Aims. The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against Staphylococcus aureus. Methods. We performed in vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10 µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated its sensitivity to killing, comparing wild type (WT) and CopAZB-deficient USA300 strains. Results. UAMS-1 demonstrated a greater sensitivity to SF compared to USA300 WT at 12 hours (p = 0.001) and 24 hours (p = 0.027). UAMS-1 died in statistically significant quantities at 24 hours (p = 0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at four (p = 0.001), 12 (p = 0.005), and 24 hours (p = 0.006). We confirmed a high sensitivity to killing with the addition of exogenous copper on both strains at four (p = 0.011), 12 (p = 0.011), and 24 hours (p = 0.011). WT and CopAZB-deficient USA300 strains significantly died in SF, demonstrating a MBC-Cu of 50 µM against USA300 WT (p = 0.011). Conclusion. SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. Adding 10 µM of copper was highly toxic, confirming its bactericidal effect. We found CopAZB proteins to be involved in copper effluxion by demonstrating the high sensitivity of mutant strains to lower copper concentrations. Thus, we propose CopAZB proteins as potential targets and use exogenous copper as a treatment alternative against S. aureus. Cite this article: Bone Joint Res 2024;13(11):632–646

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

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. 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. 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. This study aimed to explore the role of small colony variants (SCVs) of Staphylococcus aureus in intraosseous invasion and colonization in patients with periprosthetic joint infection (PJI). Methods. A PJI diagnosis was made according to the MusculoSkeletal Infection Society (MSIS) for PJI. Bone and tissue samples were collected intraoperatively and the intracellular invasion and intraosseous colonization were detected. Transcriptomics of PJI samples were analyzed and verified by polymerase chain reaction (PCR). Results. SCVs can be isolated from samples collected from chronic PJIs intraoperatively. Transmission electron microscopy (TEM) and immunofluorescence (IF) showed that there was more S. aureus in bone samples collected from chronic PJIs, but much less in bone samples from acute PJIs, providing a potential mechanism of PJI. Immunofluorescence results showed that SCVs of S. aureus were more likely to invade osteoblasts in vitro. Furthermore, TEM and IF also demonstrated that SCVs of S. aureus were more likely to invade and colonize in vivo. Cluster analysis and principal component analysis (PCA) showed that there were substantial differences in gene expression profiles between chronic and acute PJI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these differentially expressed genes were enriched to chemokine-related signal pathways. PCR also verified these results. Conclusion. Our study has shown that the S. aureus SCVs have a greater ability to invade and colonize in bone, resulting in S. aureus remaining in bone tissues long-term, thus explaining the pathogenesis of chronic PJI. Cite this article: Bone Joint Res 2022;11(12):843–853

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. Methods. Mature biofilms of S. aureus (ATCC 29213) were grown on titanium alloy (Ti6Al4V) coupons for seven days and were subjected to the following treatments or their combinations: antibiotics, mechanical cleaning, or heat shock by induction heating of 60°C for one minute. Experiments were repeated at least five times. Results. In the untreated biofilm, growth up to 1.8×10. 11. colony-forming units (CFU)/cm. 2. was observed. Treatment with ciprofloxacin, flucloxacillin, vancomycin, cefuroxime, and amoxicillin all with rifampicin gave 6.0 log, 6.1 log, 1.4 log, 4.8 log, and 3.6 log reduction in CFU/cm. 2. , respectively. Mechanical cleaning alone resulted in 4.9 log reduction and induction heating in 7.3 log reduction. There was an additional effect of ciprofloxacin, flucloxacillin, and induction heating when used in combinations. There was no additional effect for mechanical cleaning. No bacterial growth could be detected after induction heating followed by seven days of ciprofloxacin with rifampicin. Conclusion. Mechanical cleaning, antibiotics, and non-contact induction heating reduced the bacterial load of mature S. aureus biofilms with approximately 5 log or more as a single treatment. The effect of mechanical cleaning on mature S. aureus biofilms was limited when used in combination with antibiotics and/or induction heating. Cite this article: Bone Joint Res 2022;11(9):629–638

