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.

Aim. We aimed to assess the incidence and the outcome of Gram-negative prosthetic-joint infections (PJI) in 3 international tertiary hospital. Method. We included patients with Gram-negative PJI at Humanitas Clinical and Research Hospital (Milan, Italy), Centre Hospitalier Universitaire Vaudois (Lausanne, Switzerland) and Hospital Parc de Salut Mar (Barcelona, Spain) between 2014 and 2018 in a retrospective cohort. We described the treatment's success rate according to Gram-negative species and type of surgical procedure. Results. In the present cohort we have 780 PJI out of which 71 (9.1%) were caused by Gram-negative bacteria (polymicrobial infection 30%, Escherichia coli 25%, Pseudomonas aeruginosa 20%, Proteus spp. 4%, Klebsiella spp. 3%, Morganella morganii 3%, Enterobacter 3%, others 12%). Gram-negative PJI were more common in females (60%) than males (40%). Sixty percent had a hip infection, 40% a knee infection, the median age was 74 years and the median ASA score was 3. It was a chronic infection in 60% of the cases and an acute one in 40%. Two-step exchange was performed in 55%, débridement and retention (DAIR) in 30%, one-step exchange in 11% and implant removal without replacement in 4% of the patients. The overall treatment success rate was 89%. The success rate was better for two-step exchange (95%) compared to DAIR (81%) and one-step exchange (87%) (p=0.068). The median antibiotic duration was 68 days and ciprofloxacin was used in 70% of the cured patients versus in 88% of the failures (p=0.388). Infections caused by Escherichia coli were associated with a lower success rate (83%) especially compared to Pseudomonas aeruginosa (93%) and polymicrobial infections (90%) (p=0.358). Finally, the success rate was better in knee PJI compared to hip PJI (97% versus 83%, p=0.121) and in females compared to males (93% versus 82%, p=0.121). Conclusions. The treatment's success of Gram-negative PJI is comparable to reported rates for all bacteria. However, our results suggest that surgical management with two-step exchange might be useful in selected patients’ groups such as those with Escherichia coli PJI. Moreover, ciprofloxacin use seems not to improve cure rate

Aims. Trained immunity confers non-specific protection against various types of infectious diseases, including bone and joint infection. Platelets are active participants in the immune response to pathogens and foreign substances, but their role in trained immunity remains elusive. Methods. We first trained the innate immune system of C57BL/6 mice via intravenous injection of two toll-like receptor agonists (zymosan and lipopolysaccharide). Two, four, and eight weeks later, we isolated platelets from immunity-trained and control mice, and then assessed whether immunity training altered platelet releasate. To better understand the role of immunity-trained platelets in bone and joint infection development, we transfused platelets from immunity-trained mice into naïve mice, and then challenged the recipient mice with Staphylococcus aureus or Escherichia coli. Results. After immunity training, the levels of pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-α), interleukin (IL)-17A) and chemokines (CCL5, CXCL4, CXCL5, CXCL7, CXCL12) increased significantly in platelet releasate, while the levels of anti-inflammatory cytokines (IL-4, IL-13) decreased. Other platelet-secreted factors (e.g. platelet-derived growth factor (PDGF)-AA, PDGF-AB, PDGF-BB, cathepsin D, serotonin, and histamine) were statistically indistinguishable between the two groups. Transfusion of platelets from trained mice into naïve mice reduced infection risk and bacterial burden after local or systemic challenge with either S. aureus or E. coli. Conclusion. Immunity training altered platelet releasate by increasing the levels of inflammatory cytokines/chemokines and decreasing the levels of anti-inflammatory cytokines. Transfusion of platelets from immunity-trained mice conferred protection against bone and joint infection, suggesting that alteration of platelet releasate might be an important mechanism underlying trained immunity and may have clinical implications. Cite this article: Bone Joint Res 2022;11(2):73–81

Aims. Gram-negative infections are associated with comorbid patients, but outcomes are less well understood. This study reviewed diagnosis, management, and treatment for a cohort treated in a tertiary spinal centre. Methods. A retrospective review was performed of all gram-negative spinal infections (n = 32; median age 71 years; interquartile range 60 to 78), excluding surgical site infections, at a single centre between 2015 to 2020 with two- to six-year follow-up. Information regarding organism identification, antibiotic regime, and treatment outcomes (including clinical, radiological, and biochemical) were collected from clinical notes. Results. All patients had comorbidities and/or non-spinal procedures within the previous year. Most infections affected lumbar segments (20/32), with Escherichia coli the commonest organism (17/32). Causative organisms were identified by blood culture (23/32), biopsy/aspiration (7/32), or intraoperative samples (2/32). There were 56 different antibiotic regimes, with oral (PO) ciprofloxacin being the most prevalent (13/56; 17.6%). Multilevel, contiguous infections were common (8/32; 25%), usually resulting in bone destruction and collapse. Epidural collections were seen in 13/32 (40.6%). In total, five patients required surgery, three for neurological deterioration. Overall, 24 patients improved or recovered with a mean halving of CRP at 8.5 days (SD 6). At the time of review (two to six years post-diagnosis), 16 patients (50%) were deceased. Conclusion. This is the largest published cohort of gram-negative spinal infections. In older patients with comorbidities and/or previous interventions in the last year, a high level of suspicion must be given to gram-negative infection with blood cultures and biopsy essential. Early organism identification permits targeted treatment and good initial clinical outcomes; however, mortality is 50% in this cohort at a mean of 4.2 years (2 to 6) after diagnosis. Cite this article: Bone Jt Open 2024;5(5):435–443

Aims. In wound irrigation, 1 mM ethylenediaminetetraacetic acid (EDTA) is more efficacious than normal saline (NS) in removing bacteria from a contaminated wound. However, the optimal EDTA concentration remains unknown for different animal wound models. Methods. The cell toxicity of different concentrations of EDTA dissolved in NS (EDTA-NS) was assessed by Cell Counting Kit-8 (CCK-8). Various concentrations of EDTA-NS irrigation solution were compared in three female Sprague-Dawley rat models: 1) a skin defect; 2) a bone exposed; and 3) a wound with an intra-articular implant. All three models were contaminated with Staphylococcus aureus or Escherichia coli. EDTA was dissolved at a concentration of 0 (as control), 0.1, 0.5, 1, 2, 5, 10, 50, and 100 mM in sterile NS. Samples were collected from the wounds and cultured. The bacterial culture-positive rate (colony formation) and infection rate (pus formation) of each treatment group were compared after irrigation and debridement. Results. Cell viability intervened below 10 mM concentrations of EDTA-NS showed no cytotoxicity. Concentrations of 1, 2, and 5 mM EDTA-NS had lower rates of infection and positive cultures for S. aureus and E. coli compared with other concentrations in the skin defect model. For the bone exposed model, 0.5, 1, and 2 mM EDTA-NS had lower rates of infection and positive cultures. For intra-articular implant models 10 and 50 mM, EDTA-NS had the lowest rates of infection and positive cultures. Conclusion. The concentrations of EDTA-NS below 10 mM are safe for irrigation. The optimal concentration of EDTA-NS varies by type of wound after experimental inoculation of three types of wound. Cite this article: Bone Joint Res 2021;10(1):68–76

Objectives. Irrigation is the cornerstone of treating skeletal infection by eliminating pathogens in wounds. A previous study shows that irrigation with normal saline (0.9%) and ethylenediaminetetraacetic acid (EDTA) could improve the removal of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) compared with normal saline (NS) alone. However, it is still unclear whether EDTA solution is effective against infection with drug-resistant bacteria. Methods. We established three wound infection models (skin defect, bone-exposed, implant-exposed) by inoculating the wounds with a variety of representative drug-resistant bacteria including methicillin-resistant S. aureus (MRSA), extended spectrum beta-lactamase-producing E. coli (ESBL-EC), multidrug-resistant Pseudomonas aeruginosa (MRPA), vancomycin-resistant Enterococcus (VRE), multidrug-resistant Acinetobacter baumannii (MRAB), multidrug-resistant Enterobacter (MRE), and multidrug-resistant Proteus mirabilis (MRPM). Irrigation and debridement were repeated until the wound culture became negative. The operating times required to eliminate pathogens in wounds were compared through survival analysis. Results. Compared with other groups (NS, castile soap, benzalkonium chloride, and bacitracin), the EDTA group required fewer debridement and irrigation operations to achieve pathogen eradication in all three models of wound infection. Conclusion. Irrigation with EDTA solution was more effective than the other irrigation fluids used in the treatment of wound infections caused by drug-resistant pathogens. Cite this article: Z. Deng, F. Liu, C. Li. Therapeutic effect of ethylenediaminetetraacetic acid irrigation solution against wound infection with drug-resistant bacteria in a rat model: an animal study. Bone Joint Res 2019;8:189–198. DOI: 10.1302/2046-3758.85.BJR-2018-0280.R3

Introduction: Platelets play a central role in hemostasis and healing processes. Upon their activation, platelet alfa-granules release over 30 cytokines including platelet-derived growth factor (PDGF), transforming growth factor-alfa (TGF-alfa), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF) and also active substances like serotonin, catecholamines, von Willebrand factor, proaccelerin, osteonectin and antimicrobial proteins. By concentrating platelets, platelet-rich plasma (PRP) with higher levels of growth factors might be reached, which could stimulate the healing processes. The activator for PRP is a mixture of thrombin and calcium chloride. After connecting these substances platelet-rich gel (PRG) is formed. Aims: In present study, we investigated in vitro antimicrobial activity of PRG after antibiotic administration. Material and Methods: 30 minutes after iv Amoxillin/ clavulanic acid administration 54 ml of whole blood was collected from each of 10 donors. PRPs were prepared with using GPS system from Biomet. In vitro laboratory susceptibility to PRG was determined by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Becton Dickinson). Baseline antimicrobial activity was assessed by measuring the zones of inhibition. Agar plates were coated with one of the following strain: Staphylococcus aureus ATCC 43300 (MRSA), Staphylococcus aureus ATCC 25923 (MSSA), Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 35218 (ESBL), Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853. Results: We tested 10 samples of PRG. Zones of inhibition produced by PRG ranged between 6 – 23 mm in diameter. PRG inhibited the growth of Staphylococcus aureus. PG also was active against Escherichia coli, Enterococcus faecalis. No activity against Klebsiella pneumoniae and Pseudomonas aeruginosa was detected. Conclusions: Our previous study showed PRG no activity against Enterococcus faecalis without antibiotic administration. In this investigation we observed PRG strong activity against this bacteria after iv Amoxicillin-clavulanic acid administration. In infections during antibiotic treatment, PRG antimicrobial properties are enhanced by antibiotics that are concentrated in plasma

Hypochlorous acid (HOCl) is a potent anti-bacterial agent which could reduce periprosthetic joint infection. Early infection complications in joint replacements are often considered to be due to local contamination at the time of surgery and result in a significant socioeconomic cost. Current theatre cleaning procedures produce “clean” operating theatres which still contain bacteria (colony forming units, CFU). Reducing this bacterial load may reduce local contamination at the time of surgery. HOCl is produced naturally in the human neutrophil and has been implicated as the primary agent involved in bacterial killing during this process. In vitro research confirms its efficacy against essentially all clinically relevant bacteria. The recent advent of commercial production of HOCl, delivered as a fog, has resulted in extensive use in the food industry. Reported lack of corrosion and high anti-bacterial potency are seen as two key factors for the use of HOCl in the orthopaedic environment. Prior work by the authors comparing human cell toxicity of HOCl, chlorhexidine and iodine solutions shows favourable results. This study evaluates use of neutral HOCl applied as a dry room fog to decrease bacteria in the operating theatre environment. Using an animal operating theatre as the test site, bacterial swabs were taken from ten 100cm. 2. sample areas before standard cleaning with detergent, after standard cleaning, and again after 60 minutes exposure to HOCl fog. After standard cleaning, 6 of 10 sample sites recorded significant bacterial growth (>10 CFU/100cm. 2. ). After exposure to HOCl fog, growth in all 10 sites was below detection limits (<10 CFU/100cm. 2. ). This was repeated with specific exposure to Staphylococcus aureus and Escherichia coli. We can conclude that HOCl is effective when used as a fogging agent to reduce bacterial loading within an operating theatre environment and as such has significant potential to reduce intraoperative contamination and periprosthetic infection

Aim. Currently, gram-negative bacteria (GNB), including multidrug-resistant (MDR-GNB) pathogens, are gaining importance in the aetiology of prosthetic joint infection (PJI). To characterize the antimicrobial resistance patterns of Gram-negative bacteria (GNB) causing hip prosthetic joint infections in elderly patients treated at a Brazilian tertiary academic hospital. Method. This is a retrospective, cross-sectional study of patients over 60 years of age undergoing hip arthroplasty from 2018 to 2023 at a tertiary academic trauma, which were diagnosed with hip prosthetic joint infection. PJI diagnosed was based on EBJIS criteria, in which intraoperative tissue cultures identified the pathogens. Demographics, reason for arthroplasty, type of implant and susceptibility patterns using disk diffusion method were analysed. Results. Overall, among 17 elderly patients diagnosed with hip infected arthroplasty, 45 bacterial isolated were identified. Debridement, irrigation, antibiotic and implant retention (DAIR) procedures due to uncontrolled infection occurred in 47.0% (n=8/17), and five patients underwent more than two DAIR surgeries. Tissue cultures yielded eleven different bacterial species, with GNB accounted for 64.4% (n=29/45) of pathogens. Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa were identified in 34.5% (n=10/29), 17.25% (n=5/29), 13.8% (n=4/29), and 13.8% (n=4/29), respectively. In the resistance profile analysis, E. coli was most sensitive to antibiotics, whereas K. pneumoniae showed resistance rates higher than 70% for cephalosporins, carbapenems, and quinolones. All A. baumannii isolates were resistant to meropenem, and 80% of these isolates were resistant to amikacin. Conclusions. This study emphasizes the role of GNB in the microbiological profile of PJI among elderly patients at a tertiary hospital in a Brazilian centre. The present study portrays a worryingly higher rates of MDR-GNB, mainly to quinolones and cephalosporins resistance which have been the cornerstone of PJI antibiotic treatment. In addition, higher rates carbapenems and aminoglycosides resistance shows a threat to antibiotic treatment of PJI. More global studies need to be carried out to show a likely change in the microbial epidemiology of PJI

Aim. Prosthetic joint infections pose a major clinical challenge. Developing novel material surface technologies for orthopedic implants that prevent bacterial adhesion and biofilm formation is essential. Antimicrobial coatings applicable to articulating implant surfaces are limited, due to the articulation mechanics inducing wear, coating degradation, and toxic particle release. Noble metals are known for their antimicrobial activity and high mechanical strength and could be a viable coating alternative for orthopaedic implants [1]. In this study, the potential of thin platinum-based metal alloy coatings was developed, characterized, and tested on cytotoxicity and antibacterial properties. Method. Three platinum-based metal alloy coatings were sputter-coated on medical-grade polished titanium discs. The coatings were characterized using optical topography and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Ion release was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). Cytotoxicity was tested according to ISO10993-5 using mouse fibroblasts (cell lines L929 and 3T3). Antibacterial surface activity, bacterial adhesion, bacterial proliferation, and biofilm formation were tested with gram-positive Staphylococcus aureus ATCC 25923 and gram-negative Escherichia coli ATCC 25922. Colony forming unit (CFU) counts, live-dead fluorescence staining, and SEM-EDS images were used to assess antibacterial activity. Results. Three different platinum-based metal alloys consisting of platinum-iridium, platinum-copper, and platinum-zirconium. The coatings were found 80 nm thick, smooth (roughness average < 60 nm), and non-toxic. The platinum-copper coating showed a CFU reduction larger than one logarithm in adherent bacteria compared to uncoated titanium. The other coatings showed a smaller reduction. This data was confirmed by SEM and live-dead fluorescence images, and accordingly, ICP-OES measurements showed low levels of metal ion release from the coatings. Conclusions. The platinum-copper coating showed low anti-adhesion properties, even with extremely low metal ions released. These platinum-based metal alloy coatings cannot be classified as antimicrobial yet. Further optimization of the coating composition to induce a higher ion release based on the galvanic principle is required and copper looks most promising as the antimicrobial compound of choice. Acknowledgments. This publication is supported by the DARTBAC project (with project number NWA.1292.19.354) of the research program NWA-ORC which is (partly) financed by the Dutch Research Council (NWO); and the AMBITION project (with project number NSP20–1-302), co-funded by the PPP Allowance made available by Health-Holland, Top Sector Life Sciences & Health to ReumaNederland

Aim. Antibacterial activity of coatings based on metal and metal oxide nanoparticles (NPs) often depends on materials and biotic targets resulting in a material-specific killing activity of selected Gram-positive and Gram-negative bacteria, including drug-resistant strains. In this perspective, the NPs loading amount, the relative elemental concentration inside the nanogranular building blocks and the deposition method are of paramount importance when the goal is to widen the antimicrobial spectrum, but at the same time to avoid high levels of metal content to limit undesired toxic effects. Aim of the present study was evaluation of the antimicrobial properties of two multielement nanogranular coatings composed of Titanium-Silver and Copper and of Magnesium-Silver and Copper. Method. Ti-Ag-Cu and Mg-Ag-Cu NPs were deposited on circular cover glasses (VWR) by Supersonic Cluster Beam Deposition. Biofilm-producer strains of Staphylococcus aureus (methicillin susceptible and resistant), Staphylococcus epidermidis (methicillin susceptible and resistant), Escherichia coli (fully susceptible and producer of extended spectrum beta lactamases), and Pseudomonas aeruginosa (susceptible and multidrug-resistant) were selected. The abilities of the selected strains to adhere, colonize and produce biofilm on the discs coated with Ti-Ag-Cu or Mg-Ag-Cu NPs were compared to uncoated circular cover glasses which were used as growth control. Cytotoxicity was also evaluated in order to assess the biocompatibility of the newly synthesized NPs. Results. In comparison to uncoated controls, both coatings showed significant anti-adhesive properties against S. aureus, S. epidermidis, and E. coli. Reduction in adhesion to Mg-Ag-Cu coated discs was observed also for P. aeruginosa isolates, although differences vs uncoated controls did not reach statistical significance. Biofilm formation was reduced on discs coated with Mg-Ag-Cu compared to Ti-Ag-Cu and, again, coatings had a milder effect on P. aeruginosa, probably due to its exceptional capability of attachment and matrix production. These results were confirmed by the evaluation of bacterial colonization on nanoparticles-coated discs by means of confocal laser scanning microscopy. A viability of 95.8% and 89.4% of cells cultured in the presence of Ti-Ag-Cu and Mg-Ag-Cu discs, respectively, when compared to negative controls was observed, thus excluding cytotoxic effects on eukaryotic cells. Conclusions. The newly synthesized Ti-Ag-Cu and Mg-Ag-Cu coatings are able to limit bacterial adhesion colonization and biofilm production, thus highlighting the safe use of multi-element families of NPs as new strategies against bacterial attachment to the surface of biomedical implants. However, further studies addressing activity against P. aeruginosa and including a wide number of isolates are warranted

Prosthetic joint infections represent complications connected to the implantation of biomedical devices. Bacterial biofilm is one of the main issues causing infections from contaminated orthopaedic prostheses. Biofilm is a structured community of microbial cells that are firmly attached to a surface and have unique metabolic and physiological attributes that induce improved resistance to environmental stresses including toxic compounds like antimicrobial molecules (e.g. antibiotics). Therefore, there is increasing need to develop methods/treatments exerting antibacterial activities not only against planktonic (suspended) cells but also against adherent cells of pathogenic microorganisms forming biofilms. In this context, metal-based coatings with antibacterial activities have been widely investigated and used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing the biofilm formation prevention efficacy. Additionally, standardized and systematic approaches to test antibacterial activity of newly developed coatings are still missing, while standard microbiological tests (e.g. soft-agar assays) are typically used that are limited in terms of simultaneous conditions that can be tested, potentially leading to scarce reproducibility and reliability of the results. In this work, we combined the Calgary Biofilm Device (CBD) as a device for high-throughput screening, together with a novel plasma-assisted technique named Ionized Jet Deposition (IJD), to generate and test new generation of nanostructured silver- and zinc-based films as coatings for biomedical devices with antibacterial and antibiofilm properties. During the experiments we tested both planktonic and biofilm growth of four bacterial strains, two gram-positive and two gram-negative bacterial strains, i.e. Staphylococcus aureus ATCC 6538P, Enterococcus faecalis DP1122 and Escherichia coli ATCC 8739 and Pseudomonas aeruginosa PAO1, respectively. The use of CBD that had the only wells covered with the metal coatings while the biofilm supports (pegs) were not sheltered allowed to selectively define the toxic effect of the metal release (from the coating) against biofilm development in addition to the toxic activity exerted by contact killing mechanism (on biofilms formed on the coating). The results indicated that the antibacterial and antibiofilm effects of the metal coatings was at least partly gram staining dependent. Indeed, Gram negative bacterial strains showed high sensitivity toward silver in both planktonic growth and biofilm formation, whereas zinc coatings provided a significant inhibitory activity against Gram positive bacterial strains. Furthermore, the coatings showed the maximal activity against biofilms directly forming on them, although, Zn coating showed a strong effect against biofilms of gram-positive bacteria also formed on uncoated pegs. We conclude that the metal-based coatings newly developed and screened in this work are efficient against bacterial growth and adherence opening possible future applications for orthopedic protheses manufacturing

Objective. To assess the beneficial use of polypropylene mesh impregnated with vancomycin in an experimental model open fractures Gustilo IIIa in rabbits. Material and Method. We worked with 15 New Zeland White rabbits. All of them were carried out under general anaesthetic, a 5-cm incision longitudinal was made at the back of the right thigh. The femur was aproached and a fracture was performed with a shear, giving rise to a multifragment fracture. The wound remained open for 6 hours with the bone exposed, in a non-surgical ambient. Subsequently underwent surgical cleaning of the open fractures in two stages. The fracture was stabilized with an intramedular pin. The animals were sorted in 3 different therapeutic groups:. Group 1: (5 rabbits) without other treatment. Group 2: (5 rabbits) a polypropylene mesh was placed around the fracture. Group 3: (5 rabbits) a polypropylene mesh with vancomycin was placed around the fracture. The wound was closed with nylon stiches. Three weeks postoperative, the animals were intervened surgically under general anesthesia, after aseptic cure and placement of surgical fields, femoral bone biopsies, soft tissue and mesh were taken. The rabbits were sacrified. The samples were sent to pathology and bacteriology labs. Results. The bacteria isolated were as follows: Escherichia coli, Pasteurella multocida, Staphylococcus spp., Clostridium spp. Mamheinia spp. The Clostridium spp. is a common contaminant in the exposed fractures present in the environment. The Pasteurella mustocida is a microorganism present in the oral cavity of rabbits, as well as Escherichia coli is a germ present in the animal's digestive tract. Mannheimia spp. It is a beta-hemolytic organism, found in the nasal flora of these animals and their pathological role is not elucidated. Staphylococcus spp. is a germ that is found in the normal flora of the animals skin. Group 1 showed a relative risk for an infection. For Group 2 the relative risk was substantially greater than 1.4, while in Group 3, the relative risk was 0.6, significantly lower than the previous two groups. The results have shown a beneficial effect of the use of impregnated polypropylene mesh with vancomycin in this animal group. Conclusion. The use of polypropylene meshes with vancomycin could be useful in the treatment of muscle and ligamentary deficits in patients with open fractures Gustillo IIIa

Objectives. There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. Methods. BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method . was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). Results. Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. . Conclusions. Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine

Aim. The efficacy of various irrigation solutions in removing microbial contamination of a surgical wound and reducing the rate of subsequent surgical site infection (SSI), has been demonstrated extensively. However, it is not known if irrigation solutions have any activity against established biofilm. This issue is pertinent as successful management of patients with periprosthetic joint infection (PJI) includes the ability to remove biofilm established on the surface of implants and necrotic tissues. The purpose of this study was to evaluate the efficacy of various irrigation solutions in eradicating established biofilm, as opposed to planktonic bacteria, in a validated in vitro model. Method. Established biofilms of Staphylococcus aureus and Escherichia coli were exposed to different irrigation solutions that included Polymyxin 500,000U/L plus bacitracin 50,000U/L, Vancomycin 1g/L, Gentamicin 80mg/L, Normal saline 0.9%, off-the-shelf Betadine 0.3%, Chlorhexidine 0.05%, Benzalkonium 1.3g/L, Sodium hypochlorite 0.125%, and Povidone-iodine 0.5%. Each experiment was conducted in a 96-well microtiter plate with a peg lid and standardized per the MBEC assay manufacturer's protocol. Following 2 minutes of solution exposure to the irrigation solution, residual biofilms were recovered by sonication. Outcome measures for antibiofilm efficacy were residual colony forming units (CFU) and optical density (690nm). Experiments were conducted in 24 replicates and the observations recorded by two blinded observers. Statistical analysis involved t-tests with Bonferonni adjustment. Results. Povidone-iodine 0.5%, Betadine 0.3%, Benzalkonium 1.3g/L, and Sodium hypochlorite 0.125% were significantly more efficacious against S.aureus biofilm versus all other solutions (p<0.001). Against E.coli biofilm, Povidone-iodine-0.5%, Benzalkonium-1.3g/L and Sodium hypochlorite-0.125% were also most effective compared to other irrigation solutions (p<0.001). Polymyxin-bacitracin, Gentamicin, Vancomycin, and Saline solutions had minimal activity against both E.coli and S.aureus biofilms (p<0.001). Similar trends were observed using both experimental endpoints (CFU and Turbidity) and both investigators (interrater reliability; r=0.99). Conclusion. This in vitro study observed that topical antibiotic solutions do not have any activity against established biofilms. Irrigations solutions containing adequate amount of povidone-iodine, betadine, sodium hypochlorite, and benzalkonium appear to have activity against established biofilm by gram positive and gram negative organisms. The use of these irrigation solutions may need to be considered in patients with established PJI

Aim. Bone and joint infections are frequent in African countries and their prevention and treatment remain a great challenge. This study aimed to determine the bacterial ecology and sensitivity of isolates to locally available antibiotics in orthopedic unit of a tertiary care hospital in Cameroun. Method. During a 12 months period, all the patients presenting with osteomyelitis or septic arthritis irrespective of the mechanism and the location were enrolled in this study. Intraoperative samples (biopsies) were taken and sent for microbiological analysis, and all strains isolated were tested for antibiotic sensitivity according to conventional methods. Results. on the 52 bacteriological analysis performed, 48 were positive. The most isolated germs were staphylococcus aureus (41.9 % of isolates), pseudomonas aeruginosa (14.5 %), Escherichia coli (14.5 %) and Klebsiella pneumonia (12.9 %). The antibiotic sensitivity pattern revealed worrying resistance rates for common and affordable antibiotics: ampicillin (94 %), amoxicillin + clavulanic acid (63.9 %), ceftazidim (65.5%), ticarcillin + clavulanate (57.4%), gentamycin (49 %), ciprofloxacin (40 %), cefuroxim (40 %), tobramycin (38.5 %). The strains of Staphylococcus aureus showed resistance to penicillin G (83%), oxacillin (25%), lincomycin (27%) and vancomycin (7%). The overall highest sensitivity rates were observed with amikacin (92 %) and imipenem (90.1%), which for many patients were the only effective locally available antibiotics. The daily cost of treatment with those two antibiotics is close to the guaranteed minimum wage in our country. Conclusions. The alarming rate of multidrug-resistant bacteria makes the long antibiotic treatment of bone infections unaffordable (in a context of lack of social insurance) for most of our patients. We advocate strong national policies for bacteriological surveillance and antibiotic misuse de-escalation to prevent antibiotic resistance

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

Protocols for processing of tissue from arthroplasty infections vary and might affect the recovery of bacteria. We compared homogenization, bead beating and enzymatic disruption for recovery of live bacteria from tissue samples. Suspensions of Staphylococcus aureus and Escherichia coli were prepared as controls. Three samples were taken from each and the first was bead beaten, the second homogenized, and Proteinase K was added for 10 and 30 minutes to the third sample before culturing. In addition, artificially inoculated pork tissue and known infected human tissue samples were processed by either homogenization or bead beating prior to cultures and results were compared. Number of cycles of bead beating and homogenization and duration of Proteinase K treatment had significant effects. Bead beating for 2 and 4 cycles reduced the yield of S.aureus to 52% and 20% of control, and E.coli to 33% and 8%. Homogenization for 2 and 4 cycles reduced S.aureus to 86% and 65% of control, and E.coli to 90% and 87%. Proteinase K for 10 minutes and 30 minutes reduced the yield of S.aureus to 75% and 33% of control, and E.coli to 91% and 49% respectively. Inoculated Pork tissue showed a reduction in S.aureus recovery of 90% for bead beating compared to homogenization, and 80% in the case of E.coli. Bead beating of infected human tissue samples reduced the yield by 58% compared to homogenization. Bead-beating is a common recommended method of processing tissue from arthroplasty cases. However, even though it produces a homogeneous sample, it does so at the cost of significant loss of viable bacteria. Homogenization and 10 minutes of Proteinase K incubation are almost equivalent, but the homogenizer is preferred being more controllable and cheaper. This should help to define guidelines for diagnosing infections using tissue samples

Aim. Phage therapy has attracted attention as a promising alternative treatment option for biofilm infections. To establish a successful phage therapy, a comprehensive stock of different phages covering a broad bacterial spectrum is crucial. We screened human and environmental sources for presence of lytic phages against selected bacteria. Methods. Saliva collected from 10 volunteers and 500 ml of sewage water were screened for the presence of lytic phages active against 20 clinical strains of Staphylococcus aureus and 10 of Escherichia coli, both isolated from patients with prosthetic joint infection. Laboratory strains of methicillin-resistant S. aureus (MRSA)*1 and E. coli*2 were also tested. Screening was performed plaque-assay to detect phages for different strains. Isolated plaques were collected and phages were enriched to determine their activity against their bacterial host strains. The activity of bacteriophages against adherent E. coli and MRSA was evaluated by crystal violet, staining bacterial biofilms grown on glass beads. Results. Six bacteriophages specific for MRSA were isolated from saliva. Bacteriophages for E. coli strains were isolated from sewage water (n=3) and saliva (n=1). All bacteriophages tested against biofilms of their bacterial host showed a reduction of the total biomass (ranging from 19% to 84%). Conclusions. Both sewage and saliva samples provided bacteriophages specific against selected bacterial strains. 24h phage treatment of E. coli and S. aureus biofilms lead to a reduction but not to a complete eradication of biofilm

Aim. Prosthetic joint infections (PJI) due to Enterobacter cloacae are rare and often severe. The aim of this study is to describe cases with E. cloacae PJI. Method. We conducted a retrospective and a monocentric study in an orthopedic unit where complex bone and joint infections are managed. From 2012 to 2016, we included patients with PJI which perioperative samples were positive with E. cloacae. We collected background, clinical, biological and microbiological data of the current infection, surgical and medical treatment, and the outcome of these patients. Results. A total of twenty patients were included which 8 were male. Location was hip in 14 cases, knee in 5 cases and ankle in one case. The median time between arthroplasty and revision for infection was 3 years. Fourteen patients had at least two surgeries for previous PJI. The median time between the last surgery and the revision for E. cloacae infection was 31 days. Eleven patients were infected by extended-spectrum beta-lactamases (ESBL) strains. Most frequently, the antibiotics used were carbapenem in 9 cases, cefepim in 7 cases, a quinolone in 7 cases and fosfomycin in 4 cases. Infection was cured in 10 cases (50%) with a median time of follow-up of 24 months. Five patients had a recurrent infection, three due to Staphylococcus epidermidis, one to Staphylococcus epidermidis and Propionibacterium acnes and one to Escherichia coli. Four patients had a relapse of E. cloacae infection. One patient died from non-infectious cause (stroke). Conclusions. PJI infections due to E.cloacae usually occur early after the last prosthetic surgery, typically in patients with complex surgical history. A poor outcome, observed in nearly half of the patients could be explained in part by an association of factors: multiple risks factors, complex infectious history, a high rate of multiple resistance to antibiotics, unfavorable skin conditions