An extensive review of the spinal and arthroplasty literature was undertaken to evaluate the effectiveness of local antibiotic irrigation during surgery. The efficacy of antibiotic irrigation for the prevention of acute post-operative infection after total joint arthroplasty was evaluated retrospectively in 2293 arthroplasties (1990 patients) between January 2004 and December 2013. The mean follow-up was 73 months (20 to 139). One surgeon performed all the procedures with minimal post-operative infection.

The intra-operative protocol included an irrigation solution of normal saline with vancomycin 1000 mg/l and polymyxin 250 000 units/l at the rate of 2 l per hour. No patient required re-admission for primary infection or further antibiotic treatment. Two morbidly obese patients (two total hip arthroplasties) developed subcutaneous fat necrosis requiring debridement and one was revised because the deep capsular sutures were contaminated by the draining subcutaneous haematoma. One patient who had undergone total knee arthroplasty had unrecognised damage to the lateral superior geniculate artery and developed a haematoma that became infected secondarily four months after the surgery and underwent revision.

The use of antibiotic irrigation during arthroplasty surgery has been highly effective for the prevention of infection in the author’s practice. However, it should be understood that any routine prophylactic use of antibiotics may result in resistant organisms, and the wise stewardship of the use of antibiotics is an important part of surgical practice.

Cite this article: Bone Joint J 2016;98-B(1 Suppl A):23–6.

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

Aim. Chemical debridement is a fundamental step during Periprosthetic joint infection (PJI) surgery. Antiseptic solutions are commonly used, but evidence on the optimal antiseptic, concentration, and irrigation time is lacking. The aim of this study is to analyze and compare the anti-biofilm capacity of povidone iodine, H. 2. 0. 2. , acetic acid and Bactisure™ after different exposure times, as well as their combinations. Method. Surgical steel discs inoculated with methicillin susceptible (MSSA) and resistant S. aureus (MRSA), P. aeruginosa, and S. epidermidis were exposed to the following antiseptic solutions: 0.3% (PI0.3) and 10% povidone iodine (PI10), H. 2. 0. 2. , 3% Acetic acid (AA3) and Bactisure™. Combinations included AA3, H. 2. 0. 2. , and PI10 in various orders. Exposure time for the antiseptics solutions was 1, 3 and 5 minutes, while combinations had a 9-minute total exposure, 3 minutes per antiseptic sequentially. All experiments were performed in triplicate and with a sterile saline control. nThe reduction in colony-forming units (CFU) was measured after sonication, and biofilm structure was analyzed via scanning electron microscopy. Results. PI showed the highest antibiofilm activity. PI0.3 eradicated bacteria on the discs after 3 and 5 minutes of exposure, but only achieved a 77.1% reduction after 1 minute. After PI10 treatment, we did not recover any bacteria regardless of exposure time. H. 2. 0. 2. , AA3, and Bactisure™ reached a significantly lower bacterial decrease at all exposure times compared to PI0.3 and PI10. AA3 was less effective against MSSA and S. epidermidis. H. 2. 0. 2. showed less activity against MRSA than PI0.3, PI10, and Bactisure™. Combinations of antiseptics starting with AA3 showed the best results in terms of CFU reduction and cell viability. Conclusions. We propose a sequential combination of AA3 + H. 2. 0. 2. + PI10 with an exposure time of 9 minutes for the chemical debridement in PJI surgery. First, AA3 performs debridement and disruption of the biofilm. Then, H. 2. 0. 2. has a bactericidal effect and increases the porosity of the cell wall, and PI10 has a final bactericidal effect. If combinations are unavailable, PI is a cost-effective alternative

