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. 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. 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

INTRODUCTION. Prosthetic joint related-infections (PJRI) are severe complications in orthopaedic surgery. Staphylococcus aureus and Staphylococcus epidermidis are the most commonly isolated pathogens from implants (1). The variable antimicrobial susceptibility found in these microorganisms, makes it necessary to perform individual susceptibility studies in order to select the best antibiotic combination for clinical management (2). MATERIAL AND METHODS. 35 staphylococcal strains (17 S. aureus, 18 S. epidermidis) were isolated from PJRI using a previously described sonication protocol (3). Biofilm-producing collection strains S. aureus 15981 (4) and S. epidermidis ATCC 35984 were also included in the study. In vitro susceptibility was evaluated against 7 antimicrobial agents: rifampin, vancomycin, ciprofloxacin, cotrimoxazole, cloxacillin, clindamycin, and daptomycin. Minimal Inhibitory Concentration (MIC) assays were determined according to EUCAST recommendations and breakpoints (5). Minimal Bactericidal Concentration (MBC) was also calculated by colony counting after plating the well contents. RESULTS. Antibiotic susceptibility assay results are shown in tables 1 and 2. It is especially remarkable the high number of meticillin-resistant S. aureus (MRSA) strains. Cotrimoxazole and clindamycin showed better results for S. aureus. Rifampin, vancomycin, and daptomycin showed a very good activity, although some resistant strains were detected for the first two. MBC values showed a strain-dependant activity of rifampin and vancomycin. Only daptomycin showed bactericidal activity against all the tested strains. DISCUSSION & CONCLUSIONS. The treatment of PJRI is still a challenge due to the variable antibiotic susceptibility and the growing number of multidrug-resistant strains. The high number of MRSA detected in our study, makes it necessary to search other antibiotics as an alternative to vancomycin, the traditional elective treatment. The high in vitro activity of daptomycin against the tested strains suggests that it could be an important alternative according to other promising results (6). Rifampin associated with other antimicrobials and cotrimoxazole, with a good in vitro activity against most MRSA strains, could be other potential strategies (7, 8)

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

To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistant Staphylococcus aureus (MSSA or MRSA) in tissue samples, using a bead-beating DNA extraction method. Twenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize the Staphylococcus genus, S. aureus, and methicillin resistance, 3 sets of 6 primers for the 16S ribosomal ribonucleic acid (rRNA) and the femA and mecA genes were used, respectively. The limit of detection and sensitivity (detection rate) of the LAMP assay for diagnosing MSSA and MRSA infection were analyzed. The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA

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

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

Aims. 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. 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

Aim. Osteomyelitis (OM) is a debilitating infection of the bone that originates from hematogenous spreading of microbes or contamination after surgery/fracture. OM is mainly caused by the opportunistic bacterium Staphylococcus aureus (SA), which can evade the host immune response, acquire antibiotic resistance and chronically colonize the musculoskeletal tissue . 1,2. , yet the underlying molecular and cellular processes are largely unclear. This study aimed to characterize the pathogenetic mechanisms of SA-OM with a focus on the long pentraxin 3 (PTX3), a soluble pattern recognition molecule and bone tissue component that is emerging as a new player in osteoimmunology . 3. and a diagnostic marker of periprosthetic joint infections, a common form of OM. 4. . Method. A murine model of OM based on intra-bone injection of SA was developed that closely mimicked surgery/trauma-related OM in humans and allowed addressing the role of PTX3 in gene-modified (Ptx3-/-) animals. Local and systemic infection and inflammation were assessed via microbiology, flow cytometry, histochemistry and microCT techniques. Results. SA-injected mice developed chronic infection with measurable levels of viable bone-resident bacteria up until 30 days from microbial challenge. The infection was confined to the treated limbs only and accompanied by extensive tissue remodelling. The bacterial load was higher in WT than Ptx3. -/-. animals at 6 and 14 days from SA injection. Accordingly, WT mice had enhanced systemic inflammation with expanded innate immune compartment in the spleen and increased serum levels of inflammatory cytokines and chemokines. PTX3 levels were higher in SA- than vehicle (PBS)-injected WT animals both in the serum and bone tissue. Furthermore, administration of a PTX3-targeting antibody reduced the bacterial burden in the bones of SA-injected WT mice. Conclusions. In a mouse model of SA-OM, genetic deficiency of PTX3 protected from infection and inflammation, pointing to this pentraxin as a crucial player in OM pathogenesis and a novel therapeutic target in bone infections. The study was approved by the Italian Ministry of Health (approval n. 520/2019-PR issued on 19/07/2019) and supported by Fondazione Beppe and Nuccy Angiolini

