Aim. Prosthetic joint infections (PJI) are a common reason for revisions in patients that underwent total arthroplasty of the hip (THA) or knee (TKA). Extensive antibiotic treatment follows while a clear understanding of target site concentrations is lacking. The aim is to investigate the target site concentrations, like bone and synovial tissue concentrations, which consequently may lead to an optimisation of the dosing regiments of cefuroxime of PJI patients suffering from pain and immobility. Dosing optimisation may lead to a reduced risk of (re-)infection and adverse effects like renal-insufficiency and therefore lower health-care costs. Method. Patients (n=26) with PJI of hip or knee undergoing a one- or two-stage revision treated with cefuroxime were included as part of the ASTERICS study. During implant removal two samples were collected 15-30 and 60-120 minutes after IV infusion of plasma, bone tissue and synovial tissue and one synovial fluid sample. Samples were analysed using a UltraPerformance Convergence Chromotography – quadruple mass spectrometry system (UPC. 2. -MS/MS). Bone tissue and synovial tissue were pulverized before analysis acquiring for bone tissue a homogenate of cortical and cancellous bone. Using nonlinear mixed effect modelling (NONMEM) a base model was developed to analyse the bone to plasma ratio of cefuroxime in osteomyelitis patients. Results. Mean bone concentrations (mg/L) of cefuroxime at 30-60 min after IV administration in the knee and hip are 21.29 (SD:11.86) and 19.06 (SD: 11.79) respectively and 8.23 (SD:4.90) and 9.67 (SD:9.75) respectively at 90-120 min after IV administration. The penetration of cefuroxime described by the bone:plasma ratio into knee and hip affected by osteomyelitis is 0.3 and 0.4 respectively within 1 hour and 0.1 for both joints within 2 hours. The results mentioned here were collected during knee operations without blood void conditions. Concentration data was used to develop a base pharmacokinetic model using NONMEM and was best described by a two-compartment model. Conclusions. Cefuroxime penetrates osteomyelitis affected bone tissue within the hour proving the usefulness of cefuroxime as prophylaxis of orthopaedic surgery and as treatment option for PJI. However, PK modelling and further simulations need to prove whether repeated cefuroxime dosing in this population is required to reach minimal inhibitory concentrations in target tissue

Aim. Dalbavancin is a lipoglycopeptide with a broad antimicrobial spectrum against Gram-positive bacteria and effect against microorganisms in biofilm in vitro. Its pharmacokinetic properties, with an exceptionally long half-life of approximately 300 hours, allow for simplified administration that may be of value in the long-term treatment of bone and joint infections, such as prosthetic joint infections (PJIs). Several case reports and case series with “off-lable” treatment with dalbavancin of PJIs exist, but the optimal dosing regimen remains to be defined. Therapeutic drug monitoring (TDM) is recommended for treatment with >2 doses of dalbavancin. In the absence of TDM, the Swedish national guidelines for bone and joint infections (2023, . www.infektion.net. ) recommends a loading dose of dalbavancin 1,500 mg on day 1 and 1,500 mg on days 8 – 14, after which from day 28 1,000 mg is given biweekly or 500 mg every week. The aim of the present study was to determine trough levels of dalbavancin in patients with long-term treatment of PJIs according to the national guidelines. Method. Twelve patients with PJI were treated with at least 6 doses of dalbavancin, of which the first two doses were 1500 mg and the following doses were 1000 every second week, and prospectively sampled biweekly for determination of serum concentrations (trough levels) of dalbavancin which was measured by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). The renal function was also examined. Results. The median serum concentration 14 days after the first dose of dalbavancin 1500 mg was 36.3 mg/L (range 6.6 – 62.4 mg/L). The median value 14 days after the second dose of 1500 mg (day 27 – 28) was 48.2 mg/L (range 12.2 – 77.3 mg/L). The trough value after the last dose of a total of 6 – 7 doses was as median 43.1 mg/L (range 26.2 – 97.5 mg/L). Three patients showed a tendency towards successive accumulation of dalbavancin during treatment. None of the patients, including those three with increasing through levels during treatment, showed any significant alteration in creatinine nor glomerular filtration rate. Conclusions. TDM during long-term treatment with dalbavancin is recommended to avoid the risk of accumulation and unnecessarily high trough values. With TDM, the dosing interval can be extended in several cases. In addition, with the support of TDM, subtherapeutic serum concentrations, with the risk of developing resistance, can be avoided

Aims

This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels.

Aims. The metabolic variations between the cartilage of osteoarthritis (OA) and Kashin-Beck disease (KBD) remain largely unknown. Our study aimed to address this by conducting a comparative analysis of the metabolic profiles present in the cartilage of KBD and OA. Methods. Cartilage samples from patients with KBD (n = 10) and patients with OA (n = 10) were collected during total knee arthroplasty surgery. An untargeted metabolomics approach using liquid chromatography coupled with mass spectrometry (LC-MS) was conducted to investigate the metabolomics profiles of KBD and OA. LC-MS raw data files were converted into mzXML format and then processed by the XCMS, CAMERA, and metaX toolbox implemented with R software. The online Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate the metabolites by matching the exact molecular mass data of samples with those from the database. Results. A total of 807 ion features were identified for KBD and OA, including 577 positive (240 for upregulated and 337 for downregulated) and 230 negative (107 for upregulated and 123 for downregulated) ions. After annotation, LC-MS identified significant expressions of ten upregulated and eight downregulated second-level metabolites, and 183 upregulated and 162 downregulated first-level metabolites between KBD and OA. We identified differentially expressed second-level metabolites that are highly associated with cartilage damage, including dimethyl sulfoxide, uric acid, and betaine. These metabolites exist in sulphur metabolism, purine metabolism, and glycine, serine, and threonine metabolism. Conclusion. This comprehensive comparative analysis of metabolism in OA and KBD cartilage provides new evidence of differences in the pathogenetic mechanisms underlying cartilage damage in these two conditions. Cite this article: Bone Joint Res 2024;13(7):362–371

Introduction. Autologous fat grafting has favourable potential as a regenerative strategy and is the current gold-standard to repair large contour defects, as needed in breast reconstruction after mastectomy and traumatic soft tissue reconstruction. Clinically, there is a limit on the volume of lipoaspirate which can be utilised to repair a soft-tissue defect. Surgical complications are the result of poor structural fidelity of lipoaspirate and graft resorption as a filling material and are hindered further by poor graft vascularisation. This study aims to develop injectable lipoaspirate-derived adipose tissue grafts with enhanced biologically and clinically-admissible structural and functional properties adopting light photocrosslinking of unmodified lipoaspirate. Methods. Patient-derived lipoaspirate was harvested and crosslinked using novel photoinitiator and exposure to visible light (wavelength 450nm) in surgery, establishing bonds between extracellular matrix (ECM) proteins within the material. The degree of crosslinking was tuned (photoinitiator concentration, light exposure, light intensity) and covalent bond formation measured using mass spectrometry. To predict patient response, SWATH-MS was used to identify differences in patient ECM and crosslinked grafts were implanted in vivo using a subcutaneous mouse model. Functional vessel formation and resorption were quantified using micro-CT and tissue-remodelling was assessed via histology. Results. There was an increase in the relative abundance of covalent bonds present with increasing degree of crosslinking. When injected, crosslinked lipoaspirate had better shape fidelity compared with native lipoaspirate – demonstrated by a smaller fibre diameter. Crosslinked lipoaspirate remained viable over long term culture and resulted in more predictable resorption profiles when implanted in vivo. Conclusions. The crosslinking approach described here is tunable and functional across different patient samples. Improving the structural properties of lipoaspirate through minimal manipulation has clinical utility for the delivery of grafts with higher shape fidelity and therefore increased graft survival when implanted

Aims. The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies. Methods. A porcine multiple trauma model was used in which two fracture treatment strategies were compared: early total care (ETC) and damage control orthopaedics (DCO). fxH was harvested and analyzed using liquid chromatography-tandem mass spectrometry. Per group, discriminating proteins were identified and protein interaction analyses were performed to further elucidate key biomolecular pathways in the early fracture healing phase. Results. The early fxH proteome was characterized by immunomodulatory and osteogenic proteins, and proteins involved in the coagulation cascade. Treatment-specific proteome alterations were observed. The fxH proteome of the ETC group showed increased expression of pro-inflammatory proteins related to, among others, activation of the complement system, neutrophil functioning, and macrophage activation, while showing decreased expression of proteins related to osteogenesis and tissue remodelling. Conversely, the fxH proteome of the DCO group contained various upregulated or exclusively detected proteins related to tissue regeneration and remodelling, and proteins related to anti-inflammatory and osteogenic processes. Conclusion. The early fxH proteome of the ETC group was characterized by the expression of immunomodulatory, mainly pro-inflammatory, proteins, whereas the early fxH proteome of the DCO group was more regenerative and osteogenic in nature. These findings match clinical observations, in which enhanced surgical trauma after multiple trauma causes dysbalanced inflammation, potentially leading to reduced tissue regeneration, and gained insights into regulatory mechanisms of fracture healing after severe trauma. Cite this article: Bone Joint Res 2024;13(5):214–225

Aims

Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA).

Meniscal injuries affect over 1.5 million people across Europe and the USA annually. Injury greatly reduces knee joint mobility and quality of life and frequently leads to the development of osteoarthritis. Tissue engineered strategies have emerged in response to a lack of viable treatments for meniscal pathologies. However, to date, constructs mimicking the structural and functional organisation of native tissue, whilst promoting deposition of new extracellular matrix, remains a bottleneck in meniscal repair. 3D bioprinting allows for deposition and patterning of biological materials with high spatial resolution. This project aims to develop a biomimetic 3D bioprinted meniscal substitute. Meniscal tissue was characterised to effectively inform the design of biomaterials for bioprinting constructs with appropriate structural and functional properties. Histology, gene expression and mass spectrometry were performed on native tissue to investigate tissue architecture, matrix components, cell populations and protein expression regionally across the meniscus. 3D laser scanning and magnetic resonance imaging were employed to acquire the external geometrical information prior to fabrication of a 3D printed meniscus. Bioink suitability was investigated through regional meniscal cell encapsulation in blended hydrogels, with the incorporation of growth factors and assessed for their suitability through rheology, scanning electron microscopy, histology and gene expression analysis. Meniscal tissue characterisation revealed regional variations in matrix compositions, cellular populations and protein expression. The process of imaging through to 3D printing highlighted the capability of producing a construct that accurately replicated meniscal geometries. Regional meniscal cell encapsulation into hydrogels revealed a recovery in cell phenotype, with the incorporation of growth factors into the bioink's stimulating cellular re-differentiation and improved zonal functionality. Meniscus biofabrication highlights the potential to print patient specific, customisable meniscal implants. Achieving zonally distinct variations in cell and matrix deposition highlights the ability to fabricate a highly complex tissue engineered construct. Acknowledgements: This work was undertaken as part of the UK Research and Innovation (UKRI)-funded CDT in Advanced Biomedical Materials

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity. Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining. HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts. Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity

Aims

Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown.

Aim. Antibiotic concentration at the infected site is a relevant information to gain knowledge about deep-seated infections. The combination of antibiotic therapy and debridement is often indicated to treat these infections. At University Hospital Basel the most frequently administered antibiotic before debridement is amoxicillin in combination with clavulanic acid. Amoxicillin is a fragile beta-lactam antibiotic that brings multiple challenges for its quantification. As for many sample materials only little material is available, the aim of this work was to establish a sensitive and reliable quantification method for amoxicillin that only requires a small sample mass. We did not quantify clavulanic acid as we focused on the drug with antibiotic action. Method. Usually discarded sample material during debridement was collected and directly frozen. The thawed tissues were prepared using simple protein precipitation and manual homogenization with micro pestles followed by a matrix cleanup with online solid-phase extraction. Separation was performed by HPLC followed by heated electrospray ionization and tandem mass spectrometry. Results. During method development, amoxicillin showed partial formation of a covalent methanol adduct when performing protein precipitation. Furthermore, multiple in-source products of amoxicillin during ionization could be observed. Adding an aqueous buffer to the samples before protein precipitation and summing up the signals of amoxicillin and its in-source acetonitrile-sodium-adduct led to successful method validation for a calibration range of 1–51 mg/kg using 10 mg of each tissue sample. The imprecision was < 8% over the entire concentration range and the bias was ≤ 10 %. The quantitative matrix effect was < 6 % in six different tissue samples. Until now we measured amoxicillin in samples from nine patients with prosthetic joint infection, bursitis, or an abscess who obtained amoxicillin between 5 hours and 15 minutes before sampling and found concentrations between 1.4 and 35 mg/kg. Conclusions. With this method, we developed a fast, simple, and sensitive quantification assay for amoxicillin in tissue samples with little material that can now be applied to different study samples

Aims

The aims of this study were to determine if an increasing serum cobalt (Co) and/or chromium (Cr) concentration is correlated with a decreasing Harris Hip Score (HHS) and Hip disability and Osteoarthritis Outcome Score (HOOS) in patients who received the Articular Surface Replacement (ASR) hip resurfacing arthroplasty (HRA), and to evaluate the ten-year revision rate and show if sex, inclination angle, and Co level influenced the revision rate.

Stratification is required to ensure that only those patients likely to benefit, receive Autologous Chondrocyte Implantation (ACI); ideally by assessing a biomarker in the blood. This study aimed to assess differences in the plasma proteome of individuals who respond well or poorly to ACI. Isobaric tag for relative and absolute quantitation (ITRAQ) mass spectrometry and label-free proteomics analyses were performed in tandem as described previously by our group (Hulme et al., 2017; 2018; 2021) using plasma collected from ACI responders (n=10) compared with non-responders (n=10) at each stage of surgery (Stage I, cartilage harvest and Stage II, cell implantation). iTRAQ using pooled plasma detected 16 proteins that were differentially abundant at baseline in ACI responders compared with non-responders (n=10) (≥±2.0 fold; p<0.05). Responders demonstrated a mean Lysholm (patient reported functional score from 0–100) improvement of 33±13 and non-responders a mean worsening of −13±13 points. The most pronounced plasma proteome shift was seen in response to Stage I surgery in ACI non-responders, with 48 proteins being differentially abundant between the two surgical procedures. We have previously noted this marked shift in response to initial surgery in the SF of ACI non-responders, several of these proteins were associated with the Acute Phase Response. One of these proteins, clusterin, could be confirmed in patients’ plasma using an independent immunoassay using individual samples. Label-free proteomic data from individual samples identified only cartilage acidic protein-1 (known to associate with osteoarthritis progression) to be significantly more abundant at Stage I in the plasma of non-responders. This study indicates that proteins can be identified within the plasma that have potential use in ACI patient stratification. Further work is required to validate the findings of this discovery-phase work in larger ACI cohorts

Preventing infections in joint replacements is a major ongoing challenge, with limited effective clinical technologies currently available for uncemented knee and hip prostheses. This research aims to develop a coating for titanium implants, consisting of a supported lipid bilayer (SLB) encapsulating an antimicrobial agent. The SLB will be robustly tethered to the titanium using self-assembled monolayers (SAMs) of octadecylphosphonic acid (ODPA). The chosen antimicrobial is Novobiocin, a coumarin-derived antibiotic known to be effective against resistant strains of Staphylococcus aureus. ODPA SAMs were deposited on TiO. 2. -coated quartz crystal microbalance (QCM) sensors using two environmentally friendly non-polar solvents (anisole and cyclopentyl methyl ether, CPME), two concentrations of ODPA (0.5mM and 1mM) and two processing temperatures (21°C and 60°C). QCM, water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and temperature-programmed desorption mass spectrometry (TPD-MS) were used to characterise the ODPA SAM. A SLB with encapsulated Novobiocin was subsequently developed on the surface of the ODPA SAM using fluorescent lipids and a solvent assisted method. The prototype implant surface was tested for antimicrobial activity against S. aureus. A well-ordered, uniform ODPA SAM was rapidly formed using 0.5 mM ODPA in CPME at 21°C during 10 min, as confirmed by high Sauerbrey mass (≍285-290 ng/cm. 2. ), high atomic percentage phosphorus (detected using XPS) and high water contact angles (117.6±2.5°). QCM measurements combined with fluorescence microscopy provided evidence of complete planar lipid bilayer formation on the titanium surface using a solvent assisted method. Incorporation of Novobiocin into the SLB resulted in reduced attachment and viability of S. aureus. Key parameters were established for the rapid, robust and uniform formation of an ODPA SAM on titanium (solvent, temperature and concentration). This allowed the successful formation of an antimicrobial SLB, which demonstrated potential for reducing attachment and viability of pathogens associated with joint replacement infections

Aims

This study aimed to investigate the role and mechanism of meniscal cell lysate (MCL) in fibroblast-like synoviocytes (FLSs) and osteoarthritis (OA).

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

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).

Background. Surgical site infection following spine surgery is associated with increased morbidity, mortality and increased cost for the health care system. The reported pooled incidence is 3%. Perioperative antibiotic prophylaxis is a key factor in lowering the risk of acquiring an infection. Previous studies have assessed perioperative cefuroxime concentrations in the anterior column of the cervical spine with an anterior surgical approach. However, the majority of surgeries are performed in the posterior column and often involve the lumbar spine. Accordingly, the objective was to compare the perioperative tissue concentrations of cefuroxime in the anterior and posterior column of the same lumbar vertebra using microdialysis in an experimental porcine model. Method. The lumbar vertebral column was exposed in 8 female pigs. Microdialysis catheters were placed for sampling in the anterior column (vertebral body) and posterior column (posterior arch) within the same vertebra (L5). Cefuroxime (1.5 g) was administered intravenously over 10 min. Microdialysates and plasma samples were continuously obtained over 8 hours. Cefuroxime concentrations were quantified by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry. Microdialysis is a catheter-based pharmacokinetic tool, that allows dynamic sampling of unbound and pharmacologic active fraction of drugs e.g., cefuroxime. The primary endpoint was the time with cefuroxime above the clinical breakpoint minimal inhibitory concentration (T>MIC) for Staphylococcus aureus of 4 µg/mL as this has been suggested as the best predictor of efficacy for cefuroxime. The secondary endpoint was tissue penetration (AUC. tissue. /AUC. plasma. ). Results. Mean T>MIC 4 µg/mL (95% confidence interval) was 123 min (105–141) in plasma, 97 min (79–115) in the anterior column and 93 min (75–111) in the posterior column. Tissue penetration (95% confidence interval) was incomplete for both the anterior column 0.48 (0.40–0.56) and posterior column 0.40 (0.33–0.48). Conclusions. Open lumbar spine surgery often involves extensive soft tissue dissection, stripping and retraction of the paraspinal muscles which may impair the local blood flow exposing the lumbar vertebra to postoperative infections. A single intravenous administration of 1.5 g cefuroxime resulted in comparable T>MIC between the anterior and posterior column of the lumbar spine. Mean cefuroxime concentrations decreased below the clinical breakpoint MIC for S. aureus of 4 µg/mL after 123 min (plasma), 97 min (anterior column) and 93 min (posterior column). This is shorter than the duration of most lumbar spine surgeries, and therefore alternative dosing regimens should be considered in posterior open lumbar spine surgeries lasting more than 1.5 hours

Aim. The current antibiotic treatment of periprosthetic joint infection (PJI) is optimized by measuring concentrations in plasma. However, it remains unclear whether effective concentrations of the antibiotics are reached at the site of PJI. Nonetheless, adequate target site concentrations are important to achieve effective eradication of the micro-organism. In order to determine the efficacy of cefuroxime and flucloxacillin in synovial fluid, synovial tissue and bone tissue in relation to the minimal inhibitory concentration (MIC) of the pathogen causing the PJI, we perform a pharmacokinetic/pharmacodynamic (PK/PD) study. Therefore, we aimed to develop validated analytical methods for analysis of cefuroxime and flucloxacillin in synovial fluid, synovial tissue and bone tissue. Method. Blank samples of synovial fluid, synovial tissue and bone tissue were obtained by orthopedic surgeons during surgery. For validation the samples of each matrix were spiked with both cefuroxime and flucloxacillin. Synovial tissue and bone tissue was pulverized with a mikro-dismembrator. Samples were kept frozen at −20°C until analysis. After a sample preparation quantification of cefuroxime and flucloxacillin in each matrix was performed on the ultra-performance convergence chromatography-tandem mass spectrometry (UPC2-MS/MS). Stable-isotope-labeled meropenem-d6 served as internal standard. The linearity, limits of quantification, accuracy and precision and carry-over were determined for all methods separately. The methods were validated according to the European Medicine Agency (EMA) and Food and Drug Administration (FDA) guidelines on bioanalytical method validation. Results. These methods were successfully validated for cefuroxime and flucloxacillin quantification in all matrices according to the EMA and FDA guidelines. The limits of quantification were adequate to cover potential cefuroxime and flucloxacillin concentration in synovial fluid, synovial tissue and bone tissue as described in literature, with a range of 1–100mg/L for synovial fluid and 1–20 µg/g for synovial tissue and bone tissue (r >0.995). Accuracy and within-run precision were validated according to acceptance values (RSD <15%). Carry over was less than 20%. Matrix effects and recovery were investigated for synovial fluid. The results were within the range of 80–120%. Conclusions. The results of the validation fall within the limits of quantification according to the EMA and FDA guidelines. Therefore, these methods can be applied during a PK/PD study to discover the exposure of antibiotics in synovial fluid, synovial tissue and bone tissue at the site of infection in patients with a PJI

Aim. S. aureus and S. epidermidis remain the leading biofilm-forming agents causing orthopedic implant-associated infections (OIAI), but other coagulase-negative Staphylococcus (CoNS) with clinical importance is emerging. Besides, few studies have assessed specific genomic traits associated with patient outcome. This is a preliminary descriptive study of phenotypic and genomic features identified in clinical isolates of S. aureus and CoNS isolates recovered from OIAIs patients that progressed to treatment failure. Methods. Ten isolates were identified by matrix-time-of-flight laser-assisted desorption mass spectrometry (MALDI-TOF-MS) and tested for antibiotic susceptibility and biofilm formation. Genotypic characteristics, including, MLST (Multi Locus Sequence Typing), SCCmec typing, virulence and resistance genes were assessed by whole-genome sequencing (WGS) that was performed on an Illumina HiSeq 2500 platform. Bioinformatics analyzes were performed using CGE, PATRIC, VFDB, CARD RGI, SnapGene, BLAST, and PubMLST. S. aureus (215, 260 and 371) isolates belonged to CC5 (ST5 and ST105, spa type t002) and carried SCCmec type I (1B), II (2A) and V(5C2), respectively. Results. They carried multiple resistance genes, with all resistant to methicillin (MRSA), and harboured mecA, blaZ. S. aureus 215 and 371 carried ermA gene and multiple genes for aminoglycosides resistance including aph(3’)-III, ant(9)-Ia, and ant(4)-Ib, and for quinolones. S. aureus 260 also carried resistance genes for tetracycline, quinolones and trimethoprim (dfrC). All MRSA were strong biofilm producers harboring the complete icaADBC and icaR operon, and also carried multiple adhesion and toxin-related virulence genes. Seven CoNS isolates comprising five species (S. epidermidis, S. haemolyticus, S. sciuri, S. capitis and S. lugdunensis) were analyzed, with mecA gene detection in five isolates. S. haemolitycus (95) and S. lugdunensis were unable to form biofilm and did not harbor the complete icaADBCR operon. S. epidermidis (216, 403) and S. haemolyticus (53,95) isolates belonged to the ST2/CC2, ST183, ST9 and ST3, respectively. High variability of adhesion genes was detected, with atl, ebp, icaADBC operon and IS256 being the most common. Conclusions. In conclusion, this study provides insights into the phenotypic and genomic analysis of Staphylococci allowing elucidation of MRSA and CoNS specific features that are associated with treatment failure in OIAIs, including genes associated with biofilm production, and resistance to β-lactam and aminoglycosides