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

Aim. Evaluate if Neutrophil Extracellular Traps related biomarkers (citrullinated histone H3 [H3Cit], cellfree DNA [cfDNA], and myeloperoxidase) are increased in synovial fluid of patients with PJI and investigate the diagnostic accuracy of NET formation biomarkers for PJI. Method. Patients who underwent hip or knee revision total joint arthroplasty were categorised into two groups according to the Second International Consensus Meeting on Musculoskeletal Infection (2018) criteria. Sixteen patients were classified as infected and 16 as non-infected. cf-DNA, myeloperoxidase and H3Cit were measured in synovial fluid collected during surgery. Sensitivity, specificity, and receiver operating characteristic (ROC) curve were calculated. Results. Patients with PJI presented significantly higher levels of synovial fluid cf-DNA (105.5 ng/ml ± 58.3 vs 1.9 ± 1.2, p>0.0001), myeloperoxidase (1575 pg/ml ± 826 vs 50.16 ± 100, p<0.0001) and citrullinated histone H3 (1.688 ± 1.214 vs 13.88 ± 24.4, p < 0.0001). In the ROC curve analyses, the area under the curve for cf-DNA, myeloperoxidase and H3cit were 1 [0.89 – 1], 0.98 [0.86 – 1], and 0.94 [0.8 – 0.99], respectively. The sensitivity for detecting PJI using synovial fluid was 100% for cf-DNA, 93,7% for myeloperoxidase, and 87,5% for H3cit. The sensitivity for cf-DNA and myeloperoxidase was 100%, and 87,5 % for H3cit. Conclusions. Our results show that neutrophils within periprosthetic microenvironment release NETs as part of the bactericidal arsenal to fight infection. These results allow a better understanding of the cellular and molecular processes that occur in this microenvironment, enabling the design of more assertive strategies for the identification of new biomarkers and for a better use of the available ones. Furthermore, novel studies are needed to define whether and how NET-related biomarkers can be useful for the diagnosis of PJI

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

Aim. Staphylococcus aureus is the first causative agent of bone and joints infections (BJI). It causes difficult-to-treat infections because of its ability to form biofilms, and to be internalized and persist inside osteoblastic cells. Recently, phage therapy has emerged as a promising therapy to improve the management of chronic BJI. In the present study, we evaluated the efficacy of an assembly of three bacteriophages previously used in a clinical case report (Ferry, 2018) against S. aureus in in vitro models of biofilm and intracellular osteoblast infection. Methods. Using HG001 S. aureus, the bactericidal activities of the assembly of the three bacteriophages (Pherecydes Pharma) used alone or in association with vancomycin or rifampicin were compared by quantifying the number of viable bacteria in mature biofilms and infected osteoblasts after 24h of exposure. Results. The activity of bacteriophages against biofilm-embedded S. aureus was dose-dependent. Synergistic effects were observed when bacteriophages were combined to antibiotics at the lowest concentrations, with no significant bactericidal activity in monotherapy. In the human osteoblast infection model, we were able to show that phage penetration into osteoblasts was only possible when the cells were infected, suggesting a S. aureus dependent Trojan horse mechanism. The intracellular inoculum in osteoblasts treated with bacteriophages or vancomycin was significantly higher than in cells treated with lysostaphin, used as control condition of rapid killing of bacteria released in the extracellular media after death of infected cells and absence of intracellular activity. These results suggest that bacteriophages are probably both i) inactive in the intracellular compartment and ii) unable to kill all bacteria released after cell lysis into the extracellular medium fast enough to prevent them from reinfecting other osteoblasts. Conversely, the intracellular inoculum recovered from cells treated with vancomycin+bacteriophages was significantly lower than the one inside cells treated with vancomycin or bacteriophages alone, suggesting that this combination allowed a better control of released bacteria in the extracellular media. Finally, bacteriophages did not increase the activity of rifampicin in this model. Conclusion. In conclusion, we showed that the bacteriophages tested were highly active against S. aureus in mature biofilm but had no activity against bacteria internalized in osteoblasts. Additional studies using animal models of BJI and well-conducted clinical trials are needed to further evaluate phage therapy and its positioning in the management of these infections

It has been reported that some of the local anaesthetic agents possess antimicrobial activity against clinically-significant bacteria. Although bupivacaine exhibits a bacteriostatic effect at concentrations above 0.25% there are concerns that it might interact with some of the other antibiotics administered to patients. Whilst these interactions may be potentially benign, the risk is that they are antagonistic and that local bupivacaine might predispose the patient to a higher risk of infection. Bupivacaine is commonly administered as a local anaesthetic following knee arthroplasy; the purpose of this study was to assess its potential interactions with gentamicin eluting from the cement used to fix the device. A strain of Saphylococcus aureus (29213) with established susceptible Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) for gentamicin was used. This organism was inoculated into four types of broth; Mueller-Hinton broth (MH), MH with different concentrations of gentamicin, MH with 0.25% and 0.125% bupivacaine and MH with various combinations of gentamicin and bupivacaine. The broths were incubated at 37C and at 0.5, 1, 2, 3, 6 and 24 hours post inoculation the number of bacteria remaining were counted. From these data kill-curves were generated describing the absolute and individual rates of killing seen with bupivacaine and gentamicin alone and when in combination. Bupivacaine showed a bacteriostatic effect only at concentrations of 0.25% and higher. All concentrations of gentamicin above or equal to the expected MBC showed bactericidal effect. However, in combination with both strengths of Bupivacaine (0.25 and 0.125%) the bacteriocidal effect of gentamicin was seen at a lower concentration and the rate of killing of bacteria was enhanced. Bupivacaine has bacteriostatic effect at concentrations above 0.25% in line with published data. In these experiments we have shown that the use of bupivacaine together with gentamicin does not reduce the bactericidal property of the antibiotic and that the bactericidal effect of gentamicin appears to be enhanced by bupivacaine. This would suggest that the local use of bupivacaine is unlikely to increase the risk of infection in patients undergoing knee arthroplasty and may actually be beneficial

Aim. Identifying the optimal agent for irrigation for periprosthetic joint infection remains challenging as there is limited data. The ideal solution should have minimal cytotoxicity while maintaining bactericidal activity. We developed a novel activated-zinc solution containing zinc-chloride (ZnCl. 2. ) and sodium-chlorite (NaClO. 2. ). The purpose of this study was 1.) to investigate the antimicrobial efficacy of 2 concentrations (“CZ1”, “CZ2”) against Staphylococcus aureus and Pseudomonas aeruginosa and 2.) to evaluate untoward effects of the solution on local wound tissue 24 hours after solution exposure in pig wound models. Method. The study was conducted and reported in accordance to ARRIVE guidelines. We created twenty-four 1.5cm wounds on the back of a Yorkshire-cross pig. Wounds were inoculated with standardized Pseudomonas and S. aureus. 8 wounds were designated as controls (inoculum without treatment), 8 treated with CZ1, and 8 with CZ2. Punch biopsies were taken 1 hour after treatment and bacteria quantified. Wound necrosis/neutrophil infiltrate was measured 24-hours post-exposure. Results. After 1-hour, the control, CZ1 and CZ2 wounds had total bacteria of 5.7, 2.8 and 3.5 logCFU/g, respectively (p=0.017). The control, CZ1 and CZ2 wounds had S. aureus of 5.3, 2.3 and 1.6 logCFU/g, respectively (p=0.009). The control, CZ1 and CZ2 wounds had Pseudomonas of 5.5, 0.3 and 0.0 logCFU/g, respectively (p=0.000). After 24 hours of exposure to CZ1 and CZ2, there was no statistically significant increased necrosis (p=0.12, p=0.31, respectively). CZ1 had increased, moderate neutrophil infiltrate (p=0.04) when compared to controls, however CZ2 was not significant (p=0.12). Conclusions. Our novel solution demonstrated 99.5–99.9% reduction in total bacteria, 99.9–99.98 % reduction in S. aureus, and 100% eradication of Pseudomonas 1-hour after exposure, without significantly increased necrosis and no-to-minimally-increased neutrophil infiltrate. This novel solution may provide another significant tool in the arsenal to treat and/or prevent PJI and other wound infections

Aim. Staphylococcus epidermidis (S. epidermidis) is one of the main pathogens responsible for bone and joint infections especially those involving prosthetic materials (PJI). Although less virulent than S. aureus, S. epidermidis is involved in chronic infections notably due to its ability to form biofilm. Moreover, it is frequently multiresistant to antibiotics. In this context, the development of additional or alternative antibacterial therapies targeting the biofilm is a priority. Method. The aim of this study was to evaluate in vitro the activity of phage lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains responsible for PJI. We determined the remaining viable bacteria inside the biofilm (counting after serial dilution and plating) and the biomass (bacteria and extracellular matrix, using crystal violet staining) after 24h of exposition to exebacase at different concentrations, alone (0.05; 0.5; 5; 50 and 150 mg/L) or in combination (5, 50 and 150 mg/L) with antibiotics commonly used to treat multi-resistant S. epidermidis PJI (rifampin (1 mg/L), vancomycin (10mg/L) and daptomycin (10mg/L)). In this study, synergy was defined as a significantly higher effect of the association in comparison to the sum of the effect of each molecule. Results. Exebacase showed a dose-dependent reduction of biomass, ranging from 11 % at 0.5 mg/L to 66 % at 150 mg/L. Exebacase showed a significant bactericidal activity at 50 and 150 mg/l, with a mean decrease of the inoculum of 0.94 and 1.7 log, respectively. In addition, synergistic effects were observed in association with i) rifampin (1 mg/L) showing a mean decrease up to 84% of the biomass and 3.5 log CFU at 150 mg/L of exebacase, ii) vancomycin (10 mg/L) showing a mean decrease up to 81% of the biomass and 2.82 log CFU at 150 mg/L of exebacase, iii) and daptomycin (10 mg/L) showing a mean decrease up to 85% of the biomass and 3.1 log CFU at 150 mg/L of exebacase. Conclusions. Exebacase showed, in vitro, synergistic activity with antibiotics against S. epidermidis biofilms. It is a promising adjuvant therapy to rifampin, vancomycin and daptomycin in the context of PJI. Further studies are needed, in vitro to understand the mechanism of action on S. epidermidis biofilm and the heterogeneity of strain behaviour and in vivo to confirm the present data

Daptomycin is a novel lipopetide antibiotic against gram-positive organisms, including multi-resistant strains. It effectively penetrates bone and has bactericidal activity within biofilms. In adults it has been demonstrated active against Staphylococcus Aureus Methicillin Sensible (MSSA) and Resistant (MRSA) bacteremia. The main side effect is a transitory myopathy that appears to be dose and frequency related. There are limited dates on daptomycin in pediatric patients. We reviewed the medical records of four children (3 males and 1 female), with a median age of 11.2 years (range 7–13 years), who received daptomycin therapy for a complicated osteomyelitis. Osteomyelitis was clinically suspected and confirmed by magnetic resonance imaging at left ankle, left tibia, left calcaneum, lumbar column. The pathogen isolated was a MSSA in all four cases. All patients received prior antibiotic treatment. Therapy was swiched to Daptomycin for first line treatment failure (in three cases) and for an adverse reaction to first line treatment (in one case). Daptomycin was prescribed at the mean dosage of 9 mg/kg/day (range 8–10 mg/kg/day) for a median time of 15 days. After 4 days therapy, all patients clinically and laboratory improved with resolution of fever and pain and decreased inflammatory indexes. No patient underwent surgery. After a median of 20 days of hospitalization, patients were discharged with oral antibiotic therapy. They received follow-up clinical evaluation for 8 months (range 6–10 months) with no sequelae. With the limits of a small population and of a retrospective and unblended study, daptomycin therapy may be useful in complicated osteomyelitis and allowed the avoidance of surgery. The good outcome of the patients was probably due to daptomycin bactericidal activity against bacteria and to its ability to penetrate into bone and synovial fluid. Daptomycin therapy has been well tolerated in all patients, even if administered at a higher dose. No side effect was reported during therapy and at a 30 days follow-up evaluation

Objective. Bacterial infection is a serious complication after joint replacement surgery. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and epidermidis(MRSE) are very difficult to eradicate in infected prosthetic joint. Therefore, the retention rate of initial prosthesis affected with such resistant microorganisms is still low. Gentian violet shows potent antibacterial activity against gram-positive cocci as minimal bactericidal concentration is less than 0.1 %. In the present study, we investigated the efficacy of treatment with gentian violet against MRSA and MRSE infections after THA, TKA, and bipolar hip hemiarthroplasty (BHP). Methods. There were 8 patients in this study; five patients with deep periprosthetic MRSA infection (2 THA, 2 BHP, 1 revision TKA); 3 patients with MRSE infection (1 revision THA, 1 BHP, 1 TKA). When infection was suspected after the surgery, we quickly obtained synovial fluid and periprosthetic soft tissue from the joint and applied to culture and microscopic examinations for detection of microorganisms. After identification of bacterial species, we immediately debrided the affected joint and washed thoroughly twice with 0.1% solution of gentian violet for 3 minutes each, followed by intra-articular multiple injection of arbekacin sulfate solution. Then we inserted an aspiration tube into the joint and administered appropriate antibiotics intravenously. If the inflammatory symptoms persisted in spite of the first treatment, we repeated the treatment until inflammation signs and intra-articular microorganisms could not be detected. Results. At first we examined the bactericidal activity of gentian violet solution against MRSA and MRSE by culture examination with or without the solution. We confirmed gram-negative bacillicould be alive but the both MRSA and MRSE could not be alive by the treatment with 0.01 % solution of gentian violet for 3 minutes. The treatment with gentian violet allowed subsidence of the infection in all patients. Furthermore, we could preserve 4 prostheses with MRSA infection; 1 THA, 1 revision TKA and 2 BHPs; 2 prostheses with MRSE infection; 1 TKA, 1BHP. However, we could not preserve 2 prostheses, 1 THA with MRSA infection and 1 revision THA with MRSE infection. Thus, the prosthetic retention rate of this study for MRSA and MRSE infections was 75 %. We performed two-stage operation for the 2 patients in whom we could not preserve the prostheses as follows: after excision of the infected total hips followed by successful reimplantation. Moreover, all of the patients in this study are able to keep the walking ability after joint replacements. Conclusion. Infection of methicillin-resistant Staphylococcus species after joint replacement surgery has tended to increase. Gentian violet exerts a potent antibacterial activity against such microorganisms at the concentration of less than 0.1 %. We obtained good retention rate by the treatment using gentian violet against MRSA and MRSE infections after the joint surgeries. In addition, both MRSA and MRSE have not showed drug resistance for gentian violet. Therefore, we suggest that gentian violet will become a promising adjunct agent for infection after joint replacement surgery not only methicillin-sensitive but also methicillin–resistant Staphylococcus species

Aim. The demonstration of the in vivo bactericidal efficacy of a new bone cement with rifampicin contained in microcapsules and its intra-articular release profile. Method. Fifteen New Zealand White rabbits were employed to reproduce periprosthetic infection by intra-articular inoculation of 10. 5. CFU/mL of Staphylococcus aureus ATCC® 29213 using as a target implant a 3D printed stainless steel tibial insert. 7 days after inoculation, the first stage of the two stage exchange was carried out and at this time the animals were divided into two study groups: group C (7 rabbits) that received a spacer with gentamicin and group R (8 rabbits) that received a spacer with gentamicin and rifampicin microcapsules. Response to infection was monitored by clinical (weight and temperature), hematological (leukocyte, lymphocyte and platelet counts) and biochemical (erythrocyte sedimentation rate) analyses at the time of inoculation, at the first stage of exchange, 4 days after first stage and weekly until the fourth week when animals were euthanized. Microbiological counts were performed at the first stage of exchange and at the end of the study. Results. 14/15 animals (93.3%) developed a PJI 1 week after the inoculation. A statistically significant elevation of the leukocyte and platelet count and a decrease in the percentage of lymphocytes (p=0.0001) was found and positive microbiological cultures. Four weeks after the placement of the spacer, no bacterial growth was found in the soft tissue or bone samples of the group with rifampicin microcapsules (group R), being these differences statistically significant with p=0.01 and 0.03 respectively. The rifampicin intra-articular release kinetics showed concentrations above the staphylococcal MIC at all time points. Conclusions. The bone cement with microencapsulated rifampicin is effective in the in vivo treatment of prosthetic joint infection due to biofilm-forming S. aureus

Aim. Most orthopedic infections are due to the microbial colonization of abiotic surfaces, which evolves into biofilm formation. Within biofilms, persisters constitute a microbial subpopulation of cells characterized by a lower metabolic-activity, being phenotipically tolerant to high concentrations of antibiotics. Due to their extreme tolerance, persisters may cause relapses upon treatment discontinuation, leading to infection recalcitrance hindering the bony tissue regeneration. Using isothermal microcalorimetry (IMC), we aimed to evaluate in vitro the presence of persisