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

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.

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.

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

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

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

Background. Two-stage revision arthroplasty is the standard treatment for chronic hip and knee periprosthetic joint infections (PJI). Accurate diagnosis of persistent infections at 2nd stage using established biomarkers and diagnostic criteria is of paramount importance. This study aimed to evaluate the diagnostic value of synovial calprotectin and alpha-defensin, and compare established diagnostic criteria from the International Consensus Meeting (ICM 2018) and the European Bone and Joint Infection Society (EBJIS 2021) to determine persistent PJI at the 2nd stage of a two-stage revision arthroplasty. Methods. We retrospectively analyzed 97 patients who underwent 100 two-stage revisions (hip: 39, knee: 61). Synovial fluid samples were assessed for calprotectin and alpha-defensin levels. ICM 2018 and EBJIS 2021 were applied to all patients undergoing 2nd stage revision. Receiver operating characteristic (ROC) curves and Youden Index were utilized to determine optimal cut-off values, and correlations between biomarkers were evaluated. The microbiological spectrum was analyzed at 2nd stage and re-revision surgery. Results. Calprotectin levels showed a sensitivity of 66.7%, specificity of 32.9%, and accuracy of 38.0% in predicting septic failure. Alpha-defensin showed sensitivity of 28.6%, specificity of 87.8%, and accuracy of 79.2%. Significant correlations included: calprotectin with PMN% (r = 0.471, p = 0.05) and alpha-defensin with WBC (r = 0.830, p < 0.01) in the successful cohort. For septic re-revisions, calprotectin and alpha-defensin were highly correlated (r = 0.969, p < 0.01). ICM correctly diagnosed persistent PJI in 26.7%, while EBJIS diagnosed 24.2%. The microbial spectrum shifted from gram-positive to gram-negative bacteria between reimplantation and re-revision surgeries. Conclusion. Synovial calprotectin and alpha-defensin demonstrated limited accuracy in ruling out persistent PJI at reimplantation. The low sensitivity of current diagnostic criteria, combined with the observed shift in microbial spectrum, underscores the challenges in diagnosing persistent PJI during 2nd stage of a two-stage revisions arthroplasty

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

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

Aim. Fracture-related infection (FRI) is a challenging complication. This study aims to investigate (1) microbial patterns in fracture-related infection (FRI), (2) the comparison of isolated pathogens in FRI patients with early, delayed, and late onset of infection and (3) antibiotic susceptibility profiles to identify effective empiric antibiotic therapy for FRI. Method. Patients treated for FRI from 2013 to 2020 were grouped into early (< 2 weeks), delayed (2– 10 weeks) and late (> 10 weeks) onset of infection. Pathogens detected during treatment were evaluated for pathogens. Antibiotic susceptibility profiles were examined with respect to broadly used antibiotics and antibiotic combinations. Results. In total 117 patients (early n=19, delated n=60, late n=38) were included in the study. Infection was polymicrobial in 10 cases (8.6%) and culture-negative in 11 cases (9.4%). Staphylococcus aureus was the most frequently detected pathogen (40.5%), followed by Staphylococcus epidermidis (17.2%) and gram-negative bacteria (16.4%). Pathogen distribution did not differ statistically significant between the groups. Highest effectiveness could be achieved by the combination of meropenem + vancomycin (95.7%) and gentamycin + vancomycin (94.0%). More than 90% of all patients would have also been covered by co-amoxiclav + glycopeptide (93.2%), ciprofloxacin + glycopeptide and piperacillin/tazobactam + glycopeptide (92.3% each) as well as ceftriaxone + glycopeptide (91.5%). Comparing the predicted efficacy of empiric antimicrobial regimens between the subgroups only revealed a statistically significant difference regarding the combination ciprofloxacin with a glycopeptide (F= 3.304, p=.04), for which more patients with an early onset of infection would have been susceptible. Conclusions. Microbiological pattern for the causative microorganism between early, delayed, and late FRI are comparable. Empiric therapy combinations such as meropenem + vancomycin, gentamycin +vancomycin or co-amoxiclav + glycopeptide are effective antibiotic strategies. To bypass unwanted side effects of systemic antibiotics and reduce the risk of antimicrobial resistance, the administration of local antibiotic carriers should be implemented in clinical practice