Aims. Infection after surgery increases treatment costs and is associated with increased mortality. Hip fracture patients have historically had high rates of methicillin-resistant Staphylococcus aureus (MRSA) colonization and surgical site infection (SSI). This paper reports the impact of routine MRSA screening and the “cleanyourhands” campaign on rates of MRSA SSI and patient outcome. Methods. A total of 13,503 patients who presented with a hip fracture over 17 years formed the study population. Multivariable logistic regression was performed to determine risk factors for MRSA and SSI. Autoregressive integrated moving average (ARIMA) modelling adjusted for temporal trends in rates of MRSA. Kaplan-Meier estimators were generated to assess for changes in mortality. Results. In all, 6,189 patients were identified before the introduction of screening and 7,314 in the post-screening cohort. MRSA infection fell from 69 cases to 15 in the post-screening cohort (p < 0.001). The ARIMA confirmed a significant reduction in MRSA SSI post-screening (p = 0.043) but no significant impact after hand hygiene alone (p = 0.121). Overall SSI fell (2.4% to 1.5%), however deep infection increased slightly (0.89% to 1.06%). ARIMA showed neither intervention affected overall SSI (“cleanyourhands” -0.172% (95% confidence interval (CI) -0.39% to 0.21); p = 0.122, screening -0.113% per year, (95% CI -0.34 to 0.12); p = 0.373). One-year mortality after deep SSI was unchanged after screening (50% vs 45%; p = 0.415). Only warfarinization (OR 3.616 (95% CI 1.366 to 9.569); p = 0.010) and screening (OR 0.189 (95% CI 0.086 to 0.414); p < 0.001) were significant covariables for developing MRSA SSI. Conclusion. While screening and decolonization may reduce MRSA-associated SSI, the benefit to patient outcome remains unclear. Overall deep SSI remains an unsolved problem that has seen little improvement over time. Preventing other hospital-associated infections should not be forgotten in the fight against MRSA. Cite this article: Bone Joint J 2021;103-B(1):170–177

Staphylococcus aureus is one of the leading causes of surgical site infection (SSI). Over the past decade there has been an increase in methicillin-resistant S. aureus (MRSA). This is a subpopulation of the bacterium with unique resistance and virulence characteristics. Nasal colonisation with either S. aureus or MRSA has been demonstrated to be an important independent risk factor associated with the increasing incidence and severity of SSI after orthopaedic surgery. Furthermore, there is an economic burden related to SSI following orthopaedic surgery, with MRSA-associated SSI leading to longer hospital stays and increased hospital costs. Although there is some controversy about the effectiveness of screening and eradication programmes, the literature suggests that patients should be screened and MRSA-positive patients treated before surgical admission in order to reduce the risk of SSI. Cite this article: Bone Joint J 2013;95-B:4–9

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

Aims. Preoperative nasal Staphylococcus aureus screening and eradication reduces surgical site infections (SSIs) but its impact on reducing early prosthetic joint infection (PJI) remains controversial. This study aims to assess the effect of preoperative nasal S. aureus screening and eradication on the incidence of early PJI in general and S. aureus-induced early PJI. Methods. All primary total hip arthroplasties (THA) and total knee arthroplasties (TKA) performed from January 2006 to April 2018 were retrospectively reviewed for the incidence of early PJI. Demographic parameters, risk factors for PJI (American Society of Anaesthesiologists classification, body mass index, smoking status, and diabetes mellitus) and implant types were collected. A preoperative screening and eradication protocol for nasal colonization of S. aureus was introduced in October 2010. The incidence of early PJI was compared before and after the implementation of the protocol. Missing data were imputed via multiple imputation by chained equations. Inverse probability weighting was used to account for differences between patients in both groups. Weighted univariate logistic regression was used to evaluate the incidence of early PJI for both groups. Results. In total, 10,486 THAs and TKAs were performed in the research period. After exclusion, a cohort of 5,499 screened cases and 3,563 non-screened cases were available for analysis. Overall, no significant reduction in early PJI was found in the screened group (odds ratio (OR) 0.78, 95% confidence interval (CI) 0.55 to 1.11; p = 0.173). However, the incidence of S. aureus-induced PJI was significantly reduced (OR 0.58, 95% CI 0.36 to 0.92; p = 0.027) in the screened group. Conclusion. A preoperative nasal S. aureus screening and eradication protocol did not significantly reduce the overall incidence of early PJI after THA or TKA. However, a decreased incidence of S. aureus-induced early PJI was established. These findings can help to establish better consensus around the value of these screening protocols. Cite this article: Bone Joint J 2020;102-B(10):1341–1348

Aims. Induction heating is a noninvasive, nonantibiotic treatment modality that can potentially be used to cause thermal damage to the bacterial biofilm on the metal implant surface. The purpose of this study was to determine the effectiveness of induction heating on killing Staphylococcus epidermidis from biofilm and to determine the possible synergistic effect of induction heating and antibiotics. Methods. S. epidermidis biofilms were grown on titanium alloy (Ti6Al4V) coupons for 24 hours (young biofilm) and seven days (mature biofilm). These coupons with biofilm were heated to temperatures of 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, and 90°C for 3.5 minutes and subsequently exposed to vancomycin and rifampicin at clinically relevant concentrations. Results. For the young biofilm, total eradication was observed at 65°C or higher for 3.5 minutes followed by 24 hours of vancomycin 10 mg/l and rifampicin 1 mg/l. For the mature biofilm, total eradication was observed at 60°C for 3.5 minutes followed by 24 hours of vancomycin 10 mg/l and rifampicin 1 mg/l. Total eradication was also observed at 60°C for 3.5 minutes followed by 24 hours of vancomycin 1 mg/l and rifampicin 1 mg/l followed by another thermal shock of 60°C for 3.5 minutes (two thermal shocks). Conclusion. Induction heating of Ti6Al4V coupons is effective in reducing bacterial load in vitro for S. epidermidis biofilms. Induction heating and antibiotics have a synergistic effect resulting in total eradication of the biofilm at 60°C or higher for clinically relevant concentrations of vancomycin and rifampicin. Cite this article:Bone Joint Res. 2020;9(4):192–199

Objectives. Staphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability. Methods. Bovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy. Results. Hla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect. Conclusion. This study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis. Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457–467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1

Objectives. Nasal carriers of Staphylococcus (S.) aureus (MRSA and MSSA) have an increased risk for healthcare-associated infections. There are currently limited national screening policies for the detection of S. aureus despite the World Health Organization’s recommendations. This study aimed to evaluate the diagnostic performance of molecular and culture techniques in S. aureus screening, determine the cause of any discrepancy between the diagnostic techniques, and model the potential effect of different diagnostic techniques on S. aureus detection in orthopaedic patients. Methods. Paired nasal swabs for polymerase chain reaction (PCR) assay and culture of S. aureus were collected from a study population of 273 orthopaedic outpatients due to undergo joint arthroplasty surgery. Results. The prevalence of MSSA nasal colonization was found to be between 22.4% to 35.6%. The current standard direct culturing methods for detecting S. aureus significantly underestimated the prevalence (p = 0.005), failing to identify its presence in approximately one-third of patients undergoing joint arthroplasty surgery. Conclusion. Modelling these results to national surveillance data, it was estimated that approximately 5000 to 8000 S. aureus surgical site infections could be prevented, and approximately $140 million to $950 million (approximately £110 million to £760 million) saved in treatment costs annually in the United States and United Kingdom combined, by using alternative diagnostic methods to direct culture in preoperative S. aureus screening and eradication programmes. Cite this article: S. T. J. Tsang, M. P. McHugh, D. Guerendiain, P. J. Gwynne, J. Boyd, A. H. R. W. Simpson, T. S. Walsh, I. F. Laurenson, K. E. Templeton. Underestimation of Staphylococcus aureus (MRSA and MSSA) carriage associated with standard culturing techniques: One third of carriers missed. Bone Joint Res 2018;7:79–84. DOI: 10.1302/2046-3758.71.BJR-2017-0175.R1

