Objectives. This study aims to investigate whether bacteria are present in intervertebral discs (IVDs) and their influence. Causality between chronic infection of the IVD and its degenerative process gained great interest recently. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in IVDs, from these 27 studies found, Cutibacterium acnes being the most abundant. However, whether bacteria identified were present in vivo or if they represent contamination remains unclear. Methods. Human IVD tissue was fixed in paraffin and Immunohistochemical stained for Gram-positive bacteria. NP cells in monolayer have been stimulated with LPS (0.1–50 µg/ml) and Peptidoglycan (0.1–50 µg/ml) for 24, 48 and 72 hrs to investigate their influence. The concentration of proinflammatory and catabolic cytokines in the media is being measured using ELISA. RNA extracted and RT-qPCR utilised for factors associated with disc degeneration matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors. Results. Bacteria were detected within IVD tissue. Bacteria was internalized by the NP cells and influenced the nuclei morphology. Preliminary results of the exposure of NP cells to bacterial components demonstrate that ADAMTS4 as well as IL-8 were showed an increase in gene expression after LPS and peptidoglycan treatment compared to the non-treated control. Underlining these results, IL-8 protein was increased in treated groups, whereas peptidoglycan treated groups showed a dose dependence. Conclusion. This study demonstrates that Gram positive bacteria are present within the IVD. The exposure of NP cells to peptidoglycans indicates that bacterial components trigger a stress response. Conflicts of Interest: No conflict of interest. Sources of Funding: This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Introduction. Multiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D. Methods. Immunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation. Results. Immunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration. Conclusion. In conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells. No conflicts of interest.  . Sources of funding. This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Cutibacterium acnes was the most abundant, followed by coagulase-negative staphylococcus. However, whether bacteria identified were present in vivo or represent perioperative contamination remains unclear. This study investigated whether bacteria are present in IVDs and what potential effects they may have on native disc cells. Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysis. Gram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats. This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration

The Global Burden of Disease Study 2019 showed a 33.4% increase in fractures and a 65.3% increase in Years lived with disability (YLD) since 1990. Although the overall rate of fracture related infection (FRI) is low, it increases to 30% in complex fractures. In addition, the implantation of foreign materials, such as fracture stabilizing implants, decreases the number of bacteria needed to cause an infection. Then, when infections do occur, they are difficult to treat and often require multiple surgeries to heal. The bacteria can persist in the canaliculi of the bony tissue, in cells, in a biofilm on material or necrotic bone or in abscess communities. In the last decades, different approaches have been pursued to modify biomaterials as well as implant surface and to develop antimicrobial surfaces or local drug release strategies. This talk will give an introduction to the problem of bony and implant associated infections and presents the development and preclinical (as well as clinical) studies of two approaches for local drug delivery

Aim. Antimicrobial resistance (AMR) aggravates an already difficult treatment of periprosthetic joint infections (PJI). The prevalence of drug-resistant pathogens varies across countries and increases over time. Regular monitoring of bacteriological analyses should be performed. Due to many factors influencing the AMR, the correct choice of antimicrobial management remains arguable. The primary purpose of this retrospective study was to identify and compare causative bacteria and to compare the incidence of antibiotic resistance between the septic revision total knee arthroplasty (TKA) and septic revision total hip arthroplasty (THA). Method. A review of all revision TKAs and revision THAs, undertaken between 2007 and 2020 in a tertiary referral centre, was performed. Included were cases meeting the consensus criteria for PJI, in which an organism has been identified. There were no major differences in tissue sampling between revision TKAs and revision THAs over time. Results. A total of 228 bacterial strains, isolated after revision TKA and THA, were analysed for their resistance to 20 different antibiotics. There was a statistically significant higher occurrence of Gram-negative bacteria (p=0.002) and Enterococcus species (p=0.026) identified after revision THAs compared to TKA. The comparison of antibiotic resistance between revision TKAs and revision THAs was statistically significant in 9 of 20 analysed antibiotics. Pathogens isolated after revision THA were much more resistant compared to pathogens isolated after revision TKA. Resistance in revision THAs was significantly higher to oxacillin (p=0.03), ciprofloxacin (p<0.001), levofloxacin (p<0.001), moxifloxacin (p=0.005), clindamycin (p<0.001), co-trimoxazole (p<0.001), imipenem (p=0.01), rifampicin (p=0.005) and tetracycline (p=0.009). There was no significantly higher resistance of pathogens isolated after revision TKAs detected. No statistically significant difference in antibiotic resistance of Gram-negative bacteria between revision TKA and revision THA was observed. Conclusions. The occurrence and the resistance of bacteria to antibiotics differs significantly between revision TKAs and revision THAs. This has implications on of the choice of empirical antibiotic in revision surgery as well as prophylactic antibiotic in primary surgery, depending on the joint that is to be replaced

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently 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 their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

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. Methods. On polystyrene, a mature (7 day-old) methicillin-resistant Staphylococcus aureus (MRSA) biofilm was developed. Thereafter, bacteria in the biofilm were exposed to rifampicin and ciprofloxacin (both 10× >MIC) for three days. Surviving bacteria in the antibiotic-treated biofilm, presumed to include persisters, were exposed to increasing doses of the antimicrobial peptides SAAP-148, acyldepsipeptide 4 (ADEP4), LL-37 and pexiganan. SAAP-148 was further tested on antibiotic-treated mature biofilms on titanium/aluminium/niobium (TAN) discs and prosthetic joint liners. Results. Daily exposure of the mature biofilm for seven days with antibiotics resulted in a 4-log reduction of MRSA without elimination of the bacteria. The surviving bacteria within the biofilm were eliminated upon subsequent exposure to SAAP-148 and pexiganan but not with LL-37 ad ADEP4. Antibiotic treatment of mature biofilms on TAN discs followed by SAAP-148 also resulted in eradication of bacteria within the biofilm. SAAP-148 also fully eliminated bacteria within antibiotic-treated mature MRSA biofilms on an ex vivo liner of a prosthetic joint. Conclusions. A novel mature biofilm model has been developed in which the efficacy of antimicrobial peptides against bacteria, including persisters, residing within a biofilm was investigated. SAAP-148 and pexiganan were highly effective against the bacteria residing in antibiotic-exposed mature MRSA biofilms. This in vitro model system will be used to analyze the effects of novel antibiotic strategies and other anti-PJI agents

Aim. Septic arthritis (SA) is considered a medical emergency. The most common etiological agents are glucose consuming bacteria, so we evaluated the clinical utility of synovial fluid (SF) glucose levels and other biochemical parameters for supporting the diagnosis of the disease and their association with a positive bacteria culture and joint destruction. Methods. Adult patients with SA diagnose were enrolled prospectively between July 2018 and October 2019. As control group, adults with knee osteoarthritis, meniscus and/or knee ligaments lesions were enrolled. SF samples were obtained from the joints by arthrocentesis/arthrotomy. Microbiological analyses of SF were performed using Brucella broth blood culture flasks, samples were incubated at 37°C with 5% CO. 2. for 24 hours. Gram stain, chocolate and blood agar were used for the identification and growth of the bacteria. SF glucose levels, pH and leukocyte esterase were measured as biochemical parameters using a glucometer and colorimetric test strips. The Outerbridge classification was used for grading the osteochondral injury. Furthermore, blood samples were collected from patients and control subjects for determining glucose levels. Results. We included 8 subjects with knee ligaments lesions, 6 with meniscus lesions and 5 with osteoarthritis as control group, as well as 20 patients with SA diagnose. The mean age of the patients was 57.8 years with a 65% of male predominance. The most common affected joint was the knee (85%). SF culture was positive in 60% of the cases and the most common etiological agent was Staphylococcus aureus (58.3%). SF glucose levels from patients were lower than the controls (P=0.0018) and showed the lowest concentration in patients with a positive culture (P=0.0004). There was also a difference between blood and SF glucose concentration from the positive culture patients (P<0.0001). Leucocyte esterase presented the highest values in positive culture patients (P=<0.0001) and a more acidic pH was found compared to the control group (P<0.0001). Regarding the osteochondral injury, the lowest concentrations of SF glucose were found in patients with a higher grade in the classification (P = 0.0046). Conclusions. SF glucose and leukocyte esterase concentrations might be a quick and cheap useful parameter for the physician for distinguishing between bacterial infection and not infected joint. In addition, the lowest SF glucose levels might give information about the joint damage due to the disease

Aims. The aim of this study was to determine if the local delivery of vancomycin and tobramycin in primary total knee arthroplasty (TKA) can achieve intra-articular concentrations exceeding the minimum inhibitory concentration thresholds for bacteria causing acute prosthetic joint infection (PJI). Methods. Using a retrospective single-institution database of all primary TKAs performed between January 1 2014 and May 7 2019, we identified patients with acute PJI that were managed surgically within 90 days of the initial procedure. The organisms from positive cultures obtained at the time of revision were tested for susceptibility to gentamicin, tobramycin, and vancomycin. A prospective study was then performed to determine the intra-articular antibiotic concentration on postoperative day one after primary TKA using one of five local antibiotic delivery strategies with tobramycin and/or vancomycin mixed into the polymethylmethacrylate (PMMA) or vancomycin powder. Results. A total of 19 patients with acute PJI after TKA were identified and 29 unique bacterial isolates were recovered. The mean time to revision was 37 days (6 to 84). Nine isolates (31%) were resistant to gentamicin, ten (34%) were resistant to tobramycin, and seven (24%) were resistant to vancomycin. Excluding one Fusobacterium nucleatum, which was resistant to all three antibiotics, all isolates resistant to tobramycin or gentamicin were susceptible to vancomycin and vice versa. Overall, 2.4 g of tobramycin hand-mixed into 80 g of PMMA and 1 g of intra-articular vancomycin powder consistently achieved concentrations above the minimum inhibitory concentrations of susceptible organisms. Conclusion. One-third of bacteria causing acute PJI after primary TKA were resistant to the aminoglycosides commonly mixed into PMMA, and one-quarter were resistant to vancomycin. With one exception, all bacteria resistant to tobramycin were susceptible to vancomycin and vice versa. Based on these results, the optimal cover for organisms causing most cases of acute PJI after TKA can be achieved with a combination of tobramycin mixed in antibiotic cement, and vancomycin powder. Cite this article: Bone Joint J 2020;102-B(6 Supple A):163–169

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

Aims. To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. Methods. EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt Staphylococcus aureus biofilms on Matrigel-coated glass and two materials widely used in orthopaedic implants (Ti-6Al-4V and highly cross-linked polyethylene (HXLPE)). To assess the efficacy of biofilm dispersion, crystal violet staining biofilm assay and colony counting after sonification and culturing were performed. The results were further confirmed and visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). We then investigated the efficacies of EDTA-NS irrigation in vivo in rat and pig models of biofilm-associated infection. Results. When 10 mM or higher EDTA-NS concentrations were used for ten minutes, over 99% of S. aureus biofilm formed on all three types of materials was eradicated in terms of absorbance measured at 595 nm and colony-forming units (CFUs) after culturing. Consistently, SEM and CSLM scanning demonstrated that less adherence of S. aureus could be observed on all three types of materials after 10 mM EDTA-NS irrigation for ten minutes. In the rat model, compared with NS irrigation combined with rifampin (Ti-6Al-4V wire-implanted rats: 60% bacteria survived; HXLPE particle-implanted rats: 63.3% bacteria survived), EDTA-NS irrigation combined with rifampin produced the highest removal rate (Ti-6Al-4V wire-implanted rats: 3.33% bacteria survived; HXLPE particle-implanted rats: 6.67% bacteria survived). In the pig model, compared with NS irrigation combined with rifampin (Ti-6Al-4V plates: 75% bacteria survived; HXLPE bearings: 87.5% bacteria survived), we observed a similar level of biofilm disruption on Ti-6Al-4V plates (25% bacteria survived) and HXLPE bearings (37.5% bacteria survived) after EDTA-NS irrigation combined with rifampin. The in vivo study revealed that the biomass of S. aureus biofilm was significantly reduced when treated with rifampin following irrigation and debridement, as indicated by both the biofilm bacterial burden and crystal violet staining. EDTA-NS irrigation (10 mM/10 min) combined with rifampin effectively removes S. aureus biofilm-associated infections both in vitro and in vivo. Conclusion. EDTA-NS irrigation with or without antibiotics is effective in eradicating S. aureus biofilm-associated infection both ex and in vivo. Cite this article: Bone Joint Res 2024;13(1):40–51

Title. Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model. Aim. To assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model. Method. All animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 10. 5. CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours. Results. 1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the mouse femur. Proper placement of all implants were confirmed with in vivo X-rays, and ex vivo photos. We empirically derive the ROI during each imaging session by aggregating the imaged volume which ranges from (636.4um × 636.4um × 151um) = 0.625 +/- 0.014 mm. 3. of bone marrow in a global GFP-transgenic mouse. 2) IV-MLSM imaging acquisition of the “race for the surface”.In vitro MPLSM images of implants partially coated with USA300LAC (RFP-MRSA) were verified by SEM image. Results from IV-MLSM of RFP-MRSA and GFP. +. host cell colonization of the contaminated implants illustrated the mutually exclusive surface coating at 3hrs, which to our knowledge is the first demonstration of “the race for the surface” between bacteria and host cells via intravital microscopy. 3) Quantifying the “race for the surface” with CFU verification of S. aureus on the implant. 3D volumetric rendering of the GFP. +. voxels and RFP+ voxels within the ROI were generated in Imaris. The voxel numbers suggeste that the fight for the surface concludes ∼3hrs post-infection, and then transitions to an aggressive MRSA proliferation phase. The results of WT control demonstrate a significant increase in CFU by 12hrs post-op for both stainless steel (P<0.01) and titanium (P<0.01). Conclusions. We developed IV-MLSM to quantify the “Race for the Surface” between host cells and contaminating S. aureus in a murine femur implant model. This race is remarkably fast, as the implant surface is completely covered with 3hrs, peak bacterial growth on the implant occurs between 2 and 12 hours and is complete by 12hrs

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

Aim. Here we describe a cohort study to determine the performance of a commercially available Fluorescence In Situ Hybridization (FISH)-kit on samples of 65 consecutive patients suspected of orthopedic implant associated infections (IAI). Culture is routinely used and has a high specificity and sensitivity but requires days to more than a week for slow growing bacteria. FISH results are available within 45–60 minutes and thus specific treatment can start immediately. In addition, previous antibiotic therapy may hinder culture while bacteria may still be detected by FISH. Method. The hemoFISH-kit from Miacom diagnostics (Dusseldorf, Germany) was used on a total of 82 joint aspirates, sonication fluids and tissue samples of 65 consecutive patients to detect and identify possible microorganisms. This FISH-kit contains a universal 16S rRNA probe and species-specific probes for bacteria commonly encountered in blood infections. FISH and culture were compared to the clinical definition of IAI. These definitions were based on the criteria described by Pro-Implant Foundation criteria for IAI after fracture fixation or prosthetic joint infection. If no criteria were described in the literature for a specific IAI then MSIS criteria were used. Results. FISH and culture was done in 33 plain tissue samples, 43 sonication fluid samples and 6 joint aspirates of 65 patients. Results are shown in table 1. In clinical infections FISH provided earlier results in 7 and 2 extra for culture-negative. In 5 IAI-negative cases FISH was false-positive. Conclusions. Faster diagnosis by FISH is appealing, however with a PPV of 64% the hemoFISH-kit is not accurate enough for clinical use. Also, blood and orthopedic infections have different common pathogens, therefor FISH could not identify all of the bacterial strains due to a lack of specific probes. An orthopedic FISH-kit could solve this problem

Aim. The risk of haematogenic periprosthetic joint infection (PJI) after dental procedures is discussed controversially. To our knowledge, no study has evaluated infections according to the origin of infection based on the natural habitat of the bacteria. We investigated the frequency of positive monomicrobial cultures involving bacteria from oral cavity in patients with suspected PJI compared to bone and joint infections without joint prosthesis. Method. In this retrospective study we included all patients with suspected PJI or bone and joint infection without endoprosthesis, hospitalized at our orthopaedic clinic from January 2009 through March 2014. Excluded were patients with superficial surgical site infections or missing data. Demographic, clinical and microbiological data were collected using a standardized case report form. Groups were compared regarding infections caused by oral bacteria. χ2 test or Fisher's exact test was employed for categorical variables and t-test for continuous variables. Results. A total of 1673 patients were included, of whom 996 (60%) had a suspected PJI and 677 (40%) an osteoarticular infection without joint endoprosthesis (control group). In patients with suspected PJI the median age (standard deviation) was 67 (14) years; 407 (41%) were males. The anatomic location of the prosthesis was hip in 522 (52%) patients, knee in 437 (44%), megaprostheses in 14 (1%), shoulder in 8 (1%) and other endoprosthesis in 15 (2%) patients. In 437 (44%) of PJI cases pathogen(s) were detected, 271 (62%) were monomicrobial and 166 (38%) polymicrobial. Of 996 patients with suspected PJI, 2.4% (n = 24) had monomicrobial infections caused by bacteria belonging to the normal oral flora, predominantly oral streptococci (n = 21). In contrast, only 0.4% (n =3) of the control group without joint prosthesis had monomicrobial infections caused by oral bacteria. This difference was statistically significant (p = 0.002), whereas the patient age (p = 0.058) and the anatomic location of the joint prosthesis (p = 0.622) did not have any effect on the infections due to oral bacteria. Conclusions. The incidence of infections caused by oral bacteria was significantly higher in patients with endoprosthesis than in other osteoarticular infections (2.4% versus 0.4%). This finding indicates that joint prostheses are at risk of haematogenous PJI originating from oral cavity. Future prospective studies need to determine the exact risk of haematogenic PJI caused by oral bacteria, as well as the potential of preventing these infections by antibiotic prophylaxis