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. Infection is one of the worst complications following total joint arthroplasty, which is often associated with significant morbidity. Currently, due to the global burden of multidrug-resistant Gram-negative bacteria (MDR-GNB) infections, few multicentre studies have described a microbiological shift from Gram-positive cocci (GPC) towards MDR-GNB PJI (prosthetic joint infection). Additionally, the emergence of MDR-GNB impacts the therapeutic options and may increase the rate of PJI treatment failure. The purpose of the present study was to describe the predisposing factors associated to failure of treatment in an orthopaedic reference hospital in Brazil from 2014 through 2019. Method. Retrospective case-control analysis of patients treated for MDR-GNB PJI over a five-year period. Data were collected from medical, surgical and laboratory records. PJI were defined according the current MSIS criteria. MDR was defined as non-susceptibility to at least one agent in three or more antimicrobial categories. Patients with PJI with at least two positive tissue cultures for MDR-GNB were selected. The control group was patient with PJI caused by multisensitive organism (GNB or GPC). Absence of signs and symptoms of infection during the follow-up period was defined as cure. Definition of failure: death, need for another course of antibiotic, or the need for another surgical procedure to control the infectious site (relapse). Results. A total of 104 patients were selected, 59 patients in the MDR-GNB PJI group and 44 in the control group. Two outcomes were compared: cure or failure. The overall 1-year survival rate was 65.3% with the median survival time being 207.08 days. In the MDR-BGN infection group the 1-year survival rate was 59.3% and the average time of survival was 141.14 days. In contrast, in the Control group the 1-year survival rate was 73.8% with an average survival time of 230.29 days (p = 0.023). HR: 2.447, IC 1.099–5.448. The independent variables in the multivariate analysis associated to treatment failure were MDR-BGN infection (p = 0.023) HR 2,447 IC 1,099 –5,448, revision surgery (p = 0.042) HR 2,027 IC: 2,027–4,061, presence of comorbidities (p = 0.048) HR 2,508 IC: 0,972- 6,469 and previous antimicrobial use in the last 3 months (p = 0.022). HR 2,132 IC: 1,096- 4,149. Conclusions. GNB-MDR PJI increases approximately 2.5 times the chance of unfavourable outcome such as death and infectious relapse compared to infections with other multisensitive microorganism

Introduction and Aims: Accumulating evidence suggests that bacterially derived endotoxins may contribute to aseptic loosening. This study determined whether lipopolysaccharide (LPS), the classical endotoxin from Gram-negative bacteria, can be detected in periprosthetic tissue from patients with aseptic loosening. We utilised an assay that detects all forms of LPS and is unaffected by beta-glucan-like molecules. Method: Periprosthetic tissue from revision total hip arthroplasty and synovia from primary total joint arthroplasty were homogenised in PBS in endotoxin-free conditions. Non-specific amidases in the homogenates were inactivated at 100 degrees C. LPS was measured using the Endospecy assay (Associate of Cap Cod). Multiple dilutions of the homogenates were assayed to maximise sensitivity, while avoiding assay inhibition assessed by spike recovery determinations. Results were corrected for colour and spike recovery. Assay results were considered positive if the absorbances were higher than the lowest standard and the LPS level was significantly greater (p< 0.05) than the PBS control. Statistical analysis was by ANOVA with Bonferroni-Dunn (Control) post-hoc tests. Results: Samples from 13 patients have been studied to date. Multiple assays of four of these samples showed no detectable LPS while nine of these samples resulted in both positive and negative assays. This inter-assay variability prevents measurement of the concentration of LPS in the samples. Nonetheless, many of the samples contain detectable amount of LPS. Thus, six out of eight samples from revision THA patients with aseptic loosening had positive assays, as did two of four primary TJA patients. LPS was also detected in a sample from a revision control. These results demonstrate that samples from THA patients with aseptic loosening and from primary TJA contain detectable amounts of LPS derived from Gram-negative bacteria. Conclusion: This conclusion is consistent with numerous studies, showing that human serum contains LPS derived from minor infections, gut flora, or dental procedures. It is likely that many of these samples also contain molecules derived from Gram-positive bacteria that have very similar biological effects as LPS. However, detection of these Gram-positive molecules await further improvements in assay specificity and sensitivity

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

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

While stable long-term clinical results have been achieved in total joint arthroplasty, periprosthetic joint infection (PJI) has been actualized as difficult issue in this decade. For accurate diagnosis, it is important to establish standard criteria such as MSIS criteria, and it is prevailing now. As an issue involving PJI, however, the existence of viable, but non-culturable (VNC) bacteria must be noticed. It is difficult to identify the VNC state infection, because microbiologic culture result shows negative and other markers tend to be negative. Here, molecular diagnosis based on polymerase chain reaction (PCR) has certain role as potential diagnostic tools for such VNC infection. We have applied a real-time PCR system for the diagnosis of PJI, which is able to detect methicillin-resistant Staphylococcus (MRS) and distinguish gram-positive from gram-negative bacteria. The prominent advantage is that PCR is the singular way to identify MRS in such culture negative cases. Recent development of full-automatic PCR system may improve the time efficiency for routine application. In this presentation, we will show the overall sensitivity and specificity of our PCR system for diagnosing PJI and discuss the current problem and future prospect

Aim. The risk for developing a periprosthetic joint infection (PJI) as a consequence of bacteremia is not clear, except for Staphylococcus aureus bacteremia, and patient-related risk factors for it are not known. The aim of this study was to investigate the risk for developing a PJI during any bacteremia and to find out possible risk factors leading to it. Method. All patients with a primary knee or hip joint replacement performed between September 2002 and December 2013 in a tertiary care hospital (n=14 378) were retrospectively followed up until December 2014. The mean follow-up time was 6.0 years (range 0–12 years). Positive blood culture results of the patients during the study period were obtained. PJIs during the study period were identified from several data sources. PJIs as a consequence of bacteremia were recorded and confirmed from patient records. Primary PJIs resulting in bacteremia were excluded. Binary logistic regression with univariate analysis was used to study potential risk factors for PJI among those with bacteremia. Results. Of the study patients, 542 (3.8%) had at least one episode of bacteremia. In total, there were 643 episodes of bacteremia. The incidence rate of bacteremia was 7.4 per 1 000 person-years. Seven percent (47/643) of the bacteremias resulted in a PJI. The risk for PJI was highest for bacteremias caused by Staphylococcus aureus (21% of bacteremias led to a PJI) and beta-hemolytic streptococci (21%), but it was low for gram-negative bacteria (1.3%). Patients with two or more bacteremias during the study period had an increased risk for developing a PJI (OR 2.29, 95%CI 1.17–4.50). Bacteremias occurring within a year from previous surgery were associated with the highest risk for developing a PJI. Chronic comorbidities, obesity, gender, joint location, indication for surgery or use of antibiotic-loaded cement did not affect the risk for PJI during bacteremia. Conclusions. The pathogen causing the bacteremia, number of bacteremias and the timing of bacteremia with respect to previous surgery affect the risk for developing a PJI as a consequence of bacteremia. Thus the type of pathogen, previous history of infections and the timing of bacteremia should be taken into account when evaluating the risk for PJI on a patient with bacteremia. On the other hand, significant patient-related risk factors for PJI during bacteremia could not be identified

Introduction. Polymicrobial infections are expected to complicate the treatment of bone and joint infections. Septic nonunions often occur after initial open fractures, which prophylactically receive broad-spectrum antibiotics. However, no data that describes frequencies of polymicrobial infections and pathogens evident in course of the treatment of septic nonunions is published. Therefore, this study aims at investigating the frequency and pathogen types in polymicrobial infections. Methods. Surgically treated Patients with long bone septic nonunion admitted between January 2010 and March 2018 were included in the study. Following parameters were examined: age, gender, American Society of Anesthesiologists (ASA) score, body mass index (BMI), and anatomical location of the infected nonunion. Microbiological culture data, polymerase-chain-reaction results of tissue samples, sonication, and joint fluid of the initial and follow-up revision surgeries were assessed. No exclusion criteria were determined. Results. The study encompassed 42 patients with a mean age of 53.9 ± 17.7 years (range, 23 – 93). Sixteen (38.1%) patients were female. In 46.3% of the patients open fractures led to septic nonunion. Twenty-six nonunions occurred at the tibia or fibula, 11 were localized at the femur, 2 at the humerus and 3 at the forearm. Only 2 patients were assessed as ASA type 1, while 26 were ASA type 2 and 12 patients ASA type 3. Mean number of performed surgeries was 6 ± 0.67 (range 2 – 21). In 6 patients (14.3%) polymicrobial infection were evident. A change of evidenced pathogens in course of the treatment occurred in 21 patients (50%). In 16 patients (38.1%) previously detected bacteria could be evidenced by microbial testing after further revision surgery. Staphylococcus aureus was most often evident (n=34, 30.6%), followed by Enterococcus species (n=25, 22.5%) and Staphylococcus epidermidis (n=18, 16.2%). Five Staphylococcus aureus were resistant to methicillin (MRSA). In patients without polymicrobial infection or further germ detection in course of the treatment 86.4% of the infections were due to Staphylococcus species. Patients with change of detected pathogens and polymicrobial infections suffered from more enterococci infections. Infections due to streptococci and gram-negative bacteria could only be evidenced in patients with polymicrobial infection and pathogen change in course of the treatment. Conclusion. The observed difference of microbiological patterns in septic nonunion may help to facilitate adjuvant local and systemic antibiotic treatment in septic nonunion patients. Reasons for the observed difference of microbiological patterns and its influence on patient outcome have still to be elucidated

Aim. Infection is one of the worst complications following total joint arthroplasty, which is often associated with significant morbidity and increased medical costs. Although Gram–positive bacteria remains the most prevalent causative agents, an increase in prosthetic joint infections (PJI) due to gram-negative bacteria (GNB) has been reported. Additionally, the emergence of multidrug resistant resistance (MDR) in GNB impacts the therapeutic options and may increase the rate of treatment failure and drug toxicity adverse effects due the prescription of harmful and toxics antimicrobial schemes. The purpose of the present study was to describe the predisposing factors associated to PJI caused by MDR-GNB in a specialized orthopedic reference hospital in Brazil from 2014 through 2018. Method. Retrospective case-control analysis of patients treated for MDR-GNB PJI over a four-year period (2014–2018). Data were collected from medical, surgical and laboratory records. PJI were defined according the criteria of MSIS. MDR was defined as non-susceptibility to at least one agent in three or more antimicrobial categories. Patients with prosthetic infection with at least two positive tissue cultures for MDR-GNB were selected. Univariate and multivariate logistic regression models were used to determine the independent risk factors associated with MDR-GNB PJI. Controls: patients with PJI with at least two positive tissue culture for non MDR- GNB. Results. A total of 104 patients were selected, 59 patients in the MDR-GNB PJI group and 44 in the control. Patients with MDR-GNB PJI were elderly (mean age of 68.36), distribution among sex was similar (49.2% female and 50.8% male) and 72.3% had one or more comorbidities. Most frequently identified comorbidities were diabetes (10.2%), malnutrition (5.5%), hypertension (4.7 %) and obesity (3.9%). Hip replacement accounted for 91.5% of the cases and 59.3% were revision arthroplasty. The mean time between the placement of the prothesis and the onset of PJI signs and symptoms was 438 days. In the univariate regression, the significant risk factors for MDR-GNB PJI were revision arthroplasty, alcoholism, nonelective arthroplasty, prior antimicrobial use, presence of concomitant infection and blood transfusion. However, in the multivariate analysis, prior use of antimicrobials (OR 9.31, CI95% 3.02–28.64) and the nonelective arthroplasty (OR 6.29, CI95% 1.75–22.6) remained as independent risk factors for MDR-GNB PJI. Conclusions. Previous use of antimicrobial and nonelective arthroplasty are important risk factors for PJI by GNB MDR