Aim. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is commonly associated with serious cases of community-onset skin and musculoskeletal infections (Co-SMSI). Molecular epidemiology analysis of CA-MRSA recovered from skin and soft tissues specimens is lacking in Latin America. This study aimed to identify phenotypic and genotypic features of MRSA isolates recovered from patients presenting Co-SMSI. Methods. Consecutive MRSA isolates recovered from Co-SMSI of patients admitted from March 2022 to January 2023 in a Brazilian teaching hospital were tested for antimicrobial resistance and characterized by their genotypic features. Identification was carried out by automated method and through MALDI-TOF MS. Antimicrobial susceptibility was tested by disk diffusion, broth microdilution and E-test strips for determination of the minimal inhibitory concentration (MIC) according to recommendations from the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Gene mecA characterization and Sccmec typing were performed by multiplex polymerase chain reaction (PCR) assay, and gene lukF detection by single PCR. Patients were prospectively followed up for two months, in order to determine their clinical characteristics and outcomes. Results. Overall, 48 Staphylococcus aureus isolates were obtained from 68 samples recovered from patients with Co-SMSI. Twenty two (42%) were phenotypically characterized as MRSA, although mecA gene was only identified in 20 of those samples. Sccmec was untypable in 12 isolates, Sccmec was type II in 4 isolates and 2 were classified as type IVa. LukF gene was identified in 5 isolates. Antimicrobial resistance profile showed that all isolates were susceptible to linezolid and vancomycin with MIC = 1 and MIC = 2 in 66,7% and 33.3%, respectively. Susceptibility to quinolones was worryingly low and none of the isolates were sensitive to usual doses of ciprofloxacin and levofloxacin, and showed increased rates of resistance to increased exposure to these drugs, as well. Isolates were both susceptible to gentamicin and tetracycline in 85% and resistance to also Sulfamethoxazole/Trimethoprim occurred in only 2 isolates. Mortality rate evaluated within 1 month of the initial evaluation was 10% among MRSA isolates. Conclusions. Our results showed that CA-MRSA isolates causing Co-SMSI demonstrated an alarming pattern of multidrug resistance, including to β-lactam and quinolones, which have been commonly prescribed as empirical therapy for patients with skin, soft tissue and musculoskeletal infections

Aim. Prosthetic joint infections (PJI) remain a great challenge in orthopedic surgery with a high mortality rate. It is particularly complicated by biofilms and infections caused by Methicillin-resistant Staphylococcus aureus (MRSA). It concurrently shields bacteria from host immune responses and confers resistance to antibiotics. This study aims to investigate the efficacy of radioimmunotherapy as an innovative therapeutic modality to address the challenges posed by MRSA and its biofilm. Method. We induced specific monoclonal antibodies 4497-IgG1 as carriers, which target wall teichoic acids (WTA) existing on MRSA and its biofilm. Radionuclides actiniumr-225 (. 225. Ac, α-emitter) and lutetium-177 (. 177. Lu, β-emitter) were conjugated with mAbs using DOTA as chelator. Quality control was assessed using thin layer chromatography and immunoreactivity assays. . 225. Ac- and . 177. Lu-labelled 4497-IgG1 were employed to evaluate the susceptibility of MRSA and its biofilm to the radioimmunotherapy in vitro. Planktonic MRSA and biofilms, at concentrations of 10. 8. and 10. 7. CFU/mL, were incubated at 37°C for 60 minutes in PBS containing either . 225. Ac-mAb (0 - 14.8 kBq) or . 177. Lu-mAb (0 - 14.8 MBq). Radiolabelled dunituximab and free radionuclides serve as isotype-matched negative control. The bacterial viability and metabolic activity were subsequently quantified using CFU and XTT assays. Results. The radiochemical purity of the . 225. Ac-mAbs and . 177. Lu-mAbs complex were determined to be 95.4% and 96.16%. Immunoreactivity fractions of them were measured at 81.8% and 80.8%. . 225. Ac-mAbs and . 177. Lu-mAbs exhibited significant and dose-dependent antimicrobial effects on both planktonic MRSA and biofilm. . 225. Ac- and . 177. Lu-4497IgG1 at doses of 7.4 kBq and 7.4 MBq resulted in more than 4-log reduction in bacterial counts. In biofilms, 2-log reduction at the highest . 225. Ac radioactivity of 14,8kBq. The . 177. Lu complex showed a strong dose-dependent effect, with a reduction of up to 4-log. The XTT assay confirmed these findings, showing a decrease in metabolic activity corresponding to a decrease in bacterial counts, and a slight increase in metabolic activity at the lower dose. Conclusions. Our study demonstrates the efficacy of . 225. Ac and . 177. Lu-labelled 4497-IgG1 antibodies in mediating dose-dependent bactericidal effects against planktonic MRSA and biofilms in vitro. This indicates that radioimmunotherapy could be a potential targeted therapeutic strategy against MRSA and its biofilm. Further research in preclinical and clinical settings is warranted to validate and refine these findings on biofilm-associated implant infections

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

Aim. Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. Herewith we introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide the biodistribution and specificity in a mouse implant infection model. Methods. 4497-IgG1targeting S. aureus Wall Teichoic Acid was labeled to Indium-111 using “CHXA” as a chelator. SPECT-CT scans were performed at 24, 72 and 120 hours after administration in Balb/cAnNCrl mice with a subcutaneous implant pre-colonized with biofilm of S. aureus. Biodistribution over the various organs of this labelled antibody was visualized and quantified using SPECT-CT imaging and compared to uptake at the target tissue with implant infection. Results. Uptake of the . 111. In-4497 mAbs (half-life 59 hours) at the infected implant gradually increased from 8.34%ID/g at 24 hours to 9.22%ID/g at 120 hours. Uptake at the heart/blood pool decreased over time from 11.60 to 7.58%ID/g whereas the uptake in other organs decreased from 7.26 to less than 4.66%ID/g at 120 hours. Conclusion. 111. In-4497 mAbs was found to specifically detect S. aureus and its biofilm with excellent and prolonged accumulation at the colonized implant site. Therefore, it holds great promise as a drug delivery system for diagnostic and bactericidal treatment of biofilm. However, high activity in the blood pool must be considered as it could pose a risk to healthy tissue

Purpose: Asymptomatic colonization with methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staph. aureus (MSSA) has been described as a risk factor for subsequent surgical site infection. Colonization of nares, either present at admission to the hospital or acquired during hospitalization, increases the risk for infection. Identifying Staph. aureus colonization in the presurgical screening process is important in reducing subsequent surgical site infection. We performed anterior nares surveillance screens of 6551 inpatient surgeries using polymerase chain reaction (PCR) assay. This rapid technology provided results in less than 24 hrs and a decolonization protocol was administered. Method: Patients admitted for orthopedic surgery were screened in the prescreening unit. The treatment intervention for MRSA and MSSA patients was 5-day application of intranasal mupirocin 2% applied twice daily. In addition, MRSA positive patients were instructed to bathe with chlorhexidine 2% for 5 days and were rescreened prior to surgery. Contact precautions were implemented if the second screen was positive for MRSA. All MRSA positives received vancomycin for surgical prophylaxis. Results: From July 17, 2006 through August 31, 2007, 6551 patients were screened; 1471 (22%) were MSSA positive and 291 (4%) were MRSA positive. Repeat nasal screens were obtained from MRSA patients prior to surgery and revealed 86% eradication. In the cohort of positive screens, there were 3 MSSA infections (0.2%) and 3 MRSA infections (1.0%). In the 4789 negative screens there were 7 infections (1 MRSA and 6 MSSA). The surgical site infection rate in the screened patients was 0.2%. In an equivalent group of unscreened patients from the prior year, the infection rate had been 0.4%. Conclusion: We have successfully implemented an MRSA and MSSA eradication program for all inpatient surgeries during the prescreening process. It has allowed for early identification of patients colonized with MRSA and MSSA, decolonization treatment, and appropriate surgical prophylaxis for MRSA. Since implementation we have documented a significant reduction in infection rates due to MRSA and MSSA. A multidisciplinary approach with strong administrative support and consistent communication was vital to the implementation of the program

Staphylococcus aureus is the most frequently isolated organism in periprosthetic joint infections. The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding of its antibacterial characteristics is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against S. aureus. SF samples were collected from patients undergoing total elective knee or hip arthroplasty. Different S. aureus strains previously found to be sensitive and resistant, UAMS-1 and USA300 WT, respectively, were used. We performed in-vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated the sensitivity to killing, comparing WT and CopAZB-deficient USA300 strains. UAMS-1 evidenced a greater sensitivity to SF when compared to USA300 WT, at 12 (p=0.001) and 24 hours (p=0.027). UAMS-1 significantly died at 24 hours (p=0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at 4 (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 4 (p=0.011), 12 (p=0.011), and 24 hours (p=0.011). Both WT and CopAZB-deficient USA300 strains significantly died in SF, evidencing a MBC-Cu of 50µM against USA300 WT (p=0.011). SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. The addition of 10µM of copper was highly toxic for both strains, confirming its bactericidal effect. We evidenced CopAZB-proteins involvement in copper effluxion by demonstrating the high sensitivity of the mutant strain to lower copper concentrations. Thus, we propose CopAZB-proteins as potential targets and the use of exogenous copper as possible treatment alternatives against S. aureus

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

Infection is one of the most serious complications of orthopedic surgery, particularly in implant-related procedures. Minimum inhibitory concentration (MIC) for identified bacteria is an important factor for successful antibiotic treatment. We investigated the MIC of antibiotics in Staphylococcus species from orthopedic infections, comparing with isolates from respiratory medicine. Staphylococcus species isolated in our laboratory from January 2013 to July 2016 were retrospectively reviewed. The MIC of vancomycin (VCM), arbekacin (ABK), teicoplanin (TEIC), linezolid (LZD), and rifampicin (RFP) was reviewed. Differences in the MIC of each antibiotic in orthopedic and respiratory samples were determined. A total of 259 isolates were evaluated (89 orthopedic, 170 respiratory). Staphylococcus aureus was the most commonly identified species (58%). In comparison with orthopedic samples, the number of isolates with a VCM MIC <0.5 μg/ml in methicillin sensitive staphylococcus aureus (MSSA) was significantly higher in respiratory isolates, while a MIC of 2 μg/ml was significantly lower (P = 0.0078). The proportion of isolates with a VCM MIC of 2 μg/ml in methicillin-resistant coagulase-negative staphylococci (MRCNS) was significantly higher in orthopedic isolates than that seen in respiratory isolates of methicillin-resistant staphylococcus aureus (MRSA; P < 0.001). When comparing MRCNS and other orthopedic Staphylococci, the rate of RFP MIC >2 μg/ml in MRCNS isolates was significantly higher (P = 0.0058). The MIC of VCM in Staphylococcus species from orthopedic infection was higher than that of respiratory samples, particularly in MRCNS from implant-related samples. MRCNS showed a significantly higher rate of resistance for RFP versus other orthopedic isolates

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