infections are among the most challenging complications in orthopaedics, as bacteria within the biofilms are protected from the host immune system and many antibiotics. Halicin exhibits broad-spectrum activity against many planktonic bacteria, and previous studies have demonstrated that halicin is also effective against Staphylococcus aureus biofilms grown on polystyrene or polypropylene substrates. However, the effectiveness of many antibiotics can be substantially altered depending on which orthopaedically relevant substrates the biofilms grow. This study, therefore, evaluated the activity of halicin against less mature and more mature S. aureus biofilms grown on titanium alloy, cobalt-chrome, ultra-high molecular weight polyethylene (UHMWPE), devitalized muscle, or devitalized bone. Methods. S. aureus-Xen36 biofilms were grown on the various substrates for 24 hours or seven days. Biofilms were incubated with various concentrations of halicin or vancomycin and then allowed to recover without antibiotics. Minimal biofilm eradication concentrations (MBECs) were defined by CFU counting and resazurin reduction assays, and were compared with the planktonic minimal inhibitory concentrations (MICs). Results. Halicin continued to exert significantly (p < 0.01) more antibacterial activity against biofilms grown on all tested orthopaedically relevant substrates than vancomycin, an antibiotic known to be affected by biofilm maturity. For example, halicin MBECs against both less mature and more mature biofilms were ten-fold to 40-fold higher than its MIC. In contrast, vancomycin MBECs against the less mature biofilms were 50-fold to 200-fold higher than its MIC, and 100-fold to 400-fold higher against the more mature biofilms. Conclusion. Halicin is a promising antibiotic that should be tested in animal models of orthopaedic infection. Cite this article: Bone Joint Res 2024;13(3):101–109

Aim. “Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material. Biofilms are bacterial communities adhered to a surface with a self-made extracellular polymeric substance that surrounds resident bacteria. In contrast to planktonic bacteria, bacteria in a biofilm are in an adherent, dormant state and are insensitive to most antibiotics. In addition, bacteria in a biofilm are protected from phagocytic cells of the immune system. Therefore, complete surgical removal and replacement of the prosthetic implant is often necessary to treat this type of infections. Neutrophils play a crucial role in clearing bacterial pathogens. They recognize planktonic bacteria via immunoglobulin (Ig) and complement opsonisation. In this project, we aim to evaluate the role of IgG and complement in the recognition and clearance of staphylococcal biofilms by human neutrophils. Furthermore, we evaluate if monoclonal antibodies (mAbs) targeting biofilm structures can enhance recognition and clearance of staphylococcal biofilms by the human immune system.”. Method. “We produced a set of 20 recombinant mAbs specific for staphylococcal antigens. Using flow cytometry and ELISA-based methods we determined the binding of these mAbs to planktonic staphylococci and in vitro staphylococcal biofilms. Following incubation with IgG/IgM depleted human serum we determined whether mAbs can react with the human complement system after binding to biofilm. Confocal microscopy was used to visualize the location of antibody binding in the biofilm 3D structure.”. Results. “We show that mAbs directed against several staphylococcal surface targets such as wall teichoic acid (a glycopolymer on the S. aureus/S. epidermidis cell wall) and polymeric-N-acetyl-glucosamine (major constituent of the S. epidermidis biofilm extracellular matrix) bind biofilms in a dose-dependent manner. This interaction was specific since no binding was observed for control antibodies (recognizing the hapten DNP). Furthermore we show that these antibodies can penetrate the complete 3D structure of an in vitro biofilm. Products of complement activation via the classical pathway were detected upon incubation with human serum and the biofilm binding mAbs.”. Conclusions. “Having established that our mAbs can bind biofilms and induce complement opsonisation via C3b deposition, we will now study if we can engineer these antibodies to enhance complement deposition. A combination of enhanced complement and antibody opsonisation may improve recognition and clearance of biofilms by phagocytic immune cells. These mAbs could be used to boost the immune system to clear implant associated infections, without the need to replace the implant via invasive surgical procedures.”

Aims. This study investigated vancomycin-microbubbles (Vm-MBs) and meropenem (Mp)-MBs with ultrasound-targeted microbubble destruction (UTMD) to disrupt biofilms and improve bactericidal efficiency, providing a new and promising strategy for the treatment of device-related infections (DRIs). Methods. A film hydration method was used to prepare Vm-MBs and Mp-MBs and examine their characterization. Biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were treated with different groups. Biofilm biomass differences were determined by staining. Thickness and bacterial viability were observed with confocal laser scanning microscope (CLSM). Colony counts were determined by plate-counting. Scanning electron microscopy (SEM) observed bacterial morphology. Results. The Vm-MBs and Mp-MBs met the experimental requirements. The biofilm biomass in the Vm, Vm-MBs, UTMD, and Vm-MBs + UTMD groups was significantly lower than in the control group. MRSA and E. coli biofilms were most notably damaged in the Vm-MBs + UTMD group and Mp-MBs + UTMD group, respectively, with mean 21.55% (SD 0.08) and 19.73% (SD 1.25) remaining in the biofilm biomass. Vm-MBs + UTMD significantly reduced biofilm thickness and bacterial viability (p = 0.005 and p < 0.0001, respectively). Mp-MBs + UTMD could significantly decrease biofilm thickness and bacterial viability (allp < 0.001). Plate-counting method showed that the numbers of MRSA and E. coli bacterial colonies were significantly lower in the Vm-MBs + UTMD group and the Mp, Mp-MBs, UTMD, Mp-MBs + UTMD groups compared to the control group (p = 0.031). SEM showed that the morphology and structure of MRSA and E. coli were significantly damaged in the Vm-MBs + UTMD and Mp-MBs + UTMD groups. Conclusion. Vm-MBs or Mp-MBs combined with UTMD can effectively disrupt biofilms and protectively release antibiotics under ultrasound mediation, significantly reducing bacterial viability and improving the bactericidal effect of antibiotics. Cite this article: Bone Joint Res 2024;13(9):441–451

Aim. Rifampicin plays an important role in the treatment of staphylococcal prosthetic joint infection, as rifampicin-containing combinations have shown a high efficacy against S. aureus biofilm infections. However, the emergence of rifampin-resistant strains is a feared complication and the use of rifampicin in those cases seems unwarranted. Therefore, we evaluated the activity of bacteriophage Sb1 in combination with different antibiotics against the biofilm of four rifampicin-resistant MRSA strains as alternative therapeutic approach. Method. Four rifampicin-resistant MRSA strains were used in this study. The MIC for all tested antibiotics was determined by Etest. Biofilms were formed on porous glass beads for 24h and exposed to Sb1 (10. 7. PFU/mL) for 24h followed by exposure to antibiotic for 24h. Viability of bacteria after antimicrobial treatment was detected by beads sonication and plating of the sonication fluids. The minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotic required to kill all cells resulting in the appearance of no colony after plating of the sonication fluid (detection limit <20 CFU/mL). The synergistic effects were observed when Sb1 combined with antibiotics used at least 2 log-reduction lower concentrations. Results. All strains were susceptible to the three antibiotics except for MRSA3, resistant to fosfomycin, according to the EUCAST breakpoints. All tested antibiotics presented high MBEC values (ranging from 64 to >1024 µg/mL) when tested alone against biofilm (Table 1). A sequential administration of Sb1 followed by vancomycin (VAN) showed no synergistic effect against any of the tested strains, whereas the combination with fosfomycin (FOF) showed synergism in 50% of the strains with improvement of the eradication activity. The combination of Sb1 with daptomycin (DAP) showed the highest synergistic effects in 100% of the strains, with a 2 or 3-log reduction in the MBEC. Conclusions. Sb1 bacteriophage in combination with daptomycin seems a promising alternative for the treatment of rifampicin-resistant MRSA biofilm infections. Table 1 (not included). Antimicrobial activities against rifampicin-resistant MRSA strains. For the table, please contact authors directly

Aim. Ciprofloxacin is recommended as anti-biofilm therapy for gram-negative periprosthetic joint infection. With ciprofloxacin monotherapy, resistance in gram-negative bacteria was observed. Therefore, we evaluated in vitro synergistic activity of fosfomycin, ciprofloxacin and gentamicin combinations against biofilms formed by E. coli and P. aeruginosa strains. Method. E. coli ATCC 25922, P. aeruginosa ATCC 27853 and 15 clinical isolates were used for this study. MIC values were determined by Etest. Biofilms were formed on porous sintered glass beads for 24h and exposed to antibiotics for further 24h. Viability of bacteria on the glass beads after antibiotic treatment was detected by cfu counting of the sonicated beads. The minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotic required to kill biofilm cells. Synergistic activity against biofilm was evaluated by calculation of the fractional inhibitory concentration index (FICI). Results. Table 1 summarizes the antimicrobial susceptibility of planktonic (MIC), biofilm bacteria (MBEC) and synergism. Among 9 E. coli isolates, the synergism was observed in 78% of isolates treated with fosfomycin/gentamicin, 44% treated with gentamicin/ciprofloxacin and 22% treated with fosfomycin/ciprofloxacin. Among 8 P. aeruginosa isolates, the synergism was observed 75% of isolates treated with gentamicin/ciprofloxacin, 63% treated with fosfomycin/gentamicin and 50% treated with fosfomycin/ciprofloxacin. Conclusions. Based on our results, fosfomycin in combination with gentamicin seems to be a promising therapeutic approach against E. coli biofilm related infections. Combination of gentamicin with ciprofloxacin represent the most optimal treatment option for P. aeruginosa biofilm. For any figures or tables, please contact the authors directly

Biofilm formation (BF) in wounds and on biomaterials is a severe complication in trauma and orthopaedic surgery. Maggot therapy is successfully applied in wounds, that are suspected for BF. This study investigated BF by Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis and Enterobacter cloacae on polyethylene, titanium and stainless steel and tested the effect on BF by maggot excretions/secretions (ES). Comb-forming models of the biomaterials were made to fit into a 96-well microtiter plate. In the wells, a suspension of 2.5 x 105 bacteria/ml and nutrient medium was pipetted. Combs were placed in the wells and incubated for 3, 5, 7, and 9 days at 37°C. The formed biofilms were stained in crystal-violet and eluted in ethanol. The optical density (OD 595 nm) was measured to quantify BF. Then, maggots excretions/secretions (ES) were collected according to a standardized method, added in different concentrations to (non-stained) mature biofilms (7 days), incubated another 24 hours and at last stained and measured. The results showed biofilm reduction by ES on all biomaterials. Biofilms formed by S. aureus were reduced to minima of 40% on PE and SSS (p< 0.001) and 50% on TI (p=0.005). The biofilm reduction for S. epidermidis was even greater on PE, SSS and TI with respectively minima of 8% (p< 0.001), 32% (p< 0.001) and 38% BF (p< 0.001). The quantity of BF by S. aureus and S. epidermidis had a comparable strength (p=ns) and was for both bacteria the greatest on polyethylene and the lowest on titanium (p< 0.001). Klebsiella oxytoca, Enterococcus faecalis and Enterobacter cloacae formed weak biofilms on all materials. Mature BF was reached between 5 to 7 days by S. epidermidis and between 7 to 9 days by S. aureus. Our previous research showed biofilm inhibition and breakdown of Pseudomonas aeruginosa by ES. This study showed that maggot ES also reduce biofilms formed by S. aureus and S. epidermidis which are frequently isolated from biomaterial-associated infections. There may be pharmacologic agents that could be developed from maggot ES. While BF on orthopaedic materials is an increasing problem, this experimental study could indicate a new treatment for BF on infected biomaterials

The aim of the present study was to assess the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL100 (EUD) microparticles against mature biofilms of polysaccharide intercellular adhesin-positive S. epidermidis. The effect of plain, daptomycin- and vancomycin-loaded PMMA and PMMA-EUD microparticles on S. epidermidis biofilms was assessed by isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). Biofilms were grown for 48h onto poly-urethane pieces of fixed dimensions. Each sample was washed with PBS in order to remove planktonic bacteria and incubated for 24h with different concentrations of acrylic microparticles (20–1.25 mg/mL). The minimal biofilm inhibitory concentration (MBIC) of the antibiotic-loaded particles was defined as the lowest concentration of particles that was able to prevent heat flow associated to the recovery of the biofilms. After incubation with the microparticles, sessile cocci were hybridized with the pan-bacterial EUB338-FITC and the staphylococci-specific STAPHY-FICT probes and stained with DAPI. Biofilm structure and metabolic state were characterized by fluorescence microscopy. According to the IMC results, plain PMMA-particles showed no effect on S. epidermidis biofilms, whereas PMMA-EUD-microparticles negatively influenced the recovery of the biofilm probably due to the highly positive charge of these particles. The MBIC of daptomycin-loaded PMMA-microparticles was 20 mg/mL, whereas vancomycin-loaded PMMA microparticles were not able to inhibit biofilm recovery. Adding EUD to the formulation reduced the MBIC of daptomycin-loaded microparticles to 1.25 mg/mL, corresponding to a 16-fold reduction. Regarding the vancomycin-loaded microparticles, EUD caused a further decrease of their antibiofilm activity. The FISH micrographs corroborated the IMC results and provided additional insights on the antibiofilm effect of these carriers. According to FISH, daptomycin-loaded PMMA-EUD microparticles were responsible for the most pronounced reduction in biofilm mass. In addition, FISH showed that both PMMA and PMMA-EUD microparticles were able to attach to the biofilms. Adding EUD to the formulations proved to be a powerful strategy to improve daptomycin-loaded microparticles antibiofilm activity. In addition, the combination of IMC and FISH was essential in order to fully assess the effect of polymeric microparticles on sessile S. epidermidis. Although the present study enabled gaining further insights on this subject, the nature of these interactions remains unclear. However, this may be a crucial aspect for the enhancement of antibiofilm activity of antibiotic-loaded polymeric microcarriers against mature biofilms. This work was supported by the Portuguese government (Fundação para a Ciência e a Tecnologia) and FEDER (grant SFRH/BD/69260/2010 and research project EXCL/CTM-NAN/0166/2012) and strategic project PEst-OE/SAU/UI4013/2011

Aim

In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules involve in biofilm. Due to ethical restrictions, the use mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections. This model organism is easy to handle, cheap and ethical restriction free and could be used for the high through put screening of antimicrobial compounds to treat biofilm. To promote the use of this model in basic research we aimed to validate this based on the typical biofilm features such as less susceptible to the antibiotics, complexity of the biofilm structure and gene expression profile of biofilms.

The December 2022 Research Roundup³⁶⁰ looks at: Halicin is effective against Staphylococcus aureus biofilms in vitro; Synovial fluid and serum neutrophil-to-lymphocyte ratio: useful in septic arthritis?; Transcutaneous oximetry and wound healing; Orthopaedic surgery causes gut microbiome dysbiosis and intestinal barrier dysfunction; Mortality in alcohol-related cirrhosis: a nationwide population-based cohort study; Self-reported resistance training is associated with better bone microarchitecture in vegan people.

Abstract

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, with cumulative treatment costs for all prosthetic joint infections estimated to be ∼ $1 billion per annum (UK and North America). Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Low intensity pulsed ultrasound is already licensed for clinical use in fracture management and thus could be translated quickly into a clinical treatment

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged with gentamicin +/− low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 180 minutes and 20 minutes, respectively. The primary outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. Assessment of cellular metabolism was conducted using a liquid-chromatography-mass spectrometry, as well as a triphenyltetrazolium chloride assay coupled with spectrophotometry.

There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was reduced to 64mg/L. Metabolic activity of biofilm-associated S. aureus was increased by 25% following ultrasound therapy (p < 0 .0001), with identification of key biosynthetic pathways activated by non-lethal dispersal.

Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin, bringing the MBEC of gentamicin to within clinically achievable concentrations. The mechanism of action was due to partial disruption of the extracellular matrix which led to an increase of nutrient availability and oxygen tension within the biofilm. This metabolic stimulus was responsible for the reversal of gentamicin tolerance in the biofilm-associated S. aureus.

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explores, but preliminary work has shown potential benefit, especially when combined with existing antibiotics.

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, bacterial biofilms were challenged by gentamicin +/− low-intensity ultrasound (1.5MHz, 30W/cm2, pulse duration 200µs/1KHz) for 20 minutes. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin.

The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L.

Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics.

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged by a low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 20 minutes and gentamicin. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L. Further studies confirmed that the mechanism of action was due to incomplete disruption of the extracellular matrix with subsequent metabolic stimulation of the dormant biofilm-associated bacteria due to increased nutrient availability and oxygen tension.

Low intensity pulsed ultrasound was associated with a 4-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations.

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged by a low-intensity ultrasound (1.5MHz, 30mW/cm², pulse duration 200µs/1KHz) for 20 minutes and gentamicin. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 10⁹ CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L. Low intensity pulsed ultrasound was associated with a 4-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations.

Aims

Prosthetic joint infection (PJI) remains the most severe complication of arthroplasty. Failure of intensive, long-term antibiotic treatment for PJI often requires removal of the implant. Antibiotic failure is thought to be caused by biofilm and persister formation. Novel anti-biofilm and anti-persister strategies are urgently needed. Here, we investigated the effects of several antimicrobial peptides on the bacteria within antibiotic-treated biofilms in an in vitro mature biofilm model on abiotic surfaces.

Aim

Irrigation is a major step during debridement surgery in the context of Prosthetic Joint Infections (PJI), but its effects on biofilms are poorly described.

The present study aims at evaluating the effect of PW alone or followed by antibiotics on MSSA and MRSA biofilms grown on Ti6Al4V coupons in-vitro.

Antimicrobial resistance (AMR) is projected to result in 10 million deaths every year globally by 2050. Without urgent action, routine orthopaedic operations could become high risk and musculoskeletal infections incurable in a “post-antibiotic era.” However, current methods of studying AMR processes including bacterial biofilm formation are 2D in nature, and therefore unable to recapitulate the 3D processes within in vivo infection.

Within this study, 3D printing was applied for the first time alongside a custom-developed bioink to bioprint 3D bacterial biofilm constructs from clinically relevant species including Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa. Bacterial viability and biofilm formation in bioprinted constructs was excellent, with confocal laser scanning microscopy (CSLM) used to demonstrate biofilm production and maturation over 28 days. Bioprinted 3D MRSA and MSSA biofilm constructs had greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Raman spectroscopy was also adapted in a novel approach to non-invasively diagnose 3D bioprinted biofilm constructs located within a joint replacement model.

In conclusion, mature bacterial biofilm constructs were reproducibly 3D bioprinted for the first time using clinically relevant bacteria. This methodology allows the study of antimicrobial biofilm penetration in 3D, and potentially aids future antimicrobial research, replicating joint infection more closely than current 2D culture models. Furthermore, by deploying Raman spectroscopy in a novel fashion, it was possible to diagnose 3D bioprinted biofilm infections within a joint replacement model.

Peri-prosthetic joint infection (PJI) is one the most devastating complications of joint arthroplasty. Although PJI is an infrequent complication (the reported incidence is 1%-2% in the United States), it is the most common indication for revision total knee arthroplasty in the Medicare population and the third most frequent indication for revision total hip arthroplasty. Moreover, the prevalence of PJI appears to be on the rise, with a projected number exceeding 60,000 to 70,000 cases in the United States by 2020.

It is estimated that more than 25% of revision procedures annually are attributed to PJI and this number is expected to increase in the upcoming years. The increase in the prevalence of obesity, diabetes, and other comorbidities among the patient population and the emergence of resistant infecting organisms are some of the reasons for the expected rise in the number of infections that medical community will witness.

The challenges that PJI present to the orthopaedic community are on many fronts. Prevention of PJI has proven to be a difficult task indeed. Effective strategies for prevention of PJI are being refined. The Center for Disease Control will be publishing its updated Surgical Site Prevention Guidelines in the next few months that consists of specific recommendations for prevention of PJI. In recent years, strides are made in introducing novel molecular techniques for diagnosis of PJI, which may stand to change our practices. The current surgical technique for management of PJI, besides the immense cost, fall short of delivering high success to the patients. The major problem in eradication of infection relates to formation of biofilm on the implant surface and internalization of the organisms by affected cells. Biofilm is a sophisticated structure comprising of organisms embedded in multiple layers of glycoccalyx that allows the organisms to evade host immunity and is impenetrable to antibiotics. These organisms are capable of communicating through molecular mechanisms such as quorum sensing that affords them advantage for survival in the host environment. In recent years strategies to prevent colonization of the implant surface, an essential first step in formation of biofilm, or biofilm disruption techniques have been introduced. A recent International Consensus meeting on PJI that assembled more than 350 experts identified some of the best practices in this field and identified areas in need of future research. Moving into the future, the field of orthopaedics in general and PJI in particular stand to benefit from the discoveries in the field of molecular diagnostics, metabolomics and epigenetics.

Aim

Determine the time concentration profile required to achieve vancomycin-mediated eradication of Staphylococcus aureus biofilm. This is critical for the identification of performance targets for local antibiotic delivery, yet has not been described.

Cement is still in common usage in primary and revision arthroplasty surgery. Infection rates in cemented arthroplasties ranges from 1–4% and poses a huge problem for the revision arthroplasty surgeon. Infection in septic implants is biofilm based and almost completely resistant to conventional anti-microbial therapy. Recent papers have questioned the efficacy of using gentamicin-loaded cement in arthroplasty as staphylococcus aureus biofilms will develop on same. The focus of this study was to investigate the efficacy of antibiotic loaded cement in preventing initial bacterial adhesion and subsequent development of a bacterial biofilm in vitro.

Three cements Simplex unloaded, Simplex with erythromycin and Simplex with tobramycin were mixed in a conventional manner, ie vacuum hand mixing in sterile conditions and then injected into pre-moulded PTFE coated cylinder moulds yielding 8 cylinders in each group. The cement cylinders were then removed and exposed to a known pathogenic strain of staphylococcus aureus ATCC—29213-NCTC 12973 in solution 3x10⁶ Colony forming units CFH/ml) for 15 minutes. The cylinders were then removed and cultured for 24 hours at 37°C in RPMI with Glutamine. Cylinders were then removed and subjected to rinsing in PBS to remove any non-adherent bacteria. Cylinders were then sonicated at 50 Hz in Ringer’s solution and adherent biofilms were serially log diluted and plated on Columbia blood agar. Colonies were counted manually. Control cylinders of unloaded cement showed 120,000 CFU/cm² of adherent bacteria whereas loaded cement erythromycin and tobramycin showed 500 and 80 CFU/cm² respectively (p< .0005 Student t-test).

This study shows that loaded cement does not prevent biofilm adhesion in its initial reversible stages whereas unloaded cement does not. This is important since most infected implants are infected at time of primary operation and cements anti-bacterial role beyond the first 48 hours remains questionable, when inflammatory encapsulation of the implant begins. We would therefore question the usage of unloaded cement in primary arthroplasty surgery.

Post-operative surgical site infection following total joint arthroplasty occurs at rates between ~ 0.2–5 %, depending on the joint and the surgeon volume, as well as various patient risk factors. Given that an estimated over 700,000 knee and hip arthroplasties are performed in the US each year this translates to thousands of patients that are affected by this serious, costly and traumatic complication. In addition, it is now recognized that clinical culturing underestimates the infection rate and that a number of aseptic loosenings might actually have an infectious etiology. We have used a combination of non-culture based molecular methods to detect bacteria associated with hardware, antimicrobial impregnated cement, reactive tissue and pus collected during revision surgery in a total elbow arthroplasty (TEA) case and a total ankle revision (TAR) case. Confocal microscopy showed live cocci in biofilm cell clusters, and fluorescent in situ hybridization (FISH) demonstrated S. aureus biofilms. Reverse transcriptase (RT)-PCR, and multiplex PCR coupled with electrospray-ionization mass spectrometry (Ibis T5000) to identify S. aureus, S. epidermidis and genes for methicillin resistance. Together our complimentary techniques comprise compelling evidence that viable biofilm bacteria played an important role in the refractory infections in these cases.

Summary Statement

Combination of antibiotics with N-acetylcisteine and sub-MIC concentration of erythromycin was evaluated in two collection and 16 clinical strains of staphylococci isolated from PJI. The results were strain-dependent, so it evidences the necessity of perform individual studies of biofilm susceptibility.

P.aeruginosa causes acute and chronic-destructive infections, particularly wound infections, or device-associated infections by colonising respiratory tubes, catheters, or implants. The pathogenicity of P.aeruginosa is largely attributed to the relative resistance towards host defence. Especially when organised as biofilms, the bacteria evade phagocytosis and killing by polymorphonuclear neutrophils (PMN).

To elucidate the evasion mechanisms, the migration of PMN towards and through P.aeruginosa biofilms was studied. Migration of PMN towards P.aeruginosa biofilms was tested using various in vitro techniques.

We found that PMN migrated towards developing P.aeruginosa biofilms, attracted by the quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Mature biofilms which no longer produced 3OC12-HSL did not attract PMN. Addition of interleukin 8, a potent chemokine, restored the migratory capacity. Once arrived at the biofilms, PMN readily attached with no important difference between developing and mature biofilms. Migration into and penetration of the films, however, was only seen with developing films. By mass spectroscopy it became obvious that a major difference between developing and mature biofilms was the composition of the extracellular polymer substance, of which alginate is a prominent component. A series of experiments with isolated alginate showed that PMN did not migrate on or into alginate-containing matrices, but remained affixed at the contact site just as they did on mature biofilms. The mechanism of this firm attachment is still under investigation; prominent up-regulation of various adhesion molecules was seen, which could provide possible explanation.

Mature biofilms, most probably due to the composition of the extracellular polymer substance, do not allow the penetration of PMN. Consequently, bacteria embedded in deeper layers of the biofilm are protected against the host response. Due to the restricted movement of PMN, the bactericidal activity of PMN is only efficient against bacteria in the immediate vicinity, explaining the inefficient host defence.

Objectives: This study evaluates the number of recurrence of acute infection following total knee arthroplasty treated with a concept of implant salvage using programmed revision surgery and specific long-term antibiotic therapy with and without additional application of antibiotics penetrating bacterial biofilms like rifampicin.

Methods: In a retrospective study, 24 patients with early infection of unconstrained total knee arthroplasty were treated according to our protocol and were followed up for a period of 4 years [range 1,2–6,2]

using a questionnaire to investigate course of disease and health-related quality of life (VAS). 7 patients were treated with and 17 patients without additional application of antibiotics penetrating bacterial biofilms.

Results: In the group of patients treated without additional application of antibiotics penetrating bacterial biofilms 11 of 17 (65%) implants were salvaged. In 5 cases revision arthroplasty and 1 arthrodesis were necessary to eradicate infection. Health-related quality of life and function of the arthroplasy were superior in the group of salvaged implants.

In the group of patients treated with additional application of antibiotics penetrating bacterial biofilms 6 of 7 (86%) implants were salvaged and reduced number of revison surgery was needed.

Conclusion: Treatment of infection with implant salvage may be one therapeutic option if the implant is not loose. Therapy with retention of the prosthesis may be indicated: in the case of early infection (< 3 weeks of ongoing symptoms), with unconstrained implants, in the case of infection by a single organism that is susceptible to antibiotic therapy, if soft tissue envelope is not affected, and if the immune system is not compromised. Early and consequent therapy with operative debridements and specific long-term antibiotic therapy are necessary to achieve implant salvage. Additional application of antibiotics penetrating bacterial biofilms such as rifampicin contribute to improve prognosis.

Due to the fact that revision arthroplasty is often associated with limited function after infection of total knee joint, retention of the implant has to be considered a therapeutic alternative in early infection.

The formation of bacterial biofilms is increasingly recognised as the leading cause of chronic infections. It limits the application of implant materials including catheters, heart valves, or orthopaedic prostheses. It is generally assumed that the infection persists because bacteria organised as biofilms escape the host defence mechanisms. Nevertheless, when studying patients with infected implants, we found a massive infiltration of leukocytes particularly polymorphonuclear neutrophils, PMN, into the site of infection, which led to the question, whether the PMN interact with the bacterial biofilm or not.

The interaction of human PMN with Staphylococcus aureus biofilms was studied in vitro.

S.aureus was cultivated on glass cover slips for various times under conditions allowing formation of biofilms. Adherence of PMN to biofilms and phagocytosis of the bacteria were observed by confocal laser scan microscopy and time lapse video microscopy.

Migration of PMN on and into the biofilm was identified as being phagocytosis, apparent as uptake of bacteria into the cell. Concominantly, in the wake of migrating PMN bacteria depleted zones appeared, which increased in size with time. In addition to phagocytosis, release from PMN of DNA and also of elastase was seen, suggesting the formation of neutrophil extracellular traps (NETs). So far, the signal for DNA release and NET formation has not been identified; of note is, however, that they occurred preferentially on established “old” biofilms and in the absence of the opsonising human serum, while phagocytosis was most efficient with developing “young” biofilms.

Taken together, our data provide evidence that bacteria in biofilms are not entirely protected against host defence but that phagocytosis is still possible, especially when the biofilm is opsonised with human serum. Whether NET formation also contributes to bacteria killing in biofilms cannot be decided as yet but remains an attractive alternative.

Aim. Bacteriophages are remerging as alternative and adjunctive therapy for fracture-related infection (FRI). However, current administration protocols involve prolonged retention of a percutaneous draining tube with potential risk of developing superinfection. In this study, we applied a cocktail of in vitro evolved biofilm-targeting phages for Methicillin-resistant Staphylococcus aureus (MRSA) in a hydrogel platform co-delivering vancomycin. In vitro synergy and antibiofilm activity was assessed and a subsequent in vivo study was performed in a mouse FRI model with MRSA. Method. Two evolved bacteriophages (MRSA-R14 and COL-R23) with improved antibiofilm activity against a clinical isolate (MRSA3) were tested in combination with vancomycin and a carboxymethylcellulose (CMC) hydrogel in vitro and in vivo. MRSA3 bacterial biofilms were formed on sterile 4 mm sintered porous glass beads at 37 °C for 24 h. Biofilms were exposed to i-phage cocktail (10. 7. PFU/ml), ii-vancomycin at concentrations of 0.5, 1, 10 and 100 times the MIC, or iii-combination of phage cocktail and vancomycin. Recovered biofilm cells, were quantified by colony counting. The stability and release profiles of phage cocktail and vancomycin in co-delivery hydrogel were assessed in vitro for 8 days and 72 hrs, respectively, and subsequently tested in the treatment of 5-day-old MRSA3 infection of a femoral plate osteotomy in mice. Results. In vitro: The cocktail of evolved phages (10. 7. PFU/ml, 1:1) combined with 0.5 MIC vancomycin achieved 99.72% reduction in MRSA3 biofilm in vitro compared to the growth control. This combination was stable in the co-delivery hydrogel over 8 days. The release profile showed that 57% of phages and 80% of vancomycin were released after 72hrs, which was identical to the performance for gels loaded with phage or antibiotic alone. In the in vivo study, the bacterial load from animals that received co-delivery hydrogel and systemic vancomycin was significantly reduced compared to controls, animals that received systemic vancomycin and animals that received co-delivery hydrogel alone (p<0.05). Conclusions. Our study demonstrates the potential of using evolved phages in combination with vancomycin and hydrogel delivery systems for the treatment of MRSA-related infections. Further research in this area may lead to the development of specific therapies for biofilm-related infection

The October 2015 Research Roundup360 looks at: Wasted implants; Biofilms revisited; Peri-operative anticoagulation not required in atrial fibrillation; Determinants in outcome following orthopaedic surgery; Patient ‘activation’ and outcomes; Neuroplasticity and nerve repair; KOOS Score in predicting injury?

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

One of the most common bacteria in orthopaedic prosthetic infections is Staphylococcus Aureus. Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion on nanofabricated materials. We hypothesise that surface nanotopography impacts the differential ability of staphylococci species to adhere via altered metabolomics and may reduce orthopaedic implant infection rate. Bacteria were grown and growth conditions optimised. Polystyrene and titanium (Ti) nanosurfaces were studied. The polystyrene surfaces had different nanopit arrays, while the Ti surfaces expressed different nanowire structures. Adhesion analysis was performed using fluorescence imaging, quantitative PCR and bacterial percentage coverage calculations. Further substitution with ‘heavy’ labelled glucose into growth medium allowed for bacterial metabolomic analysis and identification of any up-regulated metabolites and pathways. Our data demonstrates reduced bacterial adhesion on specific nanopit polystyrene arrays, while nanowired titanium showed increased bacterial adhesion following qPCR (P<0.05) and percentage coverage calculations (P<0.001). Further metabolomic analysis identified significantly increased intensity counts of specific metabolites (Pyruvate, Aspartate, Alanine and Carbamoyl aspartate). Our study shows that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. The identification of metabolic pathways involved in adhesion may allow for a targeted approach to biofilm eradication in S. aureus. This is of significant benefit to both the patient and the surgeon, and may well extend far beyond the realms of orthopaedics

The most common bacteria in orthopaedic prosthetic infections are Staphylococcus, namely Staphylococcus Epidermidis (SE) and Staphylococcus Aureus (SA). Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion and biofilm formation on nanofabricated materials. Bacteria studied were clinically relevant from an orthopaedic perspective, SA and SE. We hypothesise that that nanosurfaces can modulate bacterial adherence and biofilm formation and may reduce orthopaedic implant infection rate. Isolated bacteria were grown and growth conditions optimised. Bacterial concentrations were calculated by using qPCR. Statistical analysis allowed identification of optimal biofilm growth conditions. These were refined on standard, non-nanopatterned surfaces, and then control and nanopatterned polystyrene (nanopits) and titanium plates (nanowires). Adhesion analysis was performed using fluorescence imaging and quantitative PCR. 4 bacterial strains were isolated and cultured. Growth kinetics based on 24hr cultures allowed isolation of optimal media for biofilm conditions (Dulbecco's Modified Eagle Medium with additional supplements). Highest bacterial concentrations were found following 2hrs incubation with Lysozyme during qPCR. Bacterial concentration significantly increased between 30, 60 and 90 minutes incubation. Differences in percentage coverage on different polysyrene nanosurfaces (nanopits) were noted varying. This was confirmed by qPCR extractions that showed different bacterial concentrations on different nanopatterns. Titanium nanowire surfaces significantly increased bacterial adhesion (P<0.05). Our study cultured and quantified bacterial biofilm and suggests that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. Clearly this is of significant benefit to the patient, the surgeon and the NHS, and may well extend far beyond the realms of orthopaedics

Introduction. Despite the routine use of irrigation, debridement and systemic antibiotics, there is a high incidence of infection in severe open fractures. The synergistic use of local and systemic antibiotics appreciably reduces infection rates although the time window within which this is effective is unknown. The aim was to determine if delaying treatment of wounds causes higher levels of infection. Methods. A defect was created in the femurs of 90 Sprague-Dawley rats and inoculated with 105CFUs Staphylococcus aureus. At 2, 6 and 24 hours following contamination, the defect was irrigated and debrided. The experimental groups had either vancomycin or tobramycin impregnated PMMA beads placed within the segmental defect. The controls received no further treatment. Two weeks after wound closure, the bacteria within the femur were quantified. Results. Delaying irrigation and debridement resulted in significantly more bacteria (p<0.01) within the control group (2 hr < 6 hr <24 hr). Both locally delivered tobramycin and vancomycin significantly reduced the bacteria (p<0.05) when administered at the earlier time points (2 and 6 hours). Locally-delivered antibiotics were ineffective when delivered at 24 hours. Conclusion. Delaying treatment of contaminated defects reduces its effectiveness to eradicate infection. This is presumably because of the biofilm formation by the bacteria. Biofilms begin to form within a couple of hours and are mature within 12 hours. Early treatment of the wound allows the surgeon to physically remove the bacteria or have antibiotics present before a mature biofilm protects the bacteria

Aims

Periprosthetic joint infections (PJIs) are among the most devastating complications after joint arthroplasty. There is limited evidence on the efficacy of different antiseptic solutions on reducing biofilm burden. The purpose of the present study was to test the efficacy of different antiseptic solutions against clinically relevant microorganisms in biofilm.

Aims. Infections of bone usually require multiple surgery and prolonged periods of treatment. One reason for problems is found in the presence of stationary phase bacteria embedded in biofilms that show increased resistance against conventional antibiotic therapy (up to 1000x MIC). Biofilms adhere to surfaces of avital material making radical debridement a prerequisite for cure. Osseous defects are common in such conditions and need to be addressed. To avoid re-infection high local antbiotic concentrations are necessary. Allograft bone may be impregnated with high loads of antibiotics using a special incubation technique. The resulting antibiotic bone compound (ABC) provides high and long lasting concentrations at the site of infection and is likely to restore bone stock simultaneously. Based on this technology we have developed a new surgical technique. Methods. 42 patients (10–67yrs) with chronic osteitis were included into a prospective study using a standardized protocol. Infection was at the humerus (1x), femur (10x), tibia (29x) or femur+tibia (2x), respectively. Treatment consisted of removal of foreign material, radical sequestrectomy and soft-tissue debridement followed by pressurized lavage. Surfaces of sclerotic bone were trimmed down to vital areas. The remaining osseous defects were filled with ABC, using an impaction technique resulting in complete dead space management. The allograft was impregnated with vancomycin, in cases with mixed pathogens combinations with tobramycin were used. Internal fixation was performed the same time whenever applicable. Sites were drained and closed immediately; rehabilitation did not differ from uninfected procedures. Results. 1 patient died shortly after surgery from cardiac failure. 41 could be followed for a minimum of 2 and a maximum of 6years (mean 3,1years). In 2 patients wound healing was unsatisfactory requiring additional coverage with a muscle flap. 2 patients showed material failure after intramedullary nailing, requiring exchange of the implant. In those cases no sign of infection was present at the time of revision. There were 3 cases with recurrence of infection, all originating from foci not detected during the index operation and becoming apparent between 3 and 12 months after surgery. Two could successfully be revised using the same technique; one refused revision and shows continuing fistulation. Radiological incorporation of allografts appeared as after conventional bone grafting, union of pseudarthroses was achieved between 2 and 6 months after (re-) stabilization. 40 patients (95,2%) were fully weight bearing, painfree and without any sign of infection at the latest follow up. Conclusion. Using antibiotic impregnated allograft bone eradication of pathogens, grafting of defects, dead space management and insertion of osteosynthetic material may be accomplished in a one stage procedure. Since the graft gradually is replaced by healthy own bone improved long term results may be expected as well as improved conditions in the case of another revision. The new technique provides for quick rehabilitation, improved results and markedly reduced costs of treatment in cases of bone infection

Aims

The aims of this study were to develop an in vivo model of periprosthetic joint infection (PJI) in cemented hip hemiarthroplasty, and to monitor infection and biofilm formation in real-time.

Metallic implants are used frequently in the operative repair of joints and fractures in orthopaedic surgery. Orthopaedic implant infection is chronic and biofilm based. Present treatment focuses on removing the infective substratum and implant surgically as well as prolonged anti-microbial therapy. Biofilms are up to 500 times more resistant than planktonic strains of bacterial flora to antibiotics. Silver coatings on polymers and nylon (catheters, heart valve cuffs, burn dressings) have shown inhibition of this biofilm formation in its adhesion stage. Our aim was to deposit effective, minute, antibacterial layers of silver on orthopaedic stainless steel and titanium K-wires and to investigate the effect of these coatings when exposed to Staphylococcus Aureus biofilms in an in vitro and in vivo environment. Combining magnetron sputtering with a neutral atom beam (Saddle Field) plasma source at 10. −4. mbar in argon gas at temperatures of 60°C, a silver coating of 99.9% purity was deposited onto stainless steel and titanium orthopaedic K-wires. Coating thickness measurements were obtained using glancing angle x-ray diffraction of glass slides coated adjacent to wires. Magnetron parameters were modified to produce varying thickness of silver. Adhesiveness was examined using Rockwell punch tests. Silver leaching experiments were carried out in phosphate buffered saline at 37°C for 48 hours and using inductive coupled plasma spectrometry to assess leached silver ions. Surface microscopy visualised physical changes in the coatings. Biofilm adhesion was determined by exposing wires to Staphylococcus Aureus ATCC 29213 – NCTC 12973 for 15 minutes to allow biofilm initiation and adhesion. Wires were then culturing for 24 hours at 37°C in RPMI. Subsequently, wires were sonicated at 50Hz in ringer’s solution and gently vortexed to dislodge biofilm. Sonicate was plated out by log dilution method on Columbia blood agar plates. Bacterial colonies were then counted and changes expressed in log factors. K-wires were coated with 1 to 50 nm of silver by running the magnetron sputtering at low currents. These coatings showed excellent adhesive properties within the 48 hours exposed with only 3.7% of silver leaching in buffered saline. The silver coated stainless steel wires showed a log 2.31 fold reduction in biofilm formation as compared to control wires (p< .001), Student t-test), the silver coated titanium wires showed a log reduction of 2.06, (p< .001, Student t-test). Animal studies demonstrated enormous difficulty in reproducing biofilm formation and showed a 0.49 log fold reduction in the titanium group when exposed to Staph Aureus (p< .01, Student t-test), the other groups showed no statistically significant reduction. We have perfected a method of depositing tiny layers of anti-bacterial silver onto stainless steel and titanium, which is anti-infective in vitro but not in vivo. Further studies involving other metal coatings such as platinum and copper are warranted

Metallic implants are used frequently in the operative repair of joints and fractures in orthopaedic surgery. Metal infection is a catastrophic complication of the surgery with patients loosing their newfound mobility and independence, associated morbidity and mortality is high. Orthopaedic implant infection is chronic and biofilm based. Present treatment focuses on removing the infective substratum and implant surgically as well as prolonged anti-microbial therapy. Biofilms are 500 times more resistant than planktonic strains of bacterial flora to antibiotics, and with evolving resistant strains this form of therapy is loosing ground. Silver coatings on polymers and nylon (catheters, heart valve cuffs, burn dressings) have shown inhibition of this biofilm formation in its adhesion stage. Our aim was to deposit effective, minute, biocompatible, anti-bacterial layers of silver on orthopaedic stainless steel K-wires. Combining magnetron sputtering with a neutral atom beam (Saddle Field) plasma source at 10. −4. mbar in argon gas at temperatures of 60°C, a silver coating of 99.9% purity was deposited onto stainless steel orthopaedic K-wires. Coating thickness measurements were obtained using glancing angle x-ray diffraction of glass slides coated adjacent to wires. Magnetron parameters were modified to produce varying thickness of silver. Adhesiveness was examined using Rockwell punch tests and tape tests. Silver leaching experiments were carried out in phosphate buffered saline at 37°C for 48hrs and using inductive coupled plasma spectrometry to assess leached silver ions. Surface microscopy visualised physical changes in the coatings. Biofilm adhesion was determined by exposing wires to Staphylococcus aureus ATCC 29213 -NCTC 12973 for 15 min to allow biofilm adhesion and initiation. Wires were then cultured for 24h at 37°C in RPMI. Subsequently wires were sonicated at 50Hz in ringer’s solution and gently vortexed to dislodge biofilm. Sonicate was plated by the log dilution method on blood agar plates. Bacterial colonies were then counted and changes expressed in log factors. Surface biofilms were visualised using scanning electron microscopy. Cytotoxicity was assessed using fibroblast cell cultures lines. K-wires were coated with 5 to 50 nm of silver by running the magnetron sputtering at low currents. These coatings showed excellent adhesive properties within the 48hr exposed with only 5% of silver leaching in buffered saline. The silver coated wires showed a log 3–4 fold reduction in biofilm formation as compared to control wires. The coatings showed no cytotoxic effects. Silver coating of medical implants has been shown in urological catheters to reduce biofilm infection. We have perfected a method of depositing thin layers of anti-bacterial silver onto stainless steel, which is both anti-infective and biocompatible. This coating could potentially add to the armourary of anti-infective agents in the elimination of infection related orthopaedic implant failure

Introduction: External fixation is used widely in the management of fractures, despite a relatively high incidence of complication, arising from pin loosening and infection. Diamond like carbon (DLC) is a low surface energy coating that can be applied to external fixator pins and may reduce biofilm formation and infection resulting in a lower incidence of pin loosening. Hydroxyapatite (HA) is well established as a coating to enhance fixation of external fixator pins. This study tests the hypothesis that HA and DLC coatings on stainless steel (SS) external fixator pin shafts modify integration of the implant with soft/hard tissues. Materials and Methods: An Orthofix external fixator was used to stabilise a tibial osteotomy with 6 self-drilling/tapping 6mm pins in 32 skeletally mature Friesland ewes. Animals were divided into four groups; SS, DLC, HA partially coated (threads only) and HA fully coated (threads and pin shaft). Pin insertion torque was measured using a torque wrench and extraction torque similarly obtained at 10 weeks when animals underwent euthanasia. Pin performance indices (PPI) were calculated as a ratio of extraction to insertion torque x100%. Pin site 2 was preserved for hard grade resin histology and subsequent pin tissue integration analysis. Pin site 3 was used for analysis of the soft tissue pin shaft interface using transmission electron microscopy. Pin site 5 was examined for the presence of biofilm formation using scanning electron microscopy. Pin site 6 was swabbed for microbiological analysis. Results: SS and DLC pins achieved significantly higher insertion torques compared to HA partially coated pins (p=0.001, 0.002). Both groups of HA coated pins demonstrated a significantly higher, extraction torque and therefore PPI for all pin site positions compared to SS and DLC (p< 0.001– 0.025). The epithelium was found not to be in contact with the pin shaft in all cases. No significant differences were found between the different pin groups for epidermal down growth and dermal contact. Both groups of HA coated pins showed a significantly higher percentage of new bone in direct contact with the embedded threads compared to SS and DLC pins (p< 0.001, p=0.004). The proportion of soft tissue in contact and within the thread, of fully coated HA pins was significantly lower compared to stainless steel (p=0.003, p=0.017), DLC (p=0.004, p=0.002) and HA partially coated pins (p=0.006, p=0.02). Biofilms were evident on all pins except those coated with DLC. More bacteria were observed on the fully HA coated pins. DLC had significantly lower number of bacterial colonies in culture compared to SS (p=0.028) and fully coated HA pins (p=0.005). Discussion: Coatings of DLC and HA do have a significant affect on hard/soft tissue reactions. However coatings do not have a significant effect on epidermal down growth or dermal attachment to the pin shaft surface. DLC coated pins had the cleanest surface with no bio-film present and significantly lower numbers of bacteria present. Fully HA coated pins despite evidence of bio-film formation, bacteria and high microbiological counts had significantly higher PPI. In addition fully coated HA pins demonstrated significantly reduced amounts of soft tissue at the pin bone interface. Therefore soft tissue reactions may affect bone integration

Aims

The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses.

Aims

Current treatments of prosthetic joint infection (PJI) are minimally effective against Staphylococcus aureus biofilm. A murine PJI model of debridement, antibiotics, and implant retention (DAIR) was used to test the hypothesis that PlySs2, a bacteriophage-derived lysin, can target S. aureus biofilm and address the unique challenges presented in this periprosthetic environment.

The December 2015 Research Roundup360 looks at: Biomarkers in periprosthetic joint infection; HbA1c and complications in arthroplasty; Getting to the bottom of biofilms; Effective antibiosis for biofilms; Stem cells and avascular necrosis; Predicting LOS in total joint arthroplasty; Long-term antibiotics reduce recurrence in periprosthetic infection