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.