Aims. The purpose of this study was to determine whether intracellular Staphylococcus aureus is associated with recurrent infection in a rat model of open fracture. Methods. After stabilizing with Kirschner wire, we created a midshaft femur fracture in Sprague-Dawley rats and infected the wound with green fluorescent protein (GFP)-tagged S. aureus. After repeated debridement and negative swab culture was achieved, the isolation of GFP-containing cells from skin, bone marrow, and muscle was then performed. The composition and viability of intracellular S. aureus in isolated GFP-positive cells was assessed. We suppressed the host immune system and observed whether recurrent infection would occur. Finally, rats were assigned to one of six treatment groups (a combination of antibiotic treatment and implant removal/retention). The proportion of successful eradication was determined. Results. Green fluorescent protein-containing cells were successfully isolated after the swab culture was negative from skin (n = 0, 0%), muscle (n = 10, 100%), and bone marrow (n = 10, 100%) of a total of ten rats. The phagocytes were predominant in GFP-positive cells from muscle (73%) and bone marrow (81%) with a significantly higher viability of intracellular S. aureus (all p-values < 0.001). The recurrent infection occurred in up to 75% of rats after the immunosuppression. The proportion of successful eradication was not associated with implant retention or removal, and the efficacy of linezolid in eradicating intracellular S. aureus is significantly higher than that of vancomycin. Conclusion. Intracellular S. aureus is associated with recurrent infection in the rat model of open fracture. Usage of linezolid, a membrane-permeable antibiotic, is an effective strategy against intracellular S. aureus. Cite this article:Bone Joint Res. 2020;9(2):71–76

Aims. To characterize the intracellular penetration of osteoblasts and osteoclasts by methicillin-resistant Staphylococcus aureus (MRSA) and the antibiotic and detergent susceptibility of MRSA in bone. Methods. Time-lapse confocal microscopy was used to analyze the interaction of MRSA strain USA300 with primary murine osteoblasts and osteoclasts. The effects of early and delayed antibiotic treatments on intracellular and extracellular bacterial colony formation and cell death were quantified. We tested the effects of cefazolin, gentamicin, vancomycin, tetracycline, rifampicin, and ampicillin, as well as agents used in surgical preparation and irrigation. Results. MRSA infiltrated bone-resident cells within 15 to 30 minutes. Penetration was most effectively prevented with early (i.e. 30 minutes) antibiotic administration. The combined administration of rifampicin with other antibiotics potentiated their protective effects against MRSA-induced cytotoxicity and most significantly reduced extracellular bacterial bioburden. Gentamicin-containing compounds were most effective in reducing intracellular MRSA bioburden. Of the surgical preparation agents evaluated, betadine reduced in vitro MRSA growth to the greatest extent. Conclusion. The standard of care for open fractures involves debridement and antibiotics within the first six hours of injury but does not account for the window in which bacteria penetrate cells. Antibiotics must be administered as early as possible after injury or prior to incision to prevent intracellular infestation. Rifampicin can potentiate the capacity of antibiotic regimens to reduce MRSA-induced cytotoxicity. Cite this article:Bone Joint Res. 2020;9(2):49–59

Objectives. Vancomycin and fosfomycin are antibiotics commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. This study compares the in vitro inhibitory effects against MRSA of articulating cement spacers impregnated with either vancomycin or fosfomycin. Methods. Vancomycin-impregnated articulating cement spacers and fosfomycin-impregnated articulating cement spacers were immersed in sterile phosphate-buffered saline (PBS) solutions and then incubated. Samples were collected for bioactivity evaluation. The aliquots were tested for MRSA inhibition with the disc diffusion method, and the inhibition zone diameters were measured. The inhibition zone differences were evaluated using the Wilcoxon Rank Sum Test. Results. The vancomycin group had significantly larger inhibition zones than the fosfomycin group from day three through to completion of the fourth week of incubation (p < 0.001). The vancomycin group exhibited a MRSA inhibition zone up to four weeks but the fosfomycin group showed an inhibition zone for only three days and after that did not show the the potential to inhibit MRSA. Conclusion. This in vitro study found that the inhibitory effect of vancomycin-impregnated articulating cement spacers against MRSA outperformed fosfomycin-impregnated articulating cement spacers. Further comparing our results to other published reports suggests there might be a limitation of the disc diffusion bioassay to show a large inhibitory zone in a high concentration of a highly soluble antibiotic. Cite this article: V. Yuenyongviwat, N. Ingviya, P. Pathaburee, B. Tangtrakulwanich. Inhibitory effects of vancomycin and fosfomycin on methicillin-resistant Staphylococcus aureus from antibiotic-impregnated articulating cement spacers. Bone Joint Res 2017;6:132–136. DOI: 10.1302/2046-3758.63.2000639

Aims. Biofilm formation is intrinsic to prosthetic joint infection (PJI). In the current study, we evaluated the effects of silver-containing hydroxyapatite (Ag-HA) coating and vancomycin (VCM) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation. Methods. Pure titanium discs (Ti discs), Ti discs coated with HA (HA discs), and 3% Ag-HA discs developed using a thermal spraying were inoculated with MRSA suspensions containing a mean in vitro 4.3 (SD 0.8) x 10. 6. or 43.0 (SD 8.4) x 10. 5. colony-forming units (CFUs). Immediately after MRSA inoculation, sterile phosphate-buffered saline or VCM (20 µg/ml) was added, and the discs were incubated for 24 hours at 37°C. Viable cell counting, 3D confocal laser scanning microscopy with Airyscan, and scanning electron microscopy were then performed. HA discs and Ag HA discs were implanted subcutaneously in vivo in the dorsum of rats, and MRSA suspensions containing a mean in vivo 7.2 (SD 0.4) x 10. 6.   or 72.0 (SD 4.2) x 10. 5.   CFUs were inoculated on the discs. VCM was injected subcutaneously daily every 12 hours followed by viable cell counting. Results. Biofilms that formed on HA discs were thicker and larger than those on Ti discs, whereas those on Ag-HA discs were thinner and smaller than those on Ti discs. Viable bacterial counts in vivo revealed that Ag-HA combined with VCM was the most effective treatment. Conclusion. Ag-HA with VCM has a potential synergistic effect in reducing MRSA biofilm formation and can thus be useful for preventing and treating PJI. Cite this article:Bone Joint Res. 2020;9(5):211–218

We investigated the incidence of cephalosporin-resistant bacteria in infected hip arthroplasties. Of 740 patients having hip replacement or related procedures performed over three years, 30 had positive bacteriological cultures from tissue removed at the time of surgery. In 18 of the 30 cultures Staphylococcus epidermidis was grown and 12 of these were methicillin-resistant. A prospective study of skin swabs taken from 100 consecutive patients at the time of admission for THR showed methicillin-resistant Staphylococcus epidermidis in 25. This cephalosporin-resistant organism was shown to be the commonest proven cause of infection, and its presence as a skin commensal raises important questions about current antibiotic prophylaxis for joint replacement

This prospective five-year study analyses the impact of methicillin-resistant Staphylococcus aureus (MRSA) on an Irish orthopaedic unit. We identified 318 cases of MRSA, representing 0.76% of all admissions (41 971). A total of 240 (76%) cases were colonised with MRSA, while 120 (37.7%) were infected. Patients were admitted from home (218; 68.6%), nursing homes (72; 22.6%) and other hospitals (28; 8.8%). A total of 115 cases (36.6%) were colonised or infected on admission. Many patients were both colonised and infected at some stage. The length of hospital stay was almost trebled because of the presence of MRSA infection. Encouragingly, overall infection rates have not risen significantly over the five years of the study despite increased prevalence of MRSA. However, the financial burden of MRSA is increasing, highlighting the need for progress in understanding how to control this resistant pathogen more effectively