In the past decades, titanium-based biomaterials have been broadly used in maxillofacial and periodontology surgery. The main aetiological agents related to complications in this procedures are Porphyromonas gingivalis, a Gram-negative anaerobic bacteria that is also responsible for the development of chronic gingivitis, and Streptococcus oralis, a Gram positive facultative anaerobic bacteria. In previous studies, we have demonstrated that the fluorine doping of titanium-based alloys reduces bacterial adherence. The aim of this study is to evaluate the bacterial adherence on fluorine-doped titanium (TiF) probes compared to chemical polishing titanium (Ti) probes. The P. gingivalis ATCC 33277 and S. oralis ATCC 9811 adherence study was performed by introducing each probe in a well of 6-well plate with 5 ml containing 106 colony forming units (CFU/ml) in sterile 0.9% NaCl and was incubated 37°C 5% CO2 for 90 minutes, in anaerobiosis in the case of P. gingivalis. After incubation, samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The statistical data were analyzed by nonparametric Wilcoxon test with a level of statistical significance of 0.05. Our results showed a significant (p<0.0053) 14.41% decrease of the adherence of P. gingivalis on TiF and an increase of 30% of dead cells. For S. oralis we did not get significant results. In conclusion, TiF can be considered a promising approach to prevent and treat infections related to maxillofacial and periodontology surgery

The passage of bacteria through surgical drapes is a potential cause of wound infection. Previous studies have shown that liquids and human albumin penetrate certain types of drapes. 12. We studied the passage of bacteria through seven different types of surgical drape and an operating tray. We also studied the effect of different wetting agents on the passage of bacteria through wet reusable woven drapes. Bacteria were grown on an overfilled whole horse blood agar plate. The plate was covered with the drape to be tested and a second agar plate was inverted and placed on the drape. After 30 minutes the second agar plate was removed, incubated and inspected for bacterial growth. The experiment was repeated removing the second plate at 60 minutes and then again at 90 minutes. The entire experiment was repeated for each drape and then for each wetting agent. Bacteria easily penetrated all the woven reusable fabrics within 30 minutes. The disposable non-woven drapes proved to be impermeable up to 90 minutes, as did the operating tray. Chlorhexidine and Povidone-Iodine were demonstrated to slow, but not stop the passage of bacteria through reusable woven drapes. Normal saline and human blood accelerated the passage of bacteria through reusable woven drapes. We recommend the use of non-woven disposable drapes or woven drapes with an impermeable operating tray, in all surgical cases

Aims. Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella. Methods. For the haematogenous infection, G. mellonella larvae were implanted with a Kirschner wire (K-wire), infected with S. aureus, and subsequently phages and/or gentamicin were administered. For the early-stage biofilm implant infection, the K-wires were pre-incubated with S. aureus suspension before implantation. After 24 hours, the larvae received phages and/or gentamicin. In both models, the larvae also received daily doses of phages and/or gentamicin for up to five days. The effect was determined by survival analysis for five days and quantitative culture of bacteria after two days of repetitive doses. Results. In the haematogenous infection, a single combined dose of phages and gentamicin, and repetitive injections with gentamicin or in combination with phages, resulted in significantly improved survival rates. In the early-stage biofilm infection, only repetitive combined administration of phages and gentamicin led to a significantly increased survival. Additionally, a significant reduction in number of bacteria was observed in the larvae after receiving repetitive doses of phages and/or gentamicin in both infection models. Conclusion. Based on our results, a single dose of the combination of phages and gentamicin is sufficient to prevent a haematogenous S. aureus implant-related infection, whereas gentamicin needs to be administered daily for the same effect. To treat early-stage S. aureus implant-related infection, repetitive doses of the combination of phages and gentamicin are required. Cite this article: Bone Joint Res 2024;13(8):383–391

Aims. Arthroplasty surgery of the knee and hip is performed in two to three million patients annually. Periprosthetic joint infections occur in 4% of these patients. Debridement, antibiotics, and implant retention (DAIR) surgery aimed at cleaning the infected prosthesis often fails, subsequently requiring invasive revision of the complete prosthetic reconstruction. Infection-specific imaging may help to guide DAIR. In this study, we evaluated a bacteria-specific hybrid tracer (. 99m. Tc-UBI. 29-41. -Cy5) and its ability to visualize the bacterial load on femoral implants using clinical-grade image guidance methods. Methods. 99m. Tc-UBI. 29-41. -Cy5 specificity for Stapylococcus aureus was assessed in vitro using fluorescence confocal imaging. Topical administration was used to highlight the location of S. aureus cultured on femoral prostheses using fluorescence imaging and freehand single photon emission CT (fhSPECT) scans. Gamma counting and fhSPECT were used to quantify the bacterial load and monitor cleaning with chlorhexidine. Microbiological culturing helped to relate the imaging findings with the number of (remaining) bacteria. Results. Bacteria could be effectively stained in vitro and on prostheses, irrespective of the presence of biofilm. Infected prostheses revealed bacterial presence on the transition zone between the head and neck, and in the screw hole. Qualitative 2D fluorescence images could be complemented with quantitative 3D fhSPECT scans. Despite thorough chlorhexidine treatments, 28% to 44% of the signal remained present in the locations of the infection that were identified using imaging, which included 500 to 2,000 viable bacteria. Conclusion. The hybrid tracer . 99m. Tc-UBI. 29-41. -Cy5 allowed effective bacterial staining. Qualitative real-time fluorescence guidance could be effectively combined with nuclear imaging that enables quantitative monitoring of the effectiveness of cleaning strategies. Cite this article: Bone Joint Res 2023;12(1):72–79

Aims. Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models. Methods. Literature research was performed on PubMed and Embase databases. Keywords used for search criteria were “bone AND biofilm”. Information on the species of the animal model, bacterial strain, evaluation of biofilm and bone infection, complications, key findings on observations, prevention, and treatment of biofilm were extracted. Results. A total of 43 studies were included. Animal models used included fracture-related infections (ten studies), periprosthetic joint infections (five studies), spinal infections (three studies), other implant-associated infections, and osteomyelitis. The most common bacteria were Staphylococcus species. Biofilm was most often observed with scanning electron microscopy. The natural history of biofilm revealed that the process of bacteria attachment, proliferation, maturation, and dispersal would take 14 days. For systemic mono-antibiotic therapy, only two of six studies using vancomycin reported significant biofilm reduction, and none reported eradication. Ten studies showed that combined systemic and topical antibiotics are needed to achieve higher biofilm reduction or eradication, and the effect is decreased with delayed treatment. Overall, 13 studies showed promising therapeutic potential with surface coating and antibiotic loading techniques. Conclusion. Combined topical and systemic application of antimicrobial agents effectively reduces biofilm at early stages. Future studies with sustained release of antimicrobial and biofilm-dispersing agents tailored to specific pathogens are warranted to achieve biofilm eradication. Cite this article: Bone Joint Res 2022;11(10):700–714

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

Aims. Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). Methods. A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR. Results. The group with LIPUS and 0.35% PI exhibited decreased levels of serum biochemical markers, improved weightbearing scores, reduced reactive bone changes, absence of viable bacteria, and decreased inflammation compared to the Control group. Despite the greater antibiofilm activity observed in the PI group compared to the LIPUS and saline group, none of the monotherapies were successful in preventing reactive bone changes or eliminating the infection. Conclusion. In the rat model of PJI treated with DAIR, LIPUS combined with 0.35% PI demonstrated stronger antibiofilm potential than monotherapy, without impairing any local soft-tissue. Cite this article: Bone Joint Res 2024;13(7):332–341

Aims. Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy. Methods. The human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP. Results. The S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts. Conclusion. Intracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage. Cite this article: Bone Joint Res 2022;11(5):327–341

Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH). Concomitant samples were cultured by standard methods. Molecular methods detected presence of bacteria in samples from 12 of 22 patients. Using clone libraries a total of 40 different bacterial species were identified including known pathogens and species not previously described in association with prosthetic joint infections. The predominant species were Propionibacterium acnes and Staphylococcus epidermidis; polymicrobial infections were found in 9 patients. Culture-based methods showed bacterial growth in 8 cases with the predominant species being S. epidermidis. Neither anaerobic bacteria (including P. acnes) nor any of the species not previously described in implant infections were isolated. Additionally, 7 of the 8 culture positive cases were monomicrobial. Overall, the results of culture-based and molecular methods showed concordance in 11 cases (hereof 9 negative by both methods) and discrepancy in 6 cases. In the remaining 5 cases, culture-based methods identified only one species or a group of bacteria (e.g., coagulase negative staphylococci or coryneform rods), while culture-independent molecular methods were able to detect several distinct bacterial species including a species within the group identified by culture. A qPCR assay was developed to assess the abundance of Propionibacterium while S. aureus was quantified by a published S. aureus qPCR assay. These quantifications confirmed the findings from the clone library approach and showed the potential of qPCR for fast detection of bacteria in orthopedic samples. Additionally, both single cells and microcolonies were visualized using FISH and confocal scanning laser microscopy. In conclusion, the molecular methods detected a more diverse bacterial flora in prosthetic joint infections than revealed by standard culture-based methods, and polymicrobial infections were more frequently observed. The pathogenesis of these microorganisms and their role in implant-associated infections needs to be determined

Correct diagnosis of infection is crucial for an adequate treatment of orthopedic implant-related infections. In the orthopedic field, infections can be difficult to diagnose(1). As a consequence, patients may suffer from an undiagnosed and untreated implant-related infection. To solve this problem, we are searching for a diagnostic method to detect these so-called low-grade infections. The technique fluorescence in situ hybridization (FISH) can detect slow-growing and even dead bacteria. Further, as FISH results are available within an hour after tissue collection it is an ideal candidate for diagnostic purposes. AIM: to evaluate the FISH technique for its potential to detect and identify orthopedic infections. Sonication fluid (SF) was collected by sonicating retrieved implants(2) from 62 patients. All samples were subjected to bacterial culture for clinical diagnostics. In addition, a commercially available FISH kit (miacom diagnostics, Germany), specifically designed for blood analysis (hemoFISH Masterpanel), was used. The kit contained 16S rRNA probes (positive control), non-sense probes (negative control), probes for Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acetinobacter spp., and Stenotrophomonas maltophilia. All FISH analyses were performed according to the protocol provided with the kit. Culture and FISH results were compared, considering culture as the gold standard. Culture resulted in 27 positive and 35 negative samples. Comparing FISH (16S rRNA probe) with culture, 24 samples tested true-positive and 32 samples true-negative. Furthermore, 3 samples tested false-negative and 3 samples false-positive. The species cultured with the highest incidence were Propionibacterium acnes and Staphylococcus epidermidis, both from 8 SF samples. As the kit did not contain a probe for Propionibacterium acnes, these strains were only detected by the 16S rRNA probe. In addition, the latter samples tested positive with the Staphylococcus spp. probe. Interestingly, 3 samples tested positive with FISH that were culture negative. This result could indicate a higher sensitivity for detection of bacteria with FISH than with culture. Before FISH can be used for diagnostic purposes, the technique needs to be optimized to prevent false-negative results, for use on other patient materials and for detection of bacterial strains relevant for the orthopedic field like Propionibacterium acnes. In conclusion, FISH holds promise to be used as a diagnostic tool for identifying orthopedic infections

Implantation of antibiotic-loaded beads is accepted as an efficient option for local antibiotic therapy in orthopedic-related infections. However, recent reports have emphasized the bacteria growth persistence on antibiotic-impregnated bone cement. Hence, the aim of this study was to elaborate if bacterial adherence and growth could be determined on explanted gentamicin- and gentamicin-vancomycin-loaded beads after infection eradication. 18 chains of antibiotic-loaded beads (11 gentamicin-, 7 gentamicin-vancomycin-loaded) were examined. Indications for primary beads implantation included postoperative infections after total hip or knee arthroplasty, rotator cuff reconstruction, chronic foot osteomyelitis, anterior cruciate ligament reconstruction and dorsal spondylodesis. Among the isolated organisms, Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) were the most frequent ones. In 4 cases (3 × S. epidermidis, 1 × MRSA) bacteria growth persistence could be determined on the beads. S. epidermidis-strains persisted only on gentamicin-loaded beads, MRSA could grow on gentamicin-vancomycin-impregnated cement. In one case, the emergence of a gentamicin-resistant S. epidermidis-strain could be observed despite preoperative susceptibility. Bacteria growth persistence on bone cement is a hazardous problem in the orthopedic surgery and should therefore be born in mind. Adherence to cement can lead to emergence of bacteria resistance despite preoperative antibiotic susceptibility and might result in clinical recurrence of infection

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative S. epidermidis are the most commonly infecting agents associated with PJI. Nowadays, Gram-negative species like Escherichia coli and Pseudomonas aeruginosa are gaining relevance. The use of TiO2 conical nanotubular doped with fluorine and phosphorous (FP-cNT) surfaces is an interesting approach to prevent surface bacterial colonization during surgery and favouring the osseointegration. Despite of there are serum markers related with PJI, to date there is described no biomaterial-related marker that allows detecting PJI. Here we describe the adherence and the bactericidal effect of FP-cNT and its capacity of marking the non-fermenting bacteria that have been in contact with it by Al. This metal is delivered by FP-cNT in non-toxic concentrations (between 25 and 29 ng/mL). F-P-cNT layers on Ti6Al4V alloy were produced as described previously by our group. Ti6Al4V chemical polishing (CP) samples without nanostructure were used as control and produced as described previously. S. aureus 15981, S. epidermidis ATCC 35984, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 strains adherence study was performed using the protocol described by Kinnari et al. in 0.9% NaCl sterile saline with a 120 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The aluminum concentration was estimated in the supernatant after incubation and in the 0.22 µm filtered supernatant by atomic absorption in graphite furnace. The statistical data were analyzed by nonparametric Kruskal-Walis test and by pairwise comparisons using the nonparametric unilateral Wilcoxon test with a level of statistical significance of p<0.05. The values are cited as medians. Our results show that the bacterial adherence of all tested species significantly decreased on FP-cNT compared to CP except P. aeruginosa ATCC 27853: 19.8% for S. aureus 15981, 45.3% for S. epidermidis ATCC 35984 and 8.1% for E. coli ATCC 25922. The bacterial viability decreased 2-fold for S. aureus 15981, and 5-fold for S. epidemidis ATCC 35984, but increased 95% for P. aeruginosa ATCC 27853 and there no was variation for E. coli ATCC 25922 on FP-cNT compared to CP. Only supernatant P. aeruginosa ATCC 27853 shows significant Al detection after 120 min incubation (p<0.05). In summary, F-P cNT is a promising biomaterial that besides favoring osseointegration and potential usefulness as drug carrier, present bactericidal, non-stick ability (at least for staphylococci and E. coli) and is able to mark P. aeruginosa with Al, which could be potentially monitored in serum and urine in patients with PJI

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

The role of biofilm in pathogenesis of several chronic human infections is widely accepted, as this structure leads pathogens to persist among the human body, being protected from the action of antibacterial molecules and drugs (1). It has been estimated that up to 65% of bacterial infections are caused by microorganisms growing in biofilms (2). Moreover, biofilm is involved in device-related orthopaedic bacterial infections, which are unaffected by vaccines and antibiotic therapies, constituting a serious problem for the human health care.

The aim of the present work was to evaluate the anti-biofilm action of a selected and patented lactobacillus strain (MD1) supernatant, both on the in-formation- biofilm and on mature biofilm produced by pathogenic bacteria.

MD1 was grown in BHI for 48 h at 37°C. After incubation, the sample was centrifuged for 5’ for 14,000 × g and the supernatant previously filtered and treated in order to obtain the anti-biofilm compounds (Special Supernatant – SS) was collected. Staphylococcus aureus and Pseudomonas aeruginosa strains were grown in BHI for 24h at 37°C. The anti-biofilm ability of the tested SS – lactobacillus strain was evaluated by a spectrophotometric method according to Christensen at al., following the incubation of pathogens and the “mature biofilm” with the lactobacillus supernatant. Confocal Laser Scanning Microscopy was used to confirm the data obtained from Crystal Violet Assay.

After the incubation of the SS with pathogens and mature biofilm, the formation of biofilm was inhibited and a significant disruption of the mature biofilm was observed. Interestingly, the same properties were observed also when the SS pH was neutralized to pH 6.5. In particular, the reduction of biofilm production and the disruption of mature biofilm was about 50–70% for all microorganisms.

The SS lactobacillus strain MD1 exhibited a relevant antibiofilm action against mature and in-formation-biofilm produced by S. aureus and P. aeruginosa strains tested in the study. Moreover, the antibiofilm action has been observed to be pH-independent, as when the supernatant was neutralized to pH 6.5, the reduction of pathogenic biofilm has been still observed. These promising results highlighted the possibility to use this SS-lactobacillus anti-biofilm property to develop a cost-effective and safety treatment able to reduce the impact of pathogenic biofilm on device-related orthopaedic bacterial infections.

The twelve matte and twelve polished surfaces of hemi-arthroplasties were contaminated with Staph. epidermidis and then irrigated with bulb or pulse irrigation. The surfaces were then quantitatively cultured using a standardized swabbing technique. Results are expressed as the percentage of contaminant bacteria recovered. The matte finish groups showed median values of 1.46 and 2.88x10. −2. while the polished finish groups showed 1.49x10. −3. and 2.83x10. −6. with bulb and pulse irrigation, respectively. The difference between irrigation types was significant (p=0.002) for both matte and polished surfaces. Pulse irrigation was more effective than bulb irrigation in removing contaminant bacteria from the prosthetic surfaces studied. Tremendous suffering is associated with infection following total joint arthroplasty. To reduce infection risk, some surgeons use pulse irrigation prior to wound closure. This practice is based on the assumption that pulse irrigation will more effectively remove adherent bacteria. However, there has been no study of the effectiveness of pulse irrigation in clearing bacteria from prosthetic surfaces. The hypothesis of this study is that pulse irrigation is more effective than bulb irrigation in removing intra-operative bacterial contaminants from prosthetic surfaces. The matte and polished surfaces of hemiarthroplasties were studied separately. Each surface was contaminated with Staph. epidermidis and then irrigated with pulse or bulb irrigation. A third group without irrigation was also studied. The surface was then swabbed three times using a standardized technique. The swab tips were quantitatively cultured. Twelve matte and twelve polished surfaces were examined using both irrigation types with corresponding non-irrigation reference values. Results are expressed as the percentage of contaminant bacteria recovered. The matte finish groups showed median values of 1.46 and 2.88x10. −2. while the polished finish groups showed 1.49x10. −3. and 2.83x10. −6. with bulb and pulse irrigation, respectively. The difference between irrigation types is significant (p=0.002) for both matte and polished surfaces. Pulse irrigation was more effective than bulb irrigation in removing contaminant bacteria from the prosthetic surfaces studied. Funding: Hip Hip Hooray, Zimmer-Sadler

Aim. Compare clinical outcomes following staged revision arthroplasty for periprosthetic joint infection (PJI) secondary to either multidrug resistant (MDR) bacteria or non-MDR (NMDR) bacteria. Method. Retrospective analysis of a prospectively collected bone infection database. Adult patients diagnosed and treated for hip or knee PJI, between January 2011 and December 2014, with minimum one-year follow-up, were included in the study. Patients were divided into two groups: MDR group (defined as resistance to 3 or more classes) and N-MDR group (defined as acquired resistance to two classes of antibiotic or less). The Charlson Comorbidity Index was used to stratify patients into low, medium and high risk. The diagnosis of PJI, and any recurrence following treatment, was made in accordance with the Musculoskeletal Infection Society criteria. Failure was defined as recurrence of infection necessitating implant removal, excision arthroplasty, arthrodesis or amputation. Results. The study population comprised 240 patients. 74 (31%) had an MDR infection. 14 patients were deceased at the time of data capture. All infections were treated by staged revision with interval antibiotic space and targeted systemic antibiotics under the supervision of a multidisciplinary team. Total number of failures in both groups was 39 (16%), 15 hips (12%) and 24 knees (21%). There were significantly more failures in the MDR group (n=24, 32%) than the non-MDR group (n=15, 9%)(p<0.0001). Using the Charlson Comorbidity Index within the N-MDR group there was no significant difference in outcomes between the low and medium groups (p=0.352), the low and high risk groups (p=1.000) and the high and medium risk groups (p=1.000). There was no statistically significant association discerned within the MDR group based on co-morbidity also. (p values = 0.1702, 0.665 and 0.1096 respectively). When comparing all cases, there was a statistically significantly higher rate of failure in patients with polymicrobial infection versus single organism infection (P<0.0001). When stratifying by the presence of an MDR organism versus an N-MDR organism, both polymicrobial sub groups showed a greater rate of failure than their single organism counterparts, however this was only significant in the MDR group and not the N-MDR group (p=0.0007 vs p=0.123). Furthermore the polymicrobial MDR group showed a statistically significant higher rate of failure versus the polymicrobial N-MDR group (p=0.002). Conclusions. The study suggests that the presence of an MDR organism may be a predictor of failure, independent of patient co-morbidity, in staged revision hip and knee arthroplasty for PJI

The first aim of the study was to investigate if bacteria were implicated in non-union of fractures of the tibia and femur, which had been treated with intramedullary nailing. The second aim was to evaluate the antimicrobial susceptibility of bacteria isolated from the retrieved intramedullary nails. Forty intramedullary nails removed from tibial and femoral fractures were retrieved for the purpose of the study. Twenty of these nails were from fractures, which had successfully united and 20 were removed from fractures which had failed to unite prior to further operative intervention. There was no evidence of clinical infection in either of the two groups. The nails were subjected to ultrasound in the research laboratory to dislodge adherent bacteria formed as biofilm from the surface of the nail. Using both standard culture techniques and non-culture techniques (Immunofluorescence microscopy and PCR analysis) any dislodged bacteria were isolated and identified. Isolated bacteria were tested for antimicrobial susceptibility to commonly used antibiotics in orthopaedic practice according to NCCLS guidelines. Bacteria were detected in 15 out of 20 [75%] of the nails removed from fractures, which had developed a non-union, and in 5 out of 20 [25%] of fractures that had united, using both standard culture techniques and non-culture techniques. The bacterial isolates identified were mainly Staphylococcus epidermidis and the Gram-positive anaerobe Proprionibacterium acnes. Vancomycin was the most effective antibiotic, with 2 out of 34 [6%] isolates being resistant. Erythromycin was the least effective, with 21 out of 34 [62%] isolates being resistant. Based on overall Minimum Bactericidal Concentrations at which 90% of all strains were killed, Vancomycin was the most active bactericidal agent tested followed in decreasing order by fucidic acid, ciprofloxacin, gentamicin, cefamandole and erythromycin. Bacteria were detected more commonly in the fracture non-union group than in the union group [p< 0.01]. Of the antibiotic agents tested Vancomycin was the most effective and Erythromycin was the least effective

Addition of antibiotics to the bone cement decreases the incidence of infection. However, the antibiotic is only partially released. Ultrasound may increase the antibiotic release and furthermore the effectiveness of the antibiotic might be enhanced by the so-called bio-acoustic effect. The objective of this study was twofold. The first aim was to evaluate to what extent antibiotic release from bone cement could be increased by ultrasound. The second aim was to investigate the viability of bacteria when antibiotic release from bone cements was combined with ultrasound. Cylindrical bone cement samples of Palacos R-G (loaded with gentamicin) and Copal (loaded with gentamicin and clindamycin) were insonated and antibiotic release was compared with uninsonated samples. In addition, identical samples were used in combination with cultures of bacteria derived from prosthesis-related infections. The viability of these bacteria was determined with and without ultrasound, using unloaded Palacos R as a control. There was a trend of increased gentamicin release under influence of ultrasound. Clindamycin release from Copal was significantly increased. Ultrasound alone did not affect bacterial viability, but the application of ultrasound in combination with antibiotic-loaded bone cements reduced both planktonic and biofilm bacterial viability. The release of antibiotics from bone cement was increased by the application of ultrasound. Antibiotic release in combination with ultrasound increases the antimicrobial efficacy against a variety of clinical isolates. The enhanced efficacy against bacteria in the biofilm mode of growth, especially against a gentamicin-resistant strain, is clinically important with regard to the treatment of infected joint prostheses. Ultrasound may also be applied in the early postoperative period to prevent infections, because planktonic bacteria present in the wound and wound area due to inevitable contamination during surgery can then be more effectively prevented from forming a biofilm

Aim. To test the hypothesis that surface skin swabs taken after skin preparation with alcoholic povidone iodine (APVPI) would not grow bacteria, whereas full thickness biopsies taken from the line of surgical incision would grow bacteria. Method. Informed consent was obtained from 44 patients undergoing primary hip (n=13) and knee (n=31) arthroplasty. Each received antimicrobial prophylaxis before skin preparation with APVPI under laminar flow. After the APVPI had dried, a skin swab and a full thickness 8mm x 4mm elliptical skin biopsy were taken from the line of incision. The skin swab was rolled in 5mL anaerobe basal broth to inactivate the APVPI, incubated at 37 degrees and checked for growth for 2 weeks. One half of the skin biopsy was snap frozen and used for gram and nitroblue tetrazolium staining. The other half was placed into 5mL of anaerobe basal broth, incubated at 37 degrees and monitored for growth for 2 weeks. Results. Forty-four skin biopsy samples and 42 corresponding swabs were collected. Fourteen of 42 surface swabs were positive for bacteria (5 Staphylococcus epidermidis, 6 Propionibacteria acnes, 1 S. aureus, 1 S. capitis, 1 S. epidermidis and P. acnes, and 1 S. warneri and P. acnes). Fifteen of 44 skin biopsies were positive for bacteria (7 P. acnes, 3 S. epidermidis, 1 S. aureus, 1 S. capitis, 1 Psuedomonas spp, 1 P. acnes and S. epidermidis, 1 S. edidermidis and S. capitis). Gram positive bacteria were seen in all gram stained sections of skin and all sections of skin were positive for live bacteria when stained with nitroblue tetrazolium. Discussion. This study shows that skin preparation with APVPI does not completely remove viable bacteria from the skin. Surgeons need to be aware of this and to adapt their surgical technique to avoid coming into contact with the patient's skin, including cut edges, when performing surgery involving implants

Aim. Data on Prosthetic joint infection (PJI) caused by multi-drug resistant (MDR) or XDR (extensively drug resistant) Gram negative bacteria (GNB) are limited. Treatment options are also restricted. We conducted a multi-national, multi-center assessment of clinical data and factors of outcome for these infections. Method. PJI were defined upon international guidelines. Data from 2000–2015 on demographics, clinical features, microbiology, surgical treatment and antimicrobial therapy was collected retrospectively. Factors associated with treatment success were evaluated by logistic regression analysis. Results. A total of 133 PJI were evaluated. Female (n=84, 61.4%) and the elderly [mean age (+/-SD) 73 (12.7)] predominated. Diabetes mellitus was the most frequent comorbidity (n=42,32.1%) followed by rheumatoid arthritis (n=14,10.7). Most PJI were early infections (84.4 %). XDR accounted for 23 cases; half of them due to Pseudomonas aeruginosa. Prevalence of MDR or XDR GNB was not different between early and late PJIs (p=0.114). Overall, P.aeruginosa (n=25, 19.1%) was followed by Klebsiella spp (n=23,17.6%) and Enterobacter spp (n=22,16.8%). PJI was located at the hip (n=85 65.6%), knee (n=41,31.3%), shoulder (n=3,2.3%) and ankle (n=1, 0.8%). Clinical characteristics included soft tissue infection (66.4%), pain (51.1%), fever (32.1%) and sinus tract(29.8%). Surgery for PJIs consisted of DAIR (debridement, antibiotics and implant retention), (n=64, 49.6%), followed by explantation of the arthroplasty (n=32, 24.8%), two-stage revision (n=16, 12,4%), one stage revision (n=9, 7%), arthrodesis (n=2, 1.6%). Median duration of antibiotic therapy was 51 days (IQR 25–75: 40–90 days). Cure after treatment was assessed in 78 patients (58.6%). No-DAIR surgical procedures in PJIs were more likely to be successful compared to DAIR surgery (75.8% vs 50%, OR 3.13, 95% CI:1.47–6.70, p=0.003)both in early or late infections. Conclusions. PJI by MDR/XDR GNB affects female, the elderly with comorbidities and previous surgery for PJI. P.aeruginosa is frequent, mostly XRD. No-DAIR procedures have higher probability of treatment success than DAIR even in early infection. Despite surgery and long-term antimicrobial administration, treatment success was less than 60%, probably reflecting the lack of effective treatment options

Soft tissue biopsies may prove culture negative in biofilm prosthetic infections. Identification of the causative bacteria could be achieved by either scraping of the prosthetic surface or by sonication of the entire implant. These techniques have not been thoroughly studied in experimental models where the biofilm is developed in vivo. In a novel rat biofilm model we compared scraping and sonication as methods for dislodging biofilm bacteria. Twenty plates of steel alloy (5×7×1mm), with a surface roughness (Ra) of 0.35 (0.19–0.51) μm, were inserted into 20 standardised pieces of sheep costae, weight: 1.2 (1.0–1.5) gram. To each bone graft was added 50 μL of a Staphylococcus epidermidis suspension containing 1.4 (1.1–1.7)×104 CFU. Ten Sprague Dawley rats were operated with implantation of the bone graft subfascially on each side of the interscapular region. After two weeks the grafts were excised. The plates were removed from the grafts and rinsed twice in saline. Aliquots of 50 μL were cultured. 10 plates were scraped, followed by vortex mixing of the knife blade; and 10 plates were sonicated at 30 kHz for five minutes. 50 μL of the saline used for a) vortex mixing of the knife blade, and b) sonication, was seeded on agar. After overnight incubation the number of CFU was counted. The total number of CFU recovered after scraping and sonication were 2(0–13) × 102 and 298(8–878) × 102, respectively (p< 0, 01). Compared to the number of CFU in the rinsing fluid, no increase was observed after scraping. For each plate that was sonicated there was a 38 (3–300) fold increase in the number of CFU. First, sonication is a superior technique for dislodging biofilm bacteria in an in vivo model, compared to scraping. Secondly, the present experimental model is a promising method for developing biofilm in vivo

Objectives. Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro?. Methods. Titanium alloy cylinders (Ti6Al4V) were exposed to PEMF from an induction heater with maximum 2000 watts at 27 kHz after being contaminated with five different types of micro-organisms: Staphylococcus epidermidis; Staphylococcus aureus; Pseudomonas aeruginosa; spore-forming Bacillus cereus; and yeast Candida albicans. The cylinders were exposed to incremental target temperatures (35°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C) for up to 3.5 minutes. Results. There was an average linear heating rate of 0.39°C per second up to the target temperature, and thereafter the target temperature was maintained until the end of the experiment. At 60°C and higher (duration 3.5 minutes), there was a 6-log reduction or higher for every micro-organism tested. At 60°C, we found that the shortest duration of effective induction heating was 1.5 minutes. This resulted in a 5-log reduction or higher for every micro-organism tested. Conclusion. Non-contact induction heating of a titanium disk is effective in reducing bacterial load in vitro. These promising results can be further explored as a new treatment modality for infections of metal orthopaedic implants. Cite this article: B. G. Pijls, I. M. J. G. Sanders, E. J. Kuijper, R. G. H. H. Nelissen. Non-contact electromagnetic induction heating for eradicating bacteria and yeasts on biomaterials and possible relevance to orthopaedic implant infections: In vitro findings. Bone Joint Res 2017;6:323–330. DOI: 10.1302/2046-3758.65.BJR-2016-0308.R1

Aims. Staphylococcus aureus is a major cause of septic arthritis, and in vitro studies suggest α haemolysin (Hla) is responsible for chondrocyte death. We used an in vivo murine joint model to compare inoculation with wild type S. aureus 8325-4 with a Hla-deficient strain DU1090 on chondrocyte viability, tissue histology, and joint biomechanics. The aim was to compare the actions of S. aureus Hla alone with those of the animal’s immune response to infection. Methods. Adult male C57Bl/6 mice (n = 75) were randomized into three groups to receive 1.0 to 1.4 × 10. 7. colony-forming units (CFUs)/ml of 8325-4, DU1090, or saline into the right stifle joint. Chondrocyte death was assessed by confocal microscopy. Histological changes to inoculated joints were graded for inflammatory responses along with gait, weight changes, and limb swelling. Results. Chondrocyte death was greater with 8325-4 (96.2% (SD 5.5%); p < 0.001) than DU1090 (28.9% (SD 16.0%); p = 0.009) and both were higher than controls (3.8% (SD 1.2%)). Histology revealed cartilage/bone damage with 8325-4 or DU1090 compared to controls (p = 0.010). Both infected groups lost weight (p = 0.006 for both) and experienced limb swelling (p = 0.043 and p = 0.018, respectively). Joints inoculated with bacteria showed significant alterations in gait cycle with a decreased stance phase, increased swing phase, and a corresponding decrease in swing speed. Conclusion. Murine joints inoculated with Hla-producing 8325-4 experienced significantly more chondrocyte death than those with DU1090, which lack the toxin. This was despite similar immune responses, indicating that Hla was the major cause of chondrocyte death. Hla-deficient DU1090 also elevated chondrocyte death compared to controls, suggesting a smaller additional deleterious role of the immune system on cartilage. Cite this article: Bone Joint Res 2022;11(9):669–678

Aims. Despite numerous studies focusing on periprosthetic joint infections (PJIs), there are no robust data on the risk factors and timing of metachronous infections. Metachronous PJIs are PJIs that can arise in the same or other artificial joints after a period of time, in patients who have previously had PJI. Methods. Between January 2010 and December 2018, 661 patients with multiple joint prostheses in situ were treated for PJI at our institution. Of these, 73 patients (11%) developed a metachronous PJI (periprosthetic infection in patients who have previously had PJI in another joint, after a lag period) after a mean time interval of 49.5 months (SD 30.24; 7 to 82.9). To identify patient-related risk factors for a metachronous PJI, the following parameters were analyzed: sex; age; BMI; and pre-existing comorbidity. Metachronous infections were divided into three groups: Group 1, metachronous infections in ipsilateral joints; Group 2, metachronous infections of the contralateral lower limb; and Group 3, metachronous infections of the lower and upper limb. Results. We identified a total of 73 metachronous PJIs: 32 PJIs in Group 1, 38 in Group 2, and one in Group 3. The rate of metachronous infection was 11% (73 out 661 cases) at a mean of four years following first infection. Diabetes mellitus incidence was found significantly more frequently in the metachronous infection group than in non-metachronous infection group. The rate of infection in Group 1 (21.1%) was significantly higher (p = 0.049) compared to Groups 2 (6.2%) and 3 (3%). The time interval of metachronous infection development was shorter in adjacent joint infections. Concordance between the bacterium of the first PJI and that of the metachronous PJI in Group 1 (21/34) was significantly higher than Group 2 (13/38; p = 0.001). Conclusion. The findings of this study suggest that metachronous PJI occurs in more than one in ten patients with an index PJI. Female patients, diabetic patients, and patients with a polymicrobial index PJI are at significantly higher risk for developing a metachronous PJI. Furthermore, metachronous PJIs are significantly more likely to occur in an adjacent joint (e.g. ipsilateral hip and knee) as opposed to a more remote site (i.e. contralateral or upper vs lower limb). Additionally, adjacent joint PJIs occur significantly earlier and are more likely to be caused by the same bacteria as the index PJI. Cite this article: Bone Joint J 2023;105-B(3):294–300

Aim: To assess the effectiveness of povidone-iodine alcoholic tincture and the alcoholic chlorhexidine gluconate solution in the eradication of bacteria in forefoot surgery, and to assess any added benefits with the use of surgical bristles. Methods: Fifty consecutive patients were prospectively enrolled into the study and randomised to receive one of two surgical skin preparations. Results: The use of povidone-iodine with prior surgical scrubbing had a better eradication rate compared to povidone-iodine alone in the interdigital web-spaces. Prior surgical scrubbing with both solutions had a better eradication rate for the skin over the 1st metatarso-phalangeal joints. But neither solution with or without the use of surgical scrubbing was superior at eradicating organisms from the medial hallucal fold. However none of these results were statistically significant. None of the patients developed any post-operative wound infection. Conclusions: Our results did not show any statistically significant advantage with either solution nor was there any apparent advantage with the use of the surgical scrub prior to the skin preparation. The authors believe that eradication of bacteria in forefoot surgery is dependant on a meticulous and methodical skin preparation technique and less so on the solution used and method of application

We modelled a 'clean' surgical wound lightly contaminated with airborne bacteria, using agar, ovine muscle and ovine adipose tissue. This was used to assess the effect on bacteria of ultraviolet C light (UVC) 1200 mu W/cm2, hydrogen peroxide 3%, povidone-iodine 1% and 10%, chlorhexidine 0.05%, pulsed jet lavage with UVC and syringe and needle lavage with chlorhexidine 0.05%. All the agents were effective on agar, but mixing with blood or plasma neutralised hydrogen peroxide and povidone-iodine 1%. All the agents were less effective on tissue specimens than on agar, but were more effective on adipose tissue than on muscle. All the antiseptics except chlorhexidine were less effective when blood or plasma was added to muscle specimens before disinfection. UVC after pulsed jet lavage had an additive effect. Syringe and needle lavage with chlorhexidine 0.05% was the most effective method tested; it reduced colony counts by 99.8% and warrants clinical investigation

Aims. The aim of this study was to determine whether the sequential application of povidone iodine-alcohol (PVI) followed by chlorhexidine gluconate-alcohol (CHG) would reduce surgical wound contamination to a greater extent than PVI applied twice in patients undergoing spinal surgery. Patients and Methods. A single-centre, interventional, two arm, parallel group randomised controlled trial was undertaken, involving 407 patients who underwent elective spinal surgery. For 203 patients, the skin was disinfected before surgery using PVI (10% [w/w (1% w/w available iodine)] in 95% industrial denatured alcohol, povidone iodine; Videne Alcoholic Tincture) twice, and for 204 patients using PVI once followed by CHG (2% [w/v] chlorhexidine gluconate in 70% [v/v] isopropyl alcohol; Chloraprep with tint). The primary outcome measure was contamination of the wound determined by aerobic and anaerobic bacterial growth from samples taken after disinfection. Results. The detection of viable bacteria in any one of the samples taken after disinfection (culture-positive) was significantly lower in the group treated with both PVI and CHG than in the group treated with PVI alone (59 (29.1%) versus 85 (41.7%), p = 0.009; odds ratio 0.574; 95% confidence interval, 0.380 to 0.866). Conclusions. Antisepsis of the skin with the sequential application of PVI and CHG more effectively reduces the contamination of a surgical wound than PVI alone. Cite this article: Bone Joint J 2017;99-B:1354–65

The implantation of endoprosthesis is a routine procedure in orthopaedics. Endoprosthesis are mainly manufactured from ceramics, polymers, metals or metal alloys. To ensure longevity of the implants they should be as biocompatible as possible and ideally have antibacterial properties, to avoid periprosthetic joint infections (PJI). Various antibacterial implant materials have been proposed, but have so far only been used sporadically in patients. PJI is one of the main risk factors for revision surgeries. The aim of the study was to identify novel implant coatings that both exhibit antibacterial properties whilst having optimal biocompatibility.

Six different novel implant coatings and surface modifications (EBM TiAl6V4, strontium, TiCuN, TiNbN, gentamicin phosphate (GP), gentamicin phosphate+cationic polymer (GP+CP)) were compared to standard CoCrMo-alloy. The coatings were further characterized with regard to the surface roughness. E. coli and S. capitis were cultured on the modified surfaces to investigate the antibacterial properties. To quantify bacterial proliferation the optical density (OD) was measured and viability was determined using colony forming units (CFU). Murine bone marrow derived macrophages (BMMs) were cultured on the surfaces and differentiated into osteoblasts to quantify the mineralisation using the alizarin red assay.

All novel coatings showed reduced bacterial proliferation and viability compared to standard CoCrMo-alloy. A significant reduction was observed for GP and GP+CP coated samples compared to CoCrMo (OD_GP,E.coli = 0.18±0.4; OD_GP+CP,E.coli = 0.13±0.3; p≤0.0002; N≥7-8). An increase in osteoblast-mediated mineralisation was observed on all surfaces tested compared to CoCrMo. Furthermore, GP and GP+CP coated samples showed a statistically significant increase (M_GP = 0.21±0.1; M_GP+CP = 0.25±0.2; p<0.0001; N≥3-6).

The preliminary data indicates that the gentamicin containing surfaces have the most effective antibacterial property and the highest osseointegrative capacity. The use of antibiotic coatings on prostheses could reduce the risk of PJI while being applied on osseointegrative implant surfaces.

To prevent nosocomial transmission (NT) of multiresistent germs (MRG) the German Robert Koch Institute (RKI) recommends to isolate patients with MRG. At a so-called normal ward isolating patients is a challenging and stressful procedure for both patients and hospital staff. The present study proposes the hypothesis that, compared to normal wards, an isolation ward reduces the nosocomial infection rate. After an isolation ward with twelve beds has been established in 2005, patients with MRG on the wards of the department for spinal cord injury as well as on the isolation ward were monitored using a prospective screening and meeting the requirements of the RKI. Apart from detecting transmitter of MRG the NT of these bacteria was identified and registered between 2006 and 2013. The total length of a patients stay in the hospital, the number of isolation days and the rate of NTs were documented. The quotient of MRG load per ward and the number of NTs per ward were compared. In the investigation period of eight years 262175 patient days, 33416 isolation days and 33 transmissions were registered. On the spinal cord injury ward 223167 of the patient days, 1120 of the isolation days and 29 of the NTs were documented. On the isolation ward 39008 of the patient days and 32296 of the isolation days with four of the transmissions were registered. The mean load of MRG resulted from the quotient of the number of days with MRG per 100 patient days. The effective nosocomial frequency of transmission resulted from the quotient of the mean load of MRG to the number of transmissions. As a result, the frequency of transmission on the isolation ward was significantly lower (p=0,001) in comparison to the spinal cord injury ward. The presented results suggest that, despite multiple higher loads of MRG, constructional measures combined with contact isolation facilitate a reduction of NT rates of MRG. The reservation must be made, however, that in case of known MRG the screening was performed under isolation conditions, with unkown MRG without meeting requirements of isolation. The present comparison of NT rates on an isolation ward and a normal spinal cord injury ward emphasizes the importance and function of an isolation ward through constructional (physical) separation and pooling of professional competency for successful management of MRG in healthcare facilities

Eradication of bacteria in forefoot surgery in necessary to prevent post-operative infections. Currently a lack of consensus exists on the optimum solution and preparation methods needed to achieve this. We compared the effect of povidine-iodine and chlorhexidine gluconate on lowering bacterial load and if any additional benefits are gained by pre-treatment with the use of a bristled brush. Fifty consecutive patients undergoing forefoot surgery were recruited into the study and randomised to receive one of two surgical skin preparations (Povidine-iodine 1% with isopropyl alcohol 23% or Chlorhexi-dine gluconate 0.5% with isopropyl alcohol 70%). In addition to the skin preparation of the foot with the randomised solution the other foot was also scrubbed with a sterile surgical bristled brush for a standardised period (3 minutes) and then painted again. Swabs were taken from three sites and analysed via qualitative and quantitative analysis. All four methods significantly decreased (p < 0.001), in all three sites, the number of colony forming units. Using two-way analysis of variance no significant interaction was observed between site of swab and method of preparation (p =0.970). This confirms that no preparation method was more superior in reducing the number of CFUs at any site than the others. We suggest that either povidone –iodine with no more that 23% isopropyl alcohol or chlorhexidine gluconate with 70% isopropyl alcohol be used for surgical preparation in forefoot surgery. No additional benefit in reduction in bacterial load is gained by scrubbing the foot prior to painting with bristles

Introduction. Open fractures occur with an annual incidence of 11.5 per 100,000 (6900 pa in UK). Infection rates, even with intravenous broad-spectrum antibiotics, remain as high as 22%. For this reason necessary bone grafting is usually delayed until soft-tissue cover of the bone injury is achieved. A biodegradable bone graft that released sustained high concentrations of antibiotics and encouraged osteogenesis, that could be implanted safely on the day of injury would reduce infection rates and avoid reoperation and secondary grafting. The non –union rate (approx 350 pa in UK) should also be reduced. Such a graft, consisting of a PLA/PGA co –polymer and containing antibiotics, is under development and here we report assessment of spectrum and duration of antimicrobial activity and effect of addition of antibiotics on mechanical properties. Methods. Varying concentrations of gentamicin, colistin, clindamycin and trimethoprim, singly and in combination, were added to the copolymer and test pieces were made. These were then tested using an established method (SPTT) which determines degree and duration of antimicrobial activity as well as risk of emerging resistance. Test bacteria were Staphylococcus epidermidis, Staphylococcus aureus, MRSA and Escherichia coli. Mechanical properties (compressive strength and porosity) were determined using established methods. Results. A combination of gentamicin (4%w/w) and clindamycin (2.5% w/w) gave best results, with inhibitory activity persisting for over 21 days (the target duration) without emergence of resistance. No significant effect of this combination/concentration on mechanical properties was found. Conclusions. The experimental PLA/PGA scaffold containing antibiotics showed activity against the common pathogens of open fractures for a period considered long enough to eradicate contamination acquired at or soon after trauma. At the optimum concentration, they had no significant effect on mechanical properties. In vivo performance is currently being investigated in a sheep model

Recent work has demonstrated that intra-operative contamination of spinal surgical wounds is relatively common. The most frequently isolated wound contaminants are Propionibacterium spp. and coagulase negative Staphylococcus spp. The aim of this study is to examine the efficacy of prophylactic antibiotics used for spinal surgery against bacterial contaminants isolated from intra-operative samples retrieved during spinal surgical procedures. Intra-operative wound samples were taken from 94 patients undergoing spinal surgery. Samples including skin, subcutaneous tissue and wound washings were processed, inoculated onto agar and incubated under both aerobic and anaerobic conditions for a period of 2 weeks. Bacterial growth was identified using commercially available biochemical test galleries. Thirty-six bacterial isolates were identified. The predominant bacteria isolated included Propionibacterium spp. (n=21) and coagulase negative Staphylococcus spp. (n=15). Each bacterial isolate was tested for its susceptibility to antibiotics used as antimicrobial prophylaxis during spinal surgery. Antibiotic sensitivities were determined in accordance with National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The antibiotic that performed best against Staphylococcus spp. isolated was ciprofloxacin with 93% of isolates being susceptible to this antibiotic. Cefamandole and cefuroxime also performed well against Staphylococcus spp. isolates. The antibiotic that performed best against Propioni-bacterium spp. isolates was cefamandole with 100% of isolates being susceptible. Cefuroxime and ciprofloxacin also performed well. The antibiotic that performed least well against bacterial isolates was erythromycin with only 76% of Propionibacterium spp. and 47% of Staphylococcus spp. exhibiting susceptibility. The results of this study demonstrate that ciprofloxacin, cefuroxime and cefamandole are effective against the majority of Propionibacterium spp. and Staphylococcus spp. isolated from within the spinal wound during surgery. The use of erythromycin in the penicillin allergic patient is questioned and ciprofloxacin proposed as a possible alternative

Considerable evidence exists that aseptic loosening is initiated by wear particles that recruit macrophages and stimulate their production of pro-inflammatory cytokines. The cytokines primarily act indirectly by inducing production of RANKL, which stimulates osteoclast differentiation, osteolysis, and inflammatory bone loss. There is also considerable evidence that activation of macrophage Toll-like Receptors (TLRs) contributes to this cascade of events. It is however controversial whether bacterially-derived immunostimulatory molecules known as Pathogen-Associated Molecular Patterns (PAMPs) can contribute to aseptic loosening by stimulating their cognate TLRs on macrophages. Priming and subsequent activation of the NLRP3 inflammasome is essential for macrophage production of mature, active IL-1β in response to wear particles. We recently confirmed that wear particles can activate pre primed NLRP3 inflammasomes in the absence of PAMPs. Thus, activation of the NLRP3 inflammasome is the only macrophage-based event in the aseptic loosening cascade that we have found to date is independent of PAMPs. In contrast, priming of the NLRP3 inflammasome by wear particles requires PAMPs as well as their cognate TLRs. These results add to the growing body of evidence that bacterially-derived PAMPs can contribute to aseptic loosening.

Aims

Here we used a mature seven-day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or antibiotics or non-contact induction heating, and which combinations could eradicate S. aureus in mature biofilms.

Aims

The surgical helmet system (SHS) was developed to reduce the risk of periprosthetic joint infection (PJI), but the evidence is contradictory, with some studies suggesting an increased risk of PJI due to potential leakage through the glove-gown interface (GGI) caused by its positive pressure. We assumed that SHS and glove exchange had an impact on the leakage via GGI.

Introduction: Aseptic loosening of THR has a multifactorial aetiology. Differentiating such cases from loosening due to low-grade infection can often be difficult. It is possible that at least some cases of ‘aseptic’ loosening may be related to unidentified bacterial infection. This study attempted to identify the frequency with which bacterial DNA could be observed in the periprosthetic membrane and synovial fluid of patients undergoing revision surgery for what was considered ‘aseptic’ loosening.

Methods: Specimens from 39 revision and 31 primary hip replacements were obtained. The latter were used as a control for environmental contamination. All revision THR cases were investigated pre-operatively for infection by CRP, ESR, WCC, Gallium Scan. Operative specimens were analysed by bacteriological culture as well as by PCR to identify the presence of the 16S bacterial ribosomal fraction. Results were analysed by Chi square test.

Results: By PCR, bacterial DNA was identified in 22 of 39 revision hip surgery specimens and 6 of 31 primary hip replacement specimens (p=0.002). By culture none of these specimens had any bacterial growth.

Conclusions: The increased frequency with which bacterial DNA has been identified in ‘aseptically’ loose revision THR is unlikely to be due solely to environmental contamination although this remains a concern. These results may have relevance for our interpretation and understanding of aseptic loosening as well for the diagnosis of prosthetic infection.

Clean air in the operating room is important during joint replacement surgery. We compared monochromatic ultraviolet radiation of 254 nm with the use of a Charnley-Howorth air enclosure by bacterial air-sampling during 113 total hip arthroplasties. Air samples were taken continuously at the edge of the wound and every 15 minutes at a site 130 cm from the operating table. We also tested the effect of occlusive clothing for all personnel. Ultraviolet light was more efficient than the ultra-clean air enclosure, and occlusive clothing on its own or in combination also produced improvement. The implications of these findings are discussed.

Aims. There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). Methods. The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites. Results. Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF. Conclusion. Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections. Cite this article: Bone Joint Res 2022;11(11):787–802

Abstract. Objectives. The objective of this study is to investigate if genomic sequencing is a useful method to diagnose orthopaedic infections. Current methods used to identify the species of bacteria causing orthopaedic infections take considerable time and the results are frequently insufficient for guiding antibiotic treatment. The aim here is to investigate if genomic sequencing is a faster and more reliable method to identify the species of bacteria causing infections. Current methods include a combination of biochemical markers and microbiological cultures which frequently produce false positive results and false negative results. Methods. Samples of prosthetic fluid were obtained from surgical interventions to treat orthopaedic infections. DNA is extracted from these samples lab and nanopore genomic sequencing is performed. Initial investigations informed that a sequencing time of 15 minutes was sufficient. The resulting genomic sequence data was classified using Basic Local Alignment Tool (BLAST) against the NCBI bacterial database and filtered by only including reads with an identity score of 90 and E-value of 1e-50. An E-value of 1e-50 suggests a high-quality result and is commonly used when analysing genomic data. This data was then filtered in R Studio to identify if any species were associated with orthopaedic infections. The results from genomic sequencing were compared to microbiology results from the hospital to see if the same species had been identified. The whole process from DNA extraction to output took approximately 2 hours, which was faster than parallel microbiological cultures. Results. In these preliminary analyses, 15 samples have been collected from patients with confirmed/suspected orthopaedic infections. To date, 11 samples from confirmed infected patients have been sequenced and a summary of the findings are presented in the table attached. As well as finding bacteria species to match microbiological cultures, genomic sequencing has also identified bacteria when culture results have been negative, but the patient is known to have an infection due to clinical indication and previous culture results. This example suggests genomic sequencing may have higher sensitivity than microbiological cultures at detecting bacteria causing orthopaedic infections. Results in table indicate the identification of bacteria from genomic sequencing that match microbiological cultures are high quality. Conclusions. Preliminary data presented using genomic sequencing suggests that the technique may be useful to identify bacterial species causing orthopaedic infections and can do so in a shorter time frame than current microbial methods. The results from genomic sequencing all produced a number of false positive results which hopefully can be reduced by improving the bioinformatic techniques used and increasing the sample number to include individuals without an infection. Further analysis will also look at identifying antibiotic resistance genes in the sequencing data and seeing if this ca be used to predict which patients will and will not respond to antibiotic treatment. The aim at the end of this project is to demonstrate if genomic sequencing is a more sensitive method to identify bacteria causing orthopaedic infections that current methods and if it can be used to guide antibiotic treatment. Include limitations, next steps and bigger picture. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Aims. Gram-negative periprosthetic joint infection (PJI) has been poorly studied despite its rapidly increasing incidence. Treatment with one-stage revision using intra-articular (IA) infusion of antibiotics may offer a reasonable alternative with a distinct advantage of providing a means of delivering the drug in high concentrations. Carbapenems are regarded as the last line of defense against severe Gram-negative or polymicrobial infection. This study presents the results of one-stage revision using intra-articular carbapenem infusion for treating Gram-negative PJI, and analyzes the characteristics of bacteria distribution and drug sensitivity. Methods. We retrospectively reviewed 32 patients (22 hips and 11 knees) who underwent single-stage revision combined with IA carbapenem infusion between November 2013 and March 2020. The IA and intravenous (IV) carbapenem infusions were administered for a single Gram-negative infection, and IV vancomycin combined with IA carbapenems and vancomycin was applied for polymicrobial infection including Gram-negative bacteria. The bacterial community distribution, drug sensitivity, infection control rate, functional recovery, and complications were evaluated. Reinfection or death caused by PJI was regarded as a treatment failure. Results. Gram-negative PJI was mainly caused by Escherichia coli (8/34), Enterobacter cloacae (7/34), and Klebsiella pneumoniae (5/34). Seven cases (7/32) involved polymicrobial PJIs. The resistance rates of penicillin, cephalosporin, quinolones, and sulfonamides were > 10%, and all penicillin and partial cephalosporins (first and second generation) were > 30%. Of 32 cases, treatment failed to eradicate infection in only three cases (9.4%), at a mean follow-up of 55.1 months (SD 25 to 90). The mean postoperative Harris Hip Score and Hospital for Special Surgery knee score at the most recent follow-up were 81 (62 to 91) and 79 (56 to 89), respectively. One patient developed a fistula, and another presented with a local rash on an infected joint. Conclusion. The use of IA carbapenem delivered alongside one-stage revision effectively controlled Gram-negative infection and obtained acceptable clinical outcomes with few complications. Notably, first- and second-generation cephalosporins and penicillin should be administrated with caution, due to a high incidence of resistance. Cite this article: Bone Joint J 2023;105-B(3):284–293

The efficacy of saline irrigation for the treatment of periprosthetic infection (PJI) is limited in the presence of infected implants. This study evaluated the efficacy of vancomycin/tobramycin-doped polyvinyl alcohol (PVA)/ceramic composites (PVA-VAN/TOB-P) after saline irrigation in a mouse pouch infection model. 3D printed porous titanium (Ti) cylinders (400, 700 and 100 µm in pore size) were implanted into mice pouches, then inoculated with S. aureus at the amounts of 1X10. 3. CFU and 1X10. 6. CFU per pouch, respectively. Mice were randomized into 4 groups (n=6 for each group): (1) no bacteria; (2) bacteria without saline wash; 3) saline wash only, and (4) saline wash+PVA-VAN/TOB-P. After seven days, pouches were washed out alone or with additional injection of 0.2 ml of PVA-VAN/TOB-P. Mice were sacrificed 14 days after pouch wash. Bacteria cultures of collected Ti cylinders and washout fluid and histology of pouch tissues were performed. The low-grade infection (1X10. 3. CFU) was more significant in 400 µm Ti cylinders than that in Ti cylinders with larger pore sizes (700 and 1000 µm (p<0.05). A similar pattern of high-grade infection (1X10. 6. CFU) was observed (p<0.05). For the end wash, the bacteria burden (0.49±0.02) in saline wash group was completely eradicated by the addition of PVA-VAN/TOB-P (0.005±0.001, p<0.05). We noticed that 400 µm Ti cylinders have the highest risk of implant infection. Our data supported that the effect of saline irrigation was very limited in the presence of contaminated porous Ti cylinders. PVA-VAN/TOB-P was biodegradable, biocompatible, and was effective in eradicating bacteria retention after saline irrigation in a mouse model of low grade and high-grade infection. We believe that PVA-VAN/TOB-P represents an alternative to reduce the risk of PJI by providing a sustained local delivery of antibiotics

Aim. Bone and joint infections (BJIs) are serious infections requiring early optimized antimicrobial therapy. BJIs can be polymicrobial or caused by fastidious bacteria, and the patient may have received antibiotics prior to sampling, which may decrease the sensitivity of culture-based diagnosis. Furthermore, culture-based diagnosis can take up to 14 days. Molecular approaches can be useful to overcome these concerns. The BioFire® system performs syndromic multiplex PCR in 1 hour, with only a few minutes of sample preparation. The BioFire® Joint Infection (JI) panel (BF-JI), recently FDA-cleared, detects both Gram-positive (n=15) and Gram-negative bacteria (n=14), Candida, and eight antibiotic resistance genes directly from synovial fluids. The aim of this study was to evaluate its performance in acute JIs in real-life conditions. Method. BF-JI was performed on synovial fluid from patients with clinical suspicion of acute JI, either septic arthritis or periprosthetic JI, in 6 French centers. The results of BF-JI were compared with the results of culture of synovial fluid and other concomitantly collected osteoarticular samples obtained in routine testing in the clinical microbiology laboratory. Results. From July 2021 to May 2022, 319 patients (including 10 children < 5y and 136 periprosthetic infections) had been included in the study. The BF-JI test was invalid for one patient (not retested). Among the 318 remaining patients, overall concordance with comparative microbiology methods was 88% (280/318): 131 samples were negative with both BF-JI and culture, and 149 samples were positive with the same microorganisms using complementary techniques. In 33 cases (10.4%), BF-JI was negative while culture was positive: 18 microorganisms were not targeted by BF-JI (including Staphylococcus epidermidis, n=10, and Cutibacterium acnes, n=2); 15 microorganisms targeted by BF-JI were obtained in culture but not by the molecular test (false-negative 4.7%). In 20 cases, BF-JI was positive while culture was not: 12 patients had received antibiotics before sampling, and 7 cases involved fragile and fastidious bacteria (Kingella kingae, n=5; Neisseria gonorrhoeae, n=2). In 6 cases, both BF-JI and culture were positive, but no yielding the same bacteria (polymicrobial specimens). Conclusions. In acute JIs, the BF-JI panel shows a good concordance with culture for the microorganisms targeted by the panel. Therefore, this molecular tool may have a place in microbiological diagnosis of acute JIs in order to confirm JI faster than culture. Moreover, it allows easy detection of difficult-to-culture bacteria. Acknowledgements. study was supported by bioMérieux, who provided all reagents

Aims. Periprosthetic joint infections (PJIs) and osteomyelitis are clinical challenges that are difficult to eradicate. Well-characterized large animal models necessary for testing and validating new treatment strategies for these conditions are lacking. The purpose of this study was to develop a rabbit model of chronic PJI in the distal femur. Methods. Fresh suspensions of Staphylococcus aureus (ATCC 25923) were prepared in phosphate-buffered saline (PBS) (1 × 10. 9. colony-forming units (CFUs)/ml). Periprosthetic osteomyelitis in female New Zealand white rabbits was induced by intraosseous injection of planktonic bacterial suspension into a predrilled bone tunnel prior to implant screw placement, examined at five and 28 days (n = 5/group) after surgery, and compared to a control aseptic screw group. Radiographs were obtained weekly, and blood was collected to measure ESR, CRP, and white blood cell (WBC) counts. Bone samples and implanted screws were harvested on day 28, and processed for histological analysis and viability assay of bacteria, respectively. Results. Intraosseous periprosthetic introduction of planktonic bacteria induced an acute rise in ESR and CRP that subsided by day 14, and resulted in radiologically evident periprosthetic osteolysis by day 28 accompanied by elevated WBC counts and histological evidence of bacteria in the bone tunnels after screw removal. The aseptic screw group induced no increase in ESR, and no lysis developed around the implants. Bacterial viability was confirmed by implant sonication fluid culture. Conclusion. Intraosseous periprosthetic introduction of planktonic bacteria reliably induces survivable chronic PJI in rabbits. Cite this article: Bone Joint Res 2021;10(3):156–165

Most patients treated at our clinical setting present during chronic osteomyelitis stage, which is anecdotally likely to be poly-microbial. Adults with poly-microbial infection have a predilection for gram-negative bacteria and anaerobes, a scenario that hypothetically leads to a higher morbidity of poly-microbial osteomyelitis following trauma. Our study looks into the epidemiology of poly-microbial osteomyelitis treated at our Tumour and Infection unit. Retrospective study of patients treated for osteomyelitis from 2016 to 2020. Records of eligible patients were retrieved for examination. Demographics such as age, sex and race were recorded. Clinical presentation, organisms cultured, including their anti-microbial sensitivities were documented. There were 63 participants in the study. 31 (49.21%) had mono-microbial osteomyelitis with 32 (50.79%) having poly-microbial osteomyelitis. Majority of the poly-microbial patients presented with a sinus (68.75%) mostly located in the tibia (50%). Multiple mixed pathogens (both gam-positive and gram-negative) were cultured in our patients and this comprised 71.21% of the total bacteria cultured. Staphylococcus Aureus was the commonest bacteria (30%) isolated, followed by Enterococcus faecalis (12%). The commonest gram-negative bacteriae cultured was Enterococcus cloacae (10%) followed by Acinetobacter baumannii (7%). Most enterobacteriacae species were sensitive to Ertapenem and Ceftazidime. A slightly higher incidence of poly-microbial osteomyelitis was found in our study than that reported in literature. Furthermore, our study demonstrated a wide variety of organisms found in poly-microbial osteomyelitis, with a large contribution made by gram-negative anaerobic rod-shaped bacteria

Aim. Rifampicin as a biofilm-active antibiotic drug has a significant role in the treatment of periprosthetic joint infection (PJI). However, rifampicin resistance is an increasing threat to PJI treatment. This study aimed to evaluate the prevalence of rifampicin resistant staphylococci over time and its association with infection-free survival after PJI in a single centre in Sweden. Methods. We included 238 PJIs in 238 patients who had undergone PJI revision surgery from 2001 to 2020 on whom the causative bacteria were staphylococci, and the agent was tested for rifampicin resistance. Data regarding agents, rifampicin resistance, treatment and outcome was obtained. Kaplan-Meier survival analysis and a Cox regression model with adjustment for age, sex, localisation (hip or knee) and type of prosthesis (primary or revision) were used to calculate infection-free survival rates and adjusted risk ratios (HRs) of the risk of treatment failure. Treatment failure was defined as any reoperation or suppression treatment with antibiotics due to prolonged infection. Results. Among the included 238 PJIs, 40 rifampicin-resistant staphylococci [93% Coagulase Negative Staphylococci (CoNS)] and 29 treatment failures were identified. The proportion of rifampicin resistant agents decreased from 25% in 2010–2015 to 12% in 2016–2020. The 2-year infection-free survival rates were 79.0% (95% CI 0.66–0.92) for the rifampicin resistant and 90% (95% CI 0.86–0.94) for the rifampicin sensitive group. Patients with PJI caused by rifampicin resistant bacteria had a significantly higher risk of treatment failure than those caused by sensitive bacteria (HR 2.5; 95% CI 1.0–6.2). Conclusions. The incidence of PJI caused by rifampicin resistant staphylococci decreased in Uppsala, Sweden over the past 20 years. PJI caused by rifampicin-resistant staphylococci has a two-fold risk for treatment failure compared to PJI caused by rifampicin-sensitive staphylococci, which stresses the importance of retaining rifampicin resistance low. Additionally, the increased risk of treatment failure when PJI is caused by a rifampicin-resistant bacteria warrants consideration of a more conservative treatment strategy

Background. Although described as a commensal bacterium with low pathogenicity, Cutibacterium acnes involvement has been reported in many clinical entities: infections associated with devices, such as shoulder prosthetic joint infections, osteosynthesis, breast implants or cerebrospinal fluid shunts. Various studies show that C. acnes grows as a biofilm, contributing to its persistence by allowing its escape from the action of the immune system and antibiotics. Purpose. Our aim was to assess the activity of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) on eight different well-characterized C. acnes strains after growth in biofilm mode. Methods. Eight susceptible strains of C. acnes were selected for this study, including two reference strains (ATCC6919 and ATCC11827) and six clinical strains. All C. acnes strains were studied using two different methods to study the biofilm production at different time points: the BioFilm Ring Test. ®. technique (early stages of adhesion) and the Crystal Violet (CV) method (mature biofilm). In a second step, the impact of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) was studied. For the CV technique, two types of tests were performed: preventive tests (addition of active substances and bacteria at the same time) and curative challenge tests (addition of active substances on a biofilm already formed after 48h). Transmission electron microscopy was performed to investigate the morphology modifications. Results. C. acnes isolates from phylotypes IA. 1. and IA. 2. , seem to produce more mature biofilm in the first stages of adhesion than other phylotypes. Curative assays were performed to evaluate the efficacy of antibiotics and Myrtacine. ®. on mature biofilm. Significant efficacy of Myrtacine. ®. at 0.03% was observed for C. acnes strains. Moreover, the combination of Myrtacine. ®. and doxycycline appears to decrease the total biofilm biomass. The effect of doxycycline as a preventive measure was minimal. On the contrary, a similar use of Myrtacine. ®. as early as 0.001% showed significant efficacy with a significant decrease in total biofilm biomass for all C. acnes strains. Transmission electron microscopy revealed a significantly decreased biofilm growth in treated bacteria with Myrtacine. ®. compared to untreated bacteria. Moreover, the total number of bacteria decreased as the concentration of Myrtacine. ®. increased suggesting also an antimicrobial effect. Conclusion. These results confirm the difference in biofilm producing ability depending on C. acnes phylotypes. These results suggest that Myrtacine. ®. may be a promising alternative antibacterial and anti-biofilm agent like peroxide de benzoyle to prevent shoulder prosthetic joint infection involving planktonic and biofilm C. acnes

Aim. The origin of surgical site and biomaterial-associated infection is still elusive. Microorganisms contaminating the wound may come from the air, the surgical team, or from the skin of the patient. Prior to surgery the skin of patients is disinfected, but bacteria deeper in the skin (e.g. in sweat glands or sebaceous glands), may not be reached. This study aims to assess a potential role of this intracutaneous bacterial reservoir in biomaterial-associated infection. Method. To study if cutaneous microbiota colonize the wound when released from the skin upon cutting, we isolated, quantified and identified aerobic and anaerobic bacteria from the skin of 99 patients undergoing trauma surgery, before and after skin disinfection, from the knife blades and from the wound directly after the first cut. Results. Ninety-nine percent of the patients were culture-positive before disinfection with chlorhexidine. Of these, 40% were still culture-positive after disinfection. Of these, 54% had a positive culture of the wound after the skin cut. Twenty percent of the patients with a negative culture after disinfection, nevertheless had a positive wound culture after cutting the skin. Staphylococcus epidermidis and Cutibacterium acnes were the most often cultured bacterial species. In 9%, more than 100 bacterial colonies were cultured from the wound, a dose that may cause biomaterial-associated infections. Conclusions. Bacteria residing in the skin and not eradicated by disinfection may enter the surgical wound upon cutting, resulting in contamination which may cause a biomaterial-associated infection. Use of two knifes likely reduces the risk of wound contamination

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Aims. Biofilm formation is one of the primary reasons for the difficulty in treating implant-related infections (IRIs). Focused high-energy extracorporeal shockwave therapy (fhESWT), which is a treatment modality for fracture nonunions, has been shown to have a direct antibacterial effect on planktonic bacteria. The goal of the present study was to investigate the effect of fhESWT on Staphylococcus aureus biofilms in vitro in the presence and absence of antibiotic agents. Methods. S. aureus biofilms were grown on titanium discs (13 mm × 4 mm) in a bioreactor for 48 hours. Shockwaves were applied with either 250, 500, or 1,000 impulses onto the discs surrounded by either phosphate-buffered saline or antibiotic (rifampin alone or in combination with nafcillin). The number of viable bacteria was determined by quantitative culture after sonication. Representative samples were taken for scanning electron microscopy. Results. The application of fhESWT led to a ten-fold reduction in bacterial counts on the metal discs for all impulse numbers compared to the control (p < 0.001). Increasing the number of impulses did not further reduce bacterial counts in the absence of antibiotics (all p > 0.289). Antibiotics alone reduced the number of bacteria on the discs; however, the combined application of the fhESWT and antibiotic administration further reduced the bacterial count compared to the antibiotic treatment only (p = 0.032). Conclusion. The use of fhESWT significantly reduced the colony-forming unit (CFU) count of a S. aureus biofilm in our model independently, and in combination with antibiotics. Therefore, the supplementary application of fhESWT could be a helpful tool in the treatment of IFIs in certain cases, including infected nonunions. Cite this article: Bone Joint Res 2021;10(1):77–84

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. The rise of multidrug-resistant bacteria and the decreasing efficacy of antibiotic therapy in successfully treating biofilm-associated infections are prompting the exploration of alternative treatment options. This study investigates the efficacy of different bioactive glass (BAG) formulations - alone or combined with vancomycin - to eradicate biofilm. Further, we study the influence of BAG on pH and osmotic pressure as important factors limiting bacterial growth. Method. Different BAG-S53P4 formulations were used for this study, including (a) BAG-powder (<45 μm), (b) BAG-granules (500–800 μm), (c) a cone-shaped BAG-scaffold and (d) two kinds of BAG-putty containing granules, with no powder (putty-A) or with additional powder (putty-B), and a synthetic binder. Inert glass beads were included as control. All formulations were tested in a concentration of 1750 g/ml in Müller-Hinton-Broth. Targeted bacteria included methicillin-resistant Staphylococcus aureus (MRSA) and epidermidis (MRSE). Vancomycin was tested at the minimum-inhibitory-concentration for each strain (1 µg/ml for MRSA; 2 μg/ml for MRSE). To investigate the antibiofilm effect of BAG alone or combined with vancomycin, 3 hour-old MRSA or MRSE biofilms were formed on porous glass beads and exposed to BAG ± vancomycin for 24h, 72h and 168h. After co-incubation, biofilm-beads were deep-washed in phosphate-buffered saline and placed in glass vials containing fresh medium. Recovering biofilm bacteria were detected by measuring growth-related heat production at 37°C for 24h by isothermal microcalorimetry. Changes in pH and osmotic pressure over time were assessed after co-incubation of each BAG formulation in Müller-Hinton-Broth for 0h, 24h, 72h and 168h. Results. All BAG formulations showed antibiofilm activity against MRSA and MRSE in a time-dependent manner, where longer incubation times revealed higher antibiofilm activity. BAG-powder and BAG-putty-B were the most effective formulations suppressing biofilm, followed by BAG-granules, BAG-scaffold and finally BAG-putty-A. The addition of vancomycin had no substantial impact on biofilm suppression. An increase in pH and osmotic pressure over time could be observed for all BAG formulations. BAG-powder reached the highest pH value of 12.5, whereas BAG-putty-A resulted in the lowest pH of 9. Both BAG-putty formulations displayed the greatest increase on osmotic pressure. Conclusions. BAG-S53P4 has demonstrated efficient biofilm suppression against MRSA and MRSE, especially in powder-containing formulations. Our data indicates no additional antibiofilm improvement with addition of vancomycin. Moreover, high pH appears to have a larger antimicrobial impact than high osmolarity. Acknowledgements. This work was supported by PRO-IMPLANT Foundation (Berlin, Germany). The tested materials were provided by Bonalive Biomaterials Ltd (Turku, Finland)

Aims. The management of periprosthetic joint infection (PJI) after total knee arthroplasty (TKA) is challenging. The correct antibiotic management remains elusive due to differences in epidemiology and resistance between countries, and reports in the literature. Before the efficacy of surgical treatment is investigated, it is crucial to analyze the bacterial strains causing PJI, especially for patients in whom no organisms are grown. Methods. A review of all revision TKAs which were undertaken between 2006 and 2018 in a tertiary referral centre was performed, including all those meeting the consensus criteria for PJI, in which organisms were identified. Using a cluster analysis, three chronological time periods were created. We then evaluated the antibiotic resistance of the identified bacteria between these three clusters and the effectiveness of our antibiotic regime. Results. We identified 129 PJIs with 161 culture identified bacteria in 97 patients. Coagulase-negative staphylococci (CNS) were identified in 46.6% cultures, followed by Staphylococcus aureus in 19.8%. The overall resistance to antibiotics did not increase significantly during the study period (p = 0.454). However, CNS resistance to teicoplanin (p < 0.001), fosfomycin (p = 0.016), and tetracycline (p = 0.014) increased significantly. Vancomycin had an 84.4% overall sensitivity and 100% CNS sensitivity and was the most effective agent. Conclusion. Although we were unable to show an overall increase in antibiotic resistance in organisms that cause PJI after TKA during the study period, this was not true for CNS. It is concerning that resistance of CNS to new antibiotics, but not vancomycin, has increased in a little more than a decade. Our findings suggest that referral centres should continuously monitor their bacteriological analyses, as these have significant implications for prophylactic treatment in both primary arthroplasty and revision arthroplasty for PJI. Cite this article: Bone Joint J 2021;103-B(6 Supple A):171–176

Aims. Biopsy of the periprosthetic tissue is an important diagnostic tool for prosthetic joint infection (PJI) as it enables the detection of the responsible microorganism with its sensitivity to antibiotics. We aimed to investigate how often the bacteria identified in the tissue analysis differed between samples obtained from preoperative biopsy and intraoperative revision surgery in cases of late PJI; and whether there was a therapeutic consequence. Methods. A total of 508 patients who required revision surgery of total hip arthroplasty (THA) (n = 231) or total knee arthroplasty (TKA) (n = 277) because of component loosening underwent biopsy before revision surgery. The tissue samples collected at biopsy and during revision surgery were analyzed according to the criteria of the Musculoskeletal Infection Society (MSIS). Results. In total, 178 (113 THA, 65 TKA) were classified as infected. The biopsy procedure had a sensitivity of 93.8%, a specificity of 97.3%, a positive predictive value (PPV) of 94.9%, a negative predictive value (NPV) of 96.7%, and an accuracy of 96.1%. Of the 178 infected patients, 26 showed a difference in the detected bacteria from the biopsy and the revision surgery (14.6%). This difference required a change to antibiotic therapy in only two cases (1.1%). Conclusion. Biopsy is a useful tool to diagnose PJI, but there may be a difference in the detected bacteria between the biopsy and revision surgery. However, this did not affect the choice of antibiotic therapy in most cases, rendering the clinical relevance of this phenomenon as low. Cite this article: Bone Joint J 2020;102-B(3):329–335

Aims. There is a considerable challenge in treating bone infections and orthopaedic device-associated infection (ODAI), partly due to impaired penetration of systemically administrated antibiotics at the site of infection. This may be circumvented by local drug administration. Knowledge of the release kinetics from any carrier material is essential for proper application. Ceftriaxone shows a particular constant release from calcium sulphate (CaSO. 4. ) in vitro, and is particularly effective against streptococci and a large portion of Gram-negative bacteria. We present the clinical release kinetics of ceftriaxone-loaded CaSO. 4. applied locally to treat ODAI. Methods. A total of 30 operations with ceftriaxone-loaded CaSO. 4. had been performed in 28 patients. Ceftriaxone was applied as a single local antibiotic in 21 operations and combined with vancomycin in eight operations, and in an additional operation with vancomycin and amphotericin B. Sampling of wound fluid was performed from drains or aspirations. Ceftriaxone concentrations were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results. A total of 37 wound fluid concentrations from 16 operations performed in 14 patients were collected. The ceftriaxone concentrations remained approximately within a range of 100 to 200 mg/l up to three weeks. The median concentration was 108.9 mg/l (interquartile range 98.8 to 142.5) within the first ten days. No systemic adverse reactions were observed. Conclusion. Our study highlights new clinical data of locally administered ceftriaxone with CaSO. 4. as carrier material. The near-constant release of ceftriaxone from CaSO. 4. observed in vitro could be confirmed in vivo. The concentrations remained below known local toxicity thresholds. Cite this article: Bone Joint Res 2022;11(11):835–842

Periprosthetic joint infections (PJI) are one of the most common reasons for orthopedic revision surgeries. In previous studies, it has been shown that silver modification of titanium (Ti-6Al-4V) surfaces by PMEDM (powder mixed electrical discharge machining) has an antibacterial effect on Staphylococcus aureus adhesion. Whether this method also influences the proliferation of bacteria has not been investigated so far. Furthermore, the effect is only limitedly investigated on the ossification processes. Therefore, the aim of this work is to investigate the antibacterial effect as well as the in vitro ossification process of PMEDM machined surfaces modified by integration of silver. In this study, we analyzed adhesion and proliferation of S. aureus in comparison to of surface roughness, silver content and layer thickness of the silver-integrated-PMEDM surfaces (N = 5). To test the in vitro ossification, human osteoblasts (SaOs-2) and osteoclasts (differentiated from murine-bone-marrow-macrophages) were cultured on the silver surfaces (N = 3). We showed that the attachment of S. aureus on the surfaces was significantly lower than on the comparative control surfaces of pure Ti-6Al-4V without incorporated silver, independently of the measured surface properties. Bacterial proliferation, however, was not affected by the silver content. No influence on the in vitro ossification was observed, whereas osteoclast formation was drastically reduced on the silver-modified surfaces. We showed that 1 to 3% of silver in the surface layer significantly reduced the adhesion of S. aureus, but not the proliferation of already attached bacteria. At the same time, no influence on the in vitro ossification was observed, while no osteoclasts were formed on the surface. Therefore, we state that PMEDM with simultaneous silver modification of the machined surfaces represents a promising technology for endoprostheses manufacturing to reduce infections while at the same time optimizing bone ingrowth

Prosthetic joint infection (PJI) is a serious complication following joint replacement. Antiseptic solutions are often used for intraoperative wound irrigation particularly in cases of revision for PJI. Antiseptic irrigation is intended to eradicate residual bacteria which may be either free floating or in residual biofilm although there is no clear clinical efficacy for its use. Also, reviewing the scientific literature there is discordance in in vitro results where some studies questions antiseptic efficacy whilst others suggest that even at low concentration antiseptic agents are effective at eradicating bacterial biofilms. The aim of this in vitro study was to establish the efficacy of undiluted antiseptic agents at eradication of a typical PJI forming biofilm and determine the importance of an antiseptic neutralisation step in this assessment. Mature Staphylococcus epidermidis biofilms grown on TiAl6V4 discs were submerged in chlorohexidine (CHL) gluconate 4%, povidone-iodine (PI) 10% or phosphate-buffered saline (PBS) control solution. The discs were then rinsed, the biofilm bacteria suspended in solution using sonication and vortexing, and the viable count (CFU/ml) of the bacterial suspensions determined. The rinse/suspension solution was either (a) PBS or (b) Dey-Engley neutralization broth (NB). When PBS was used to rinse/suspend the biofilm a highly significant, 7.5 and 4.1, mean log reduction in biofilm vitality was observed from the control, for CHL 4% and PI 10%, respectively. However, when NB was the rinse/suspension solution the apparent antiseptic biofilm eradication efficacy was replaced with a statistically significant but clinically irrelevant less the one log-reduction in biofilm vitality. Clinical antiseptic agents are ineffective at eradicating S. epidermidis biofilm in an in vitro PJI model and absence of a neutralisation step gives the false impression of efficacy. Antiseptics alone are an ineffective treatment for biofilm related PJI and no substitute for meticulous debridement

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently 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 their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

Aim. To describe the risk factors, microbiology and treatment outcome polymicrobial prosthetic joint infections (PJI) compared to monomicrobial PJI. Methods. Between January 2011 and December 2021, a total of 536 patients were diagnosed with PJI at our institution. Clinical records were revised, and 91(16.9%) had an isolation of two or more pathogens. Age, sex, previous conditions, Charlson comorbidity score, previous surgery, PJI diagnosis and surgical and antibiotic treatment, from the index surgery onwards were reviewed and compared between groups. Results. Polymicrobial PJI success rate was 57.1%, compared to 85.3% of the monomicrobial PJI(p=0.0036). There were no statistically significative differences between acute and chronic infections. In terms of related risk factors, revision surgery(p=0.0002), fracture(p=0.002), tobacco(p=0.0031) and Body Mass Index (BMI) between 20–25(p=0.0021) were associated to monomicrobial PJI, whereas overweight(p=0.005) and obesity(p=0.02) were linked to polymicrobial PJI. Regarding pathogens, the most common microorganism isolated in monomicrobial was S.aureus (33.5%), followed by S. epidermidis(20%) and gram negative bacilli (12.2%); while S. epidermidis(56%), gram negative bacilli (41.8%) and E.colli (30.8%) were the most frequent in the polymicrobial PJI. Enterococci(p=0.0008), S. epidermidis(p=0.007), E.colli (p=0.0008), gram negative bacilli (p=0.00003) and atypical bacteria (p=0.00001) statistically significative linked to polymicrobial PJI; while S.aureus (p=0.018) was related to monomicrobial PJI. Conclusion. Polymicrobial PJI showed worse outcome compared to monomicrobial PJI in our cohort. In terms of risk factors, overweight, obesity and some pathogens like gram negative bacilli, atypical bacteria, enterococci, S. epidermidis and E.colli were associated with Polymicrobial PJI

We developed a novel silorane-based biomaterial (SBB) for use as an orthopedic cement. SBB is comprised of non-toxic silicon-based monomers, undergoes non-exothermic polymerization, and has weight-bearing strength required of orthopedic cements. We sought to compare the antibiotic release kinetics of this new cement to that of commercially available PMMA bone cement. We also evaluated each material's inherent propensity to support the attachment of bacteria under both static and dynamic conditions. One gram of either rifampin or vancomycin was added to 40g batches of PMMA and SBB. Pellets were individually soaked in PBS. Eluate was collected and tested daily for 14 days using HPLC. Compressive strength and modulus were tested over 21 days. Bioassays were used to confirm the bioactivity of the antibiotics eluted. We measured the growth and maturation of staphylococcus aureus (SA) biofilm on the surface of both PMMA and SBB disks over the course of 72 hours in a static well plate and in a dynamic biofilm reactor (CDC Biofilm Reactor). N=4 at 24, 48, and 72 hours. A luminescent strain of SA (Xen 29) was employed allowing imaging of bacteria on the discs. SBB eluted higher concentrations of vancomycin than did PMMA over the course of 14 days (p<0.001). A significant 55.1% greater day 1 elution was observed from SBB. Silorane cement was able to deliver rifampin in clinically favorable concentrations over 14 days. On the contrary, PMMA was unable to deliver rifampin past day 1. The incorporation of rifampin into PMMA severely reduced its mechanical strength (p<0.001) and modulus (p<0.001). Surface bacterial radiance of PMMA specimens was significantly greater than that of SBB specimens at all time points (p<0.05). The novel silorane-based cement demonstrated superior antibiotic release and, even without antibiotic incorporation, demonstrated an innate inhabitation to bacterial attachment and biofilm

There is a lack of carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotic for Staphylococcus aureus deep bone infections (DBIs). RIF is also associated with systemic side effects, and known for causing rapid development of antibiotic resistance when given as monotherapy. We evaluated a clinically usedbi-phasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). It was hypothesized that this combined approach could provide improved biofilm eradication and prevent the development of RIF resistance. Methods: 1) Biofilm eradication: Using a modified crystal violet staining biofilm quantification method, the antibiotics released at different time points (Day 1, 3, 7, 14, 21, 28 and 35) from the hemispherical pellets of CaS/HA(500 mg)-VAN (24.57 mg) / GEN (10.35 mg) composites with or without RIF (8.11 mg) were tested for their ability to disrupt the preformed 48-h old biofilms of S. aureus ATCC 25923, and S. aureus clinical strain P-3 in 96-well microtitre plate. For each tested group of antibiotic fractions, five separate wells were used (n=5). 2) Testing for resistance development: Similar to the method mentioned above the 48-h biofilm embeded bacteria exposed to antibiotic fractions from different time points continuously for 7 days. The biofilms remained were then tested for RIF resistant strains of bacteria. Overall, there was clear antibiofilm biofilm activity observed with CaS/HA-VAN/GEN+RIF combinations compared with CaS/HA-VAN/GEN alone. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with combinations of CaS/HA-VAN/GEN+RIF. Enhanced antibiofilm effects without development of RIF resistance indicates that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for additional local delivery in clinically demanding DBIs. Acknowledgement: We deeply acknowledge the Royal Fysiographic Society of Lund, Landshövding Per Westlings Minnesfond and the Stina and Gunnar Wiberg fond for financial support

Irrigation with antiseptic agents, antibiotics, and surfactants are used for treatment and prevention of infections. Despite desirable microbicidal actions, studies have demonstrated cytotoxic effects on host tissue that may impair healing. This study investigated the extent of tissue damage caused by commonly used irrigation solutions in the presence or absence of infection. Air pouches created in 60 balb/c mice were divided into two groups (n=30): infected with Staphylococcus aureus and control. One week later the infected group was subdivided into 5 subgroups (n=6) based on irrigation solutions and by day 0 (immediately) and day7 after irrigation (n=3). Solutions included Saline, Bacitracin, Clorpactin, Irrisept and Bactisure. In infected group wash fluid was collected for quantitative analysis of bacterial growth. At the specified times mice were sacrificed, pouch tissue sent for histology, and sections analyzed for inflammation, necrosis, and edema. Inflammation decreased in infected vs sterile pouches for all solutions except Bacitracin day 0 and for all solutions day 7 with significance in all except Bacitracin (p<0.05). On day 0, necrosis increased in infected vs sterile pouches in Bacitracin (p=0.006), Irrisept (p=0.18), or Bactisure (p=0.07); however, on day 7, necrosis significantly decreased in infected pouches for all solutions (p<0.05) except for Clorpactin (p=0.18). Edema decreased in infected vs sterile pouches on day 0 for all solutions with significance in saline, Irrisept, and Bacitracin (p<0.05). On day 7, infected pouches had decreased edema in saline, Bacitracin, and Bactisure (p<0.05) and increased in Irrisept (p<0.05) and Clorpactin (p=0.069) compared to sterile pouches. Bacterial culture of washouts demonstrated that Clorpactin, Irrisept and Bactisure controlled the infection, whereas saline and Bacitracin showed bacterial multiplication 3.9 × 10^7 CFU/ml and 6.7 × 10^7 CFU/ml respectively. Bacitracin wash showed significantly more bacteria growth compared to Clorpactin (p=0.024), Irrisept (p=0.025) and Bactisure (p=0.025). Tissue damage varied with irrigation solutions and the presence or absence of infection. Presence of bacteria appeared to lead to less tissue inflammation and edema. Tissue necrosis varied over time with different solutions. Surgeons must weigh risks and benefits when selecting solutions and determining when to irrigate

Aim. Identifying the optimal agent for irrigation for periprosthetic joint infection remains challenging as there is limited data. The ideal solution should have minimal cytotoxicity while maintaining bactericidal activity. We developed a novel activated-zinc solution containing zinc-chloride (ZnCl. 2. ) and sodium-chlorite (NaClO. 2. ). The purpose of this study was 1.) to investigate the antimicrobial efficacy of 2 concentrations (“CZ1”, “CZ2”) against Staphylococcus aureus and Pseudomonas aeruginosa and 2.) to evaluate untoward effects of the solution on local wound tissue 24 hours after solution exposure in pig wound models. Method. The study was conducted and reported in accordance to ARRIVE guidelines. We created twenty-four 1.5cm wounds on the back of a Yorkshire-cross pig. Wounds were inoculated with standardized Pseudomonas and S. aureus. 8 wounds were designated as controls (inoculum without treatment), 8 treated with CZ1, and 8 with CZ2. Punch biopsies were taken 1 hour after treatment and bacteria quantified. Wound necrosis/neutrophil infiltrate was measured 24-hours post-exposure. Results. After 1-hour, the control, CZ1 and CZ2 wounds had total bacteria of 5.7, 2.8 and 3.5 logCFU/g, respectively (p=0.017). The control, CZ1 and CZ2 wounds had S. aureus of 5.3, 2.3 and 1.6 logCFU/g, respectively (p=0.009). The control, CZ1 and CZ2 wounds had Pseudomonas of 5.5, 0.3 and 0.0 logCFU/g, respectively (p=0.000). After 24 hours of exposure to CZ1 and CZ2, there was no statistically significant increased necrosis (p=0.12, p=0.31, respectively). CZ1 had increased, moderate neutrophil infiltrate (p=0.04) when compared to controls, however CZ2 was not significant (p=0.12). Conclusions. Our novel solution demonstrated 99.5–99.9% reduction in total bacteria, 99.9–99.98 % reduction in S. aureus, and 100% eradication of Pseudomonas 1-hour after exposure, without significantly increased necrosis and no-to-minimally-increased neutrophil infiltrate. This novel solution may provide another significant tool in the arsenal to treat and/or prevent PJI and other wound infections

Aim. Several local antibiotic-eluting drug delivery systems have been developed to treat bacterial bone infections. However, available systems have significant shortcomings, including suboptimal drug-release profiles with a burst followed by subtherapeutic release, which may lead to treatment failure and selection for drug resistance. Here, we present a novel injectable, biocompatible, in situ-forming depot, termed CarboCells, which can be fine-tuned for the desired antibiotic-release profile. The CarboCell technology has flexible injection properties that allow surgeons to accurately place antibiotic-eluting depots within and surrounding infectious sites in soft tissue and bones. The CarboCell technology is furthermore compatible with clinical image-guided injection technologies. These studies aimed to determine the therapeutic potential of CarboCell formulations for treatment of implant-associated osteomyelitis by mono- and dual antimicrobial therapy. Methods. The solubility and stability of several antibiotics were determined in various CarboCell formulations, and in vitro drug release was characterized. Lead candidates for antimicrobial therapy were selected using a modified semi-solid biofilm model with 4-day-matured Staphylococcus aureus biofilm (osteomyelitis-isolate, strain S54F9). Efficacy was investigated in a rat implant-associated osteomyelitis model established in the femoral bone by intraosseous implantation of a stainless-steel pin with 4-day-old in vitro-matured S. aureus biofilm. CarboCells were injected subcutaneously at the femur, and antimicrobial efficacy was evaluated 7 days post-implantation. Lead formulations were subsequently tested in a well-established translational implant-associated tibial S. aureus osteomyelitis pig model. Infection was established for 7 days before revision surgery consisting of debridement, washing, implantation of a new stainless-steel pin, and injection of antibiotic-releasing CarboCells into the debrided cavity and in the surrounding bone- and soft-tissue. Seven days post-revision, pigs were euthanized, and samples were collected for microbial and histopathological evaluation. Results. Lead antimicrobial agents were soluble in high concentrations and were stable in CarboCell formulations. Three combinations completely eradicated bacteria in the in vitro semi-solid biofilm model. In the rat osteomyelitis model, CarboCell formulations of the lead combinations also eradicated bacteria in bone and implant in several rats and significantly reduced infection in all treated rats. In the pig model, CarboCell antimicrobial monotherapy demonstrated promising therapeutic efficacy, including complete eradication of infection in bone and implants in several pigs and significantly reduced bacterial burden in others. Conclusions. Using the CarboCell technology for antimicrobial delivery exert substantial loco-regional efficacy. The attractive sustained high-dose antibiotic release profile combined with the flexible injection technology allows surgeons to accurately place effective drug-eluting depots in key areas not accessible to competing technologies

Favoring osseointegration and avoiding bacterial contamination are the key challenges in the design of implantable devices for orthopedic applications. To meet these goals, a promising route is to tune the biointerface of the devices, that can regulate interactions with the host cells and bacteria, by using nanostructured antibacterial and bioactive coatings. Indeed, the selection of adequate metal-based coatings permits to discourage infection while avoiding the development of bacterial resistance and nanostructuring permits to tune the release of the antimicrobial compounds, allowing high efficacy and decreasing possible cytotoxic effects. In addition, metal-doped calcium phosphates-based nanostructured coatings permit to tune both composition and morphology of the biointerfaces, allowing to regulate host cells and bacteria response. To tune the biointerfaces of implantable devices, nanostructured coatings can be used, but their use is challenging when the substrate is heat-sensitive and/or porous. Here, we propose the use of Ionized Jet Deposition (IJD) to deposit metallic and ion-doped calcium phosphates materials onto different polymeric substrates, without heating and damaging the substrate morphology. 3D printed scaffolds in polylactic acid (PLA) and polyurethane (PU), and electrospun matrices in polycaprolactone (PCL) and PLA were used as substrates. Biogenic apatite (HA), ion doped (zinc, copper and iron) tricalcium phosphate (TCP) and silver (Ag) coatings were obtained on porous and custom-made polymeric substrates. Chemical analyses confirmed that coatings composition matches that of the target materials, both in terms of main phase (HA or TCP) and ion doping (presence of Cu, Zn or Fe ion). Deposition parameters, and especially its duration time, influence the coating features (morphology and thickness) and substrate damage. Indeed, SEM/EDS observations show the presence of nanostructured agglomerates on substrates surface. The dimensions of the aggregates and the thickness of the coating films increase increasing the deposition time, without affecting the substrate morphology (no porosity alteration or fibers damaging). The possible substrate damage is influenced by target and substrate material, but it can be avoided modulating deposition time. Once the parameters are optimized, the models show suitable in vitro biological efficacy for applications in bone models, regenerative medicine and infection. Indeed, HA-based coatings favor cells adhesion on printed and electrospun fibers. For antibacterial applications, the ion doped TCP coatings can reduce the bacterial growth and adhesion (E.coli and S.aureus) on electrospun matrices. To conclude, it is possible achieve different properties applying nanostructured coatings with IJD technique on polymeric substrates, modulating deposition conditions to avoid substrate damage

Aim. Infections represent a serious threat to the successful utilization of implants in modern medicine. Implant-associated infections are difficult to treat, because they involve biofilms that protect bacteria from the immune system and harbour antibiotic-tolerant persister cells. In this work, we developed an antibody-drug conjugate (ADC) containing the anti-neoplastic drug mitomycin C (MMC) as a novel treatment paradigm for implant-associated infections. MMC was chosen as it is a potent antimicrobial against biofilms and its synthesis into an ADC was chosen to alleviate toxicity. Following development and synthesis of the ADC, stability and release of MMC was measured. We then used the ADC to kill bacteria in suspension and in biofilms, in vitro and in vivo. Method. Mitomycin C was conjugated to a commercially available antibody against S. aureus via a disulfide linkage, with a drug release occurred via thiol-disulfide exchange. ADC as tested against S. aureus under various growth conditions (planktonic, persisters and biofilm). In vitro toxicity of ADC vs MMC was measured using a human cell line (MOLT-4). Finally, two independent in vivo experiments were performed in a murine implant-associated osteomyelitis model. In experiment one ADC treatment was compared NaCl, vancomycin and vancomycin + ADC (n=10 for all groups). Subsequently, ADC was compared to NaCl, the antibody used in the ADC construction, MMC and a novel ADC constructed with a non-S. aureus antibody (n=10 for all groups). All treatments were started day 7 post inoculation and were administered for 3 days. CFU enumeration was done following sonication to quantify bacterial load. Results. Drug release could be triggered on demand with N-acetyl cysteine and release occurred, once in contact with free thiols on S. aureus cell surface. The ADCs exhibited a concentration-dependent antimicrobial effect against S. aureus with doses exceeding 0.5 mg/l reducing amount of CFU to below detection limit (p< 0.001). 15 minutes exposure to ADC resulted in an approx. 2 log CFU/ml reduction compared to untreated biofilms (p < 0.01). In vivo ADC treatment was effective compared to NaCl treatment and the vancomycin treatment (p≤ 0.001). Further ADC and MMC treatment were comparable in efficacy, but both were superior than NaCl, pure antibody and the non-specific ADC (p≤ 0.05). Finally, in vitro cytotoxicity was significantly lower for ADC than MMC. Conclusions. In this study we have demonstrated that ADCs can be a novel treatment approach to combat implant-associated infections caused by S. aureus

Aim. Fungal periprosthetic joint infections are difficult to treat and often associated with a limited outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules for bacteria. However, in vivo modeling of biofilm-associated fungi models are very rare. Furthermore, due to ethical restrictions, 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 with bacteria. This model organism was not used for fungi biofilm infection yet. Thus, we aimed to establish G. mellonella as in vivo model to study fungal implant infections using Candida albicans as model organism and to test anti-fungal medication. Method. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. For the infection process, implants were pre-incubated in specified fungal growth culture Candida albicans at 1×10. 7. CFU/ml for 30 min at 150 rpm shaking conditions. Later, these implants were washed with 10ml PBS and implanted in the larvae as mentioned. To analyze the susceptibility of the implant-associated fungal infections towards anti fungal compounds, the larvae were treated with amphotericin B, fluconazole and voriconazole after 24h of implantation. The effect of anti-fungal compounds was measured in terms of survival observation for 5 days and fungal load in larvae on 2. nd. day. To reveal the fungal biofilm formation on implant, the implants were removed on day 3 and processed for SEM analysis. Results. Pre-incubated K-wire caused the Candida infection and observed the death of the larvae. The treatment with antifungal compounds recovered the larvae from the implant-infection, except in case of Voriconazole. However, the recovery with treatment of anti fungal compounds was not effective as the larvae with planktonic infection, which highlights typical biofilm phenotype. Further, the treatment with anti-fungal compounds with Amphotericin B and Fluconazole reduced the fungal load in larvae tissue. The SEM analysis revealed the formation fungal biofilm with hyphae and spores associated with larvae tissue on implant surface. Conclusions. The results from survival analysis, antifungal treatment and SEM analysis are very promising to use of G. mellonella as in vivo model to study fungal infections on implanted materials. Our study highlights the use of G. mellonella larvae as alternative in vivo model to study implant-associated fungal infections that reduces the use of the higher mammals

Aim. To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5–15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum. Method. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5–15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry. In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 10. 4. CFU S. aureus (S54F9), Group B: seven days old 5–15 µm aggregate-inoculum; 10. 4. CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed. Results. The separation of aggregates into different size fractions was done successfully by the filtering method. Isothermal microcalorimetry showed, a delayed Time-to-peak metabolic activity of the aggregate-inoculum compared to the planktonic inoculum. S. aureus was isolated from subcutis, bone and implants from all animals in groups A and B. Both group A animals showed osteomyelitis at gross inspection with suppuration and sequestration, while groups B and C animals had no macroscopic lesions. From CT scans, both group A animals also showed positive signs of osteomyelitis, i.e., osteolysis, while only one animal in group B did, and none in group C. Histopathological examination of the bones showed more extensive inflammation in group A animals compared to those in group B, which showed more osteoid formation. Conclusions. Formation and separation of low metabolism bacterial aggregates into different size fractions was possible. The aggregates can be used as inoculum in the porcine IAO model, with microbiological re-isolation from both implants and tissue. Furthermore, the aggregates caused a less aggressive IAO, than the planktonic counterparts. Using aggregated bacteria as inoculum appears to be more relevant to the clinical situation of infecting bacteria

Aim. To describe the management of PJI due to S. aureus in CRIOAcs in 2019 and to particularly focus on the evaluation of the efficacy of DAIR regarding control of infection and risk factors for failure up to 12 months. Method. Thirteen CRIOAcs were selected to participate to the study. Data concerning the management of all the PJI in the year 2019 were retrospectively collected and registered in eCRFs. Inclusion criteria were: ≥ 18 years old patients with S. aureus ± other bacteria (in per surgical procedure sample); knee or hip PJI and with clinical signs of infection. Patients treated with bacteriophages were excluded. All eligible patients were notified by an information letter. Patients treated by the DAIR procedure were selected, and rate of control of infection (no inflammatory local signs or no new surgical procedure or no S. aureus in case of puncture) was analyzed using Kaplan Meier method and risk factors for failure at 12 months were assessed using Cox regression model. Results. A total of 978 PJI were managed in the 9 CRIOAcs, including 238 hip and knee PJI due to S. aureus and 79 to S. aureus plus another bacteria. Among all of them, 154 were managed with DAIR, and 100 fulfilled inclusion criteria, notifying no opposition to their data collection. The median age was 73.0 years; 57% were male, the median Charlson score was 4.0; 66% had hip PJI. A total of 45 failure were observed during the period studied. At 12 months, the control rate was 58. 7% [36.5–75.4], 49.3% [34.3–62.7] in in early and late PJI respectively according to Tsukuyama classification and 49.6% [30.5–66.1], 54.1% [37.7 – 68.0] in early and delayed/late PJI respectively according to Zimmerli classification, 56.6% [39.5–70.5] in case of mobile part exchange, 53.4% [35.3–68.5] for MRSA PJI and 63.4% [50.5–73. 8] in patients treated with rifampicin. No rifampicin intake was the only significative risk factor for failure in univariate analysis (HR=0.31 (0.17–0.57), p=0.0002), and remained significant after adjustment on Charlson score (aHR=0.34 (0.18–0.64), p=0.0008). Conclusions. The DAIR procedure is frequently performed in patients with acute and late PJI, and is associated with a high rate of failure, especially for patients who cannot receive rifampin. There is a strong rational to assess the use of bacteriophages during the DAIR, as bacteriophages have antibiofilm activity in vitro, and could improve the efficacy of the DAIR to control the disease

Objectives. The purpose of this study was to examine the bactericidal efficacy of hydrogen peroxide (H. 2. O. 2. ) on Cutibacterium acnes (C. acnes). We hypothesize that H. 2. O. 2. reduces the bacterial burden of C. acnes. Methods. The effect of H. 2. O. 2. was assessed by testing bactericidal effect, time course analysis, growth inhibition, and minimum bactericidal concentration. To assess the bactericidal effect, bacteria were treated for 30 minutes with 0%, 1%, 3%, 4%, 6%, 8%, or 10% H. 2. O. 2. in saline or water and compared with 3% topical H. 2. O. 2. solution. For time course analysis, bacteria were treated with water or saline (controls), 3% H. 2. O. 2. in water, 3% H. 2. O. 2. in saline, or 3% topical solution for 5, 10, 15, 20, and 30 minutes. Results were analyzed with a two-way analysis of variance (ANOVA) (p < 0.05). Results. Minimum inhibitory concentration of H. 2. O. 2. after 30 minutes is 1% for H. 2. O. 2. prepared in saline and water. The 3% topical solution was as effective when compared with the 1% H. 2. O. 2. prepared in saline or water. The controls of both saline and water showed no reduction of bacteria. After five minutes of exposure, all mixtures of H. 2. O. 2. reduced the percentage of live bacteria, with the topical solution being most effective (p < 0.0001). Maximum growth inhibition was achieved with topical 3% H. 2. O. 2. . Conclusion. The inexpensive and commercially available topical solution of 3% H. 2. O. 2. demonstrated superior bactericidal effect as observed in the minimum bactericidal inhibitory concentration, time course, and colony-forming unit (CFU) inhibition assays. These results support the use of topical 3% H. 2. O. 2. for five minutes before surgical skin preparation prior to shoulder surgery to achieve eradication of C. acnes for the skin. Cite this article: P. Hernandez, B. Sager, A. Fa, T. Liang, C. Lozano, M. Khazzam. Bactericidal efficacy of hydrogen peroxide on Cutibacterium acnes. Bone Joint Res 2019;8:3–10. DOI: 10.1302/2046-3758.81.BJR-2018-0145.R1

Objectives. Prosthetic joint infection (PJI) is the most common cause of arthroplasty failure. However, infection is often difficult to detect by conventional bacterial cultures, for which false-negative rates are 23% to 35%. In contrast, 16S rRNA metagenomics has been shown to quantitatively detect unculturable, unsuspected, and unviable pathogens. In this study, we investigated the use of 16S rRNA metagenomics for detection of bacterial pathogens in synovial fluid (SF) from patients with hip or knee PJI. Methods. We analyzed the bacterial composition of 22 SF samples collected from 11 patients with PJIs (first- and second-stage surgery). The V3 and V4 region of bacteria was assessed by comparing the taxonomic distribution of the 16S rDNA amplicons with microbiome sequencing analysis. We also compared the results of bacterial detection from different methods including 16S metagenomics, traditional cultures, and targeted Sanger sequencing. Results. Polymicrobial infections were not only detected, but also characterized at different timepoints corresponding to first- and second-stage exchange arthroplasty. Similar taxonomic distributions were obtained by matching sequence data against SILVA, Greengenes, and The National Center for Biotechnology Information (NCBI). All bacteria isolated from the traditional culture could be further identified by 16S metagenomics and targeted Sanger sequencing. Conclusion. The data highlight 16S rRNA metagenomics as a suitable and promising method to detect and identify infecting bacteria, most of which may be uncultivable. Importantly, the method dramatically reduces turnaround time to two days rather than approximately one week for conventional cultures. Cite this article: M-F. Chen, C-H. Chang, C. Chiang-Ni, P-H. Hsieh, H-N. Shih, S. W. N. Ueng, Y. Chang. Rapid analysis of bacterial composition in prosthetic joint infection by 16S rRNA metagenomic sequencing. Bone Joint Res 2019;8:367–377. DOI: 10.1302/2046-3758.88.BJR-2019-0003.R2

Aims. Prompt and sufficient broad-spectrum empirical antibiotic treatment is key to preventing infection following open tibial fractures. Succeeding co-administration, we dynamically assessed the time for which vancomycin and meropenem concentrations were above relevant epidemiological cut-off (ECOFF) minimal inhibitory concentrations (T > MIC) in tibial compartments for the bacteria most frequently encountered in open fractures. Low and high MIC targets were applied: 1 and 4 µg/ml for vancomycin, and 0.125 and 2 µg/ml for meropenem. Methods. Eight pigs received a single dose of 1,000 mg vancomycin and 1,000 mg meropenem simultaneously over 100 minutes and 10 minutes, respectively. Microdialysis catheters were placed for sampling over eight hours in tibial cancellous bone, cortical bone, and adjacent subcutaneous adipose tissue. Venous blood samples were collected as references. Results. Across the targeted ECOFF values, vancomycin displayed longer T > MIC in all the investigated compartments in comparison to meropenem. For both drugs, cortical bone exhibited the shortest T > MIC. For the low MIC targets and across compartments, mean T > MIC ranged between 208 and 449 minutes (46% to 100%) for vancomycin and between 189 and 406 minutes (42% to 90%) for meropenem. For the high MIC targets, mean T > MIC ranged between 30 and 446 minutes (7% to 99%) for vancomycin and between 45 and 181 minutes (10% to 40%) for meropenem. Conclusion. The differences in the T > MIC between the low and high targets illustrate how the interpretation of these results is highly susceptible to the defined MIC target. To encompass any trauma, contamination, or individual tissue differences, a more aggressive dosing approach may be considered to achieve longer T > MIC in all the exposed tissues, and thereby lower the risk of acquiring an infection after open tibial fractures. Cite this article: Bone Joint Res 2022;11(2):112–120

Introduction and Objective. Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. Materials and Methods. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined. Results. The resulting hybrid coating of about 1 μm in thickness is composed of amorphous calcium phosphate and carbonated poorly crystalline hydroxyapatite, wrapped within chitosan/hyaluronic acid polysaccharide complex. Hybrid coated membrane possesses excellent bioactivity and capability of inducing an overwhelmingly positive response of MSCs and monocytes in favor of bone regeneration. Furthermore, the antibacterial experiments showed that the hybrid coating provides contact-killing properties by disturbing the cell wall integrity of Gram-positive and Gram-negative bacteria. Its combination with MSCs, able to release antibacterial agents and mediators of the innate immune response, constitutes an excellent strategy for fighting bacteria. A preclinical in vivo study was therefore conducted in rat calvaria bone defect. μCT reconstructions showed that hybrid coated membrane favored bone regeneration, as we observed a two-fold increase in bone volume / total volume ratios vs. uncoated membrane. The histological characterizations revealed the presence of mineralized collagen (Masson's Trichrome and Von Kossa stain), and immunohistochemistry analysis highlighted a bone vascularization at 8 weeks post-implantation. However, second harmonic generation analysis showed that the newly formed collagen was not fully organized. Despite a significant increase in the elastic modulus of the newly formed bone with hybrid coated membrane (vs. uncoated membrane), the obtained values were lower than those for native bone (approximately 3 times less). Conclusions. These significant data shed light on the regenerative potential of such bioinspired hybrid coating, providing a suitable environment for bone regeneration and vascularization, as well as an ideal strategy to prevent bone implant-associated infections

Introduction and Objective. Found in bone-associated prosthesis, Cutibacterium acnes (C. acnes) is isolated in more than 50% of osteoarticular prosthesis infections, particularly those involving shoulder prostheses. Ongoing controversies exist concerning the origin of C. acnes infection. Few reports construct a reasonable hypothesis about probable contaminant displaced from the superficial skin into the surgical wound. Indeed, despite strict aseptic procedures, transecting the sebaceous glands after incision might result in C. acnes leakage into the surgical wound. More recently, the presence of commensal C. acnes in deep intra-articular tissues was reported. C. acnes was thus detected in the intracellular compartment of macrophages and stromal cells in 62.5% of the tested patients who did not undergo skin penetration. Among bone stromal cells, mesenchymal stem cells (MSCs) are predominantly found in bone marrow and periosteum. MSCs are the source of osteogenic lines of cells capable of forming bone matter. In this study, the pathogenicity of C. acnes in bone repair context was investigated. Materials and Methods. Human bone marrow derived MSCs were challenged with C. acnes clinical strains harvested from non-infected bone site (Cb). The behaviour of Cb strain was compared to C. acnes took from orthopaedic implant-associated infection (Ci). The infective capabilities of both strains was determined following gentamicin-based antibiotic protection assay. The morphology and ultrastructural analysis of infected MSCs was performed respectively through CLSM pictures of Phalloidin. ®. stained MSCs cytoskeleton and DAPI labelled Cb, and transmission and scanning electron microscopies. The virulence of intracellular Ci and Cb (Ci-MSCs and Cb-MSCs) was investigated by biofilm formation on non-living bone materials; and the immunomodulatory response of infected MSCs was investigated (PGE-2 and IDO secretion detected by ELISA). Bone cells (osteoblasts and PMA differentiated macrophages) were then challenged with Cb-MSCs and Ci-MSCs. Intracellular accumulation of ROS within infected macrophages was assessed by flow cytometry after 2 h of infection and the catalase production by Cb-MSC and Ci-MSC was evaluated. Statistical analyses were performed using Mann & Whitney test. Results. Following MSCs infection by C. acnes, the rate of viable bacteria inside MSCs was about 4% and 6% for Cb and Ci, respectively. Cb showed however a lower invasiveness in comparison to Ci (0.6-fold, p=0.01), confirming the higher pathogenicity of Ci. The ultrastructural and morphology analysis of infected MSCs confirmed the presence of bacteria free in MSCs cytoplasm, localized between F-actin fibers of MSCs, which preserved their elongated morphology. Considering the high level of secreted immunomodulatory mediators (PGE-2 and IDO), our results suggest that Cb-infected MSCs could promote a transition of macrophages from a primarily pro-inflammatory M1 to a more anti-inflammatory M2 phenotype. In comparison with Cb, Cb-MSCs increased significantly the formation of biofilm on TA6V and PEEK but reduced the biofilm formation on 316L SS. Ci-MSCs showed a significant increase in biofilm formation on PEEK vs Ci, while no difference in biofilm formation was noticed on TA6V and 316L SS. Regarding the ability of MSCs bacteria to infect osteoblasts, our results showed a higher infective capabilities of Cb-MSCs versus Cb (>2-fold, p=0.02), while no difference was noticed between Ci and Ci-MSCs. Along with an increase in catalase production by Cb-MSCs, we noticed its higher persistence to macrophage degradation. Conclusions. Taken together, our results demonstrate a shift in commensal Cb to pathogenic following infection. Indeed, Cb- MSCs acquires features that (i) increase biofilm formation on orthopedic based materials, (ii) increase the osteoblast infection and (iii) develop resistance to the macrophage degradation, through the increase of catalase production. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections

Introduction. Management of periprosthetic joint infection (PJI) after total knee arthroplasty (TKA) is a challenging task. Antibiotic management remains elusive due to differences in epidemiology and resistance between countries and reports. Increasing resistance of bacteria is a concern. The aim of this study was to investigate the development of antibiotic resistance of main causative bacteria in a single center. Methods. A retrospective review of all revision TKA between 2006 and 2018 in a tertiary referral center was performed. Included were cases meeting the consensus criteria for PJI with at least two positive cultures. Three chronological groups were created using a two-step cluster analysis. Results. In 129 cases of PJI there were 161 different bacteria identified by growth in culture. Of all bacteria 76.4% were gram positive, 23.6% were gram negative. Coagulase-negative Staphylococci (CNS) were diagnosed in 46.6% cultures, followed by Staphylococcus aureus in 19.8% of cultures. Overall antibiotic resistance (p=0.454) and Staphylococcus aureus resistance (p=0.788) have not increased during the study period. CNS resistance to teicoplanin (4% to 44%; p<0.001), fosfomycin (16% to 44%, p=0.016) and tetracycline (0% to 28%, p=0.014) increased significantly. Vancomycin had an 84.4% overall sensitivity and 100% CNS sensitivity and remained the most effective agent. Conclusion. The increasing resistance to several antibiotics over a little more than a decade is concerning. This study suggests that referral centres should continuously monitor obtained cultures as this has significant implications for both prophylactic treatment in primary as well as empirical antibiotic treatment in PJI. Resistance profiles may differ between centres and are subject to dynamic changes

Aim. In cases of prosthetic joint infections the sensitivity of bacterial cultivation of tissue samples is not 100%. In fact, the reported sensitivity based on standardized criteria and rigorous tissue sampling technique probably differs between 86 to 89%. It has been claimed that sonication of explanted prostheses with subsequent culturing of sonication fluid can increase the sensitivity of the test compared to culturing of tissue samples. To what degree bacteria embedded in biofilm is dislodged during the sonication process has to our knowledge not been fully elucidated. We studied the effect of sonication as a method to dislodge biofilm embedded Staphylococcus epidermidis in vitro. Method. 46 steel plates were colonized with biofilm forming S. epidermidis ATCC 35984 in TSB with 1% glucose aerobically at 37°C for 24 hours. Plates were cleansed for non-adherent bacteria before microscopy. Biofilm embedded bacteria were stained with LIVE/DEAD ™ BacLight ™ Bacterial Viability Kit for microscopy and visualized under vital conditions using EVOS™ FL Auto 2 Imaging System (epifluorescence) and an inverse confocal laser scanning microscope LSM510 (CLSM). All steel plates were subjected to epifluorescence microscopy before and after sonication. CLSM and SEM were used to confirm the presence of biofilm embedded bacteria after sonication. Pictures from epifluorescence microscopy were processed for image analysis with help of a macro application (Fiji) and the data was expressed as biofilm coverage rate (BCR). The sonication was performed using a BactoSonic® Bandolin sonicator and the applied effect in each glass test tube (40 kHz, 800W) was measured with a Bruel og Kjær 8103 hydrophone. The amount of bacteria in the sonication fluid was quantified by counting the number of colony forming units (CFU). Three steel plates acted as negative controls. Results. The BCR was highly variable on the plates after sonication. The biofilm was eradicated from the majority of the plates but a considerable number of plates still had biofilm attached to the surface in a highly variable manner. The amount of bacteria in the sonication fluid correlated poorly with BCR on corresponding plates. Conclusions. Our conclusion is that the ability of sonication to dislodge biofilm embedded S. epidermidis in vitro is not as effective as current opinion might suggest. After sonication biofilm still adhere to a significant number of plates in a highly varying manner. This prompts the need to investigate the effect of sonication on biofilm embedded bacteria formed in vivo

Aim. Periprosthetic joint infections (PJI) are increasing due to our elderly population with the need of a joint prosthesis. These infections are difficult to treat, because bacteria form biofilms within one day on the orthopedic implant surface. Notably, most of the current available antibiotics do not penetrate the biofilm or are not active against the sessile forms of bacteria. Therefore, prevention is key. In the current paradigm, bacteria from the skin surface or dermis - such as Staphylococcus aureus, coagulase-negative staphylococci, or Cutibacterium sp. – contaminate the periimplant tissue during surgery. Cutibacterium avidum, which has increasingly been reported in hip PJIs, colonizes the skin in the groin area in 32.3%. We were wondering if standard skin antisepsis before hip arthroplasty is effective to eliminate C. avidum colonization in the surgical field. Method. In a single-center, prospective study, we preoperatively screened all patients undergoing a hip arthroplasty through a direct anterior approach for different skin bacteria in the groin area. Only in patients colonized with C. avidum, we intraoperatively searched for persistent bacterial growth during and after triple skin antisepsis with povidone-iodine/alcohol. For that, we collected skin scrapings after first and third antisepsis and biopsies from the dermis at the surgical incision and evaluated bacterial growth and species. In addition, thin sections of the dermis biopsies were submitted to Fluorescence in situ Hybridization (FISH) using pan-bacteria probe EUB338. Results. From October 2018 until March 2019, 53 patients (47.2% female) were screened. Patients were mainly colonized with coagulase-negative staphylococci (41, 77.4%; 41), C. avidum (12, 22.6%), and Cutibacterium acnes (8, 15.1%). Intraoperative skin antisepsis of patients colonized with C. avidum was ineffective to eliminate any bacteria in 75% (5 out of 7) after the first and 28.6% (2 out of 7) after the third antisepsis. Focusing on C. avidum, antisepsis was ineffective in 43% (3 out of 7) and 14% (1 out of 7), respectively. Dermis biopsies were all culture negative, but FISH showed positive ribosome-rich bacteria in 50%. Conclusions. We show in our ongoing study that the commensal C. avidum resists the standard skin antisepsis and bacteria visually persist in the dermis as demonstrated by FISH technique. Standard skin antisepsis is of limited effectiveness, resulting in a risk for intraoperatively acquired PJIs. Thus, new and more effective techniques to improve skin antisepsis are urgently needed

Aim. Bone and Joint Infections (BJIs) present with non-specific symptoms and can be caused by a wide variety of bacteria and fungi, including many anaerobes and microorganisms that can be challenging to culture or identify by traditional microbiological methods. Clinicians currently rely primarily on culture to identify the pathogen(s) responsible for infection. The BioFire. ®. FilmArray. ®. Bone and Joint Infection (BJI) Panel (BioFire Diagnostics, Salt Lake City, UT) was designed to detect 15 gram-positive (seven anaerobes), 14 gram-negative bacteria (one anaerobe), two yeast, and eight antimicrobial resistance (AMR) genes from synovial fluid specimens in an hour. The objective of this study was to evaluate the performance of an Investigational Use Only (IUO) version of the BioFire BJI Panel (BBJIP) compared to conventional used as reference methods. Method. In a monocentric study, leftover synovial fluid specimens were collected in a single institution including 4 hospitals and tested using conventional bacterial culture (Standard of Care (SoC)) according to routine procedures following French national recommendations. Specimen has been placed in a refrigerator (4°C) as soon as possible after collection and stored for less than or equal to 7 days before enrollment. Performance of the IUO version of the BBJIP was determined by comparison to SoC for species identification. Results. To date, 201 specimens have been collected and tested using BBJIP. A total of 39 pathogens were obtained in culture. Compared to SoC culture, the overall PPA was 89.7% (35 TP, 4 FN (SA, 1; Strepto Spp, 2; P. micra, 1) and the overall NPA was 99.7% with 16 FP for a total of 5374 bacterial targets screened. Two complementary molecular tests using home-made PCR are underway to definitively conclude about the FN et FP for BBJIP observed in the preset study. Conclusions. The BioFire BJI Panel appears as a promising, sensitive, specific, and robust test for rapid detection of 31 microorganisms (including anaerobes) and eight AMR genes in synovial fluid specimens. The number of pathogens and resistance markers included in the BioFire BJI Panel, together with a reduced time-to-result and increased diagnostic yield compared to culture, is expected to aid in the management of BJIs

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

Aim. Bacteriophages, viruses specific of bacteria, are receiving substantial attention as alternative antibacterial agents to treat bacteria frequently multi-resistant to antibiotics and/or able to form biofilms, such as staphylococci. The latter are responsible for very difficult to treat bone and joint infections (BJIs). In this context, our consortium aims to develop a production of therapeutic phages in accordance with the will of ANSM (French National Agency for the Safety of Medicines and Health Products) to encourage the development of a national academic platform for phage therapy. We report the isolation and characterization of new anti-Staphylococcus phages as well as the evaluation of their activity on a collection of clinical strains of S. aureus (SA) and coagulase-negative staphylococci (CNS) in order to assess their therapeutic potential. Method. Seventeen phages were isolated from wastewater samples. Their identification was obtained by Illumina whole genome sequencing. To evaluate their spectrum of activity, 30 genetically characterized SA strains representative of the main genetic backgrounds as well as 32 strains belonging to 7 CNS species responsible for BJIs were included. The spot test technique, based on the determination of the Efficiency Of Plating ratio, was used (EOP, ratio between the phage titer obtained on a tested strain/titer on a reference strain, close to 1 if high sensitivity to the phage). Results. All isolated phages belonged to the Myoviridae family: 14/17 and 3/17 to the Kayvirus and Silviavirus genera respectively. Silviavirus phages were more active on SA strains (EOP>0.001 for 73–90% of strains) than Kayvirus phages (EOP>0.001 for 13–70% of strains, except for V1SA21: 80%). In total, 83% of strains were susceptible to the phage with the broadest spectrum in each genus, their combination representing a promising opportunity to prevent the emergence of resistance. Kayvirus phages had polyvalent activity on several CNS species (maximum 47% of tested strains), mainly S. lugdunensis, S. capitis and S. caprae, whereas Silviavirus phages were only active on 6–12% of the tested strains. Conclusions. We report the characterization of a large collection of novel phages with complementary spectra against a collection of SA and CNS strains. Further work is currently focused on i) the isolation of anti-S. epidermidis phages, bacterial species against which the present collection of phages was insufficiently active, while it is a major pathogen in this context, ii) the development of production and purification protocols in order to meet the requirements of ANSM for human use

Aim. Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella. Method. The bacteriophages (191219) were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages, and S. aureus EDCC 5055(1×10. 7. CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37. o. C and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analysing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well plate. The eradication of biofilm was analysed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts at 2h, 4h and 24h time points of post treatment. In addition, we have analysed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Finally, experiments are performed to prove the effect of bacteriophages to clear in vivo biofilm using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis. Results. Our results demonstrate the in vitro efficacy of bacteriophages against planktonic S. aureus. Transmission electron microscopy (TEM) experiments revealed severe infection of bacteria by bacteriophages. Bacteriophages also eradicated in a dose-dependent manner in vitro S. aureus biofilm formation and were active against intracellular S. aureus in an osteoblastic cell line. TEM analysis visualized the effect of the bacteriophages on S. aureus inside the osteoblasts with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria infection model, single administration of phages failed to show improvement in survival rates, but exhibited some synergistic effects with gentamicin or rifampicin, which was not statistically significant. Conclusions. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections. Further preclinical and clinical trials are required to establish adequate treatment protocols

Aim. There is an ongoing controversy whether the observed benefit of infection risk reduction by ALBC outweighs the risk of possible antimicrobial resistance development. Methods. The scientific & clinical literature in PubMed, Medline and Embase has been systematically reviewed with the keywords “antibiotic resistance”, “antibiotic loaded bone cement”, “local antibiotics”, “bacterial colonization” and “joint infection”. In total 28 relevant publications were found with the majority of them reporting laboratory results. Only 7 papers focused on clinical septic situations & patient data. Results. Although rare as consequence of the initially high drug concentrations in situ, experimental and clinical studies demonstrated survival of resistant bacteria on ALBC with subsequent bacterial re-colonisation of the biomaterial. This was most notable for coagulase-negative staphylococci (CoNS). Bacterial survival in presence of ALBC represents a selection process of already pre-existing high-level resistant mutants and not antibiotic resistance induction. The use of antibiotic combinations with gentamicin in bone cement is associated with a markedly lower risk of survival of resistant bacteria. This is particularly important in patients at high infection risks and in septic revision cases. There is no clinical evidence for a widespread increase of clinically important gentamicin resistancies in the orthopaedic ward because of routine use of ALBC. On an individual basis, the benefit of a lower infection probability with combined systemic & local antibiotic application should outweigh the risk of selecting pre-existing resistant bacteria. Each prevented infection case means that a complex and extended antibiotic therapy with risk of antibiotic resistance development over time has been avoided for a patient. In those cases where pre-existing resistant bacteria have survived the prophylactic exposure to antibiotics in bone cement, they remain in vast majority still susceptible to the clinically important antibiotics used for treatment of prosthetic joint infections. Conclusions. The benefit of a lower infection probability with ALBC should outweigh the risk of selecting resistant bacteria against the particular antibiotic used in bone cement. A trend towards broad resistance development which may complicate treatment of infection cases was not found

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. Methods. Sprague-Dawley rats underwent cemented hip hemiarthroplasty via the posterior approach with pre- and postoperative gait assessments. Infection with Staphylococcus aureus Xen36 was monitored with in vivo photoluminescent imaging in real-time. Pre- and postoperative gait analyses were performed and compared. Postmortem micro (m) CT was used to assess implant integration; field emission scanning electron microscopy (FE-SEM) was used to assess biofilm formation on prosthetic surfaces. Results. All animals tolerated surgery well, with preservation of gait mechanics and weightbearing in control individuals. Postoperative in vivo imaging demonstrated predictable evolution of infection with logarithmic signal decay coinciding with abscess formation. Postmortem mCT qualitative volumetric analysis showed high contact area and both cement-bone and cement-implant interdigitation. FE-SEM revealed biofilm formation on the prosthetic head. Conclusion. This study demonstrates the utility of a new, high-fidelity model of in vivo PJI using cemented hip hemiarthroplasty in rats. Inoculation with bioluminescent bacteria allows for non-invasive, real-time monitoring of infection. Cite this article: Bone Joint J 2021;103-B(7 Supple B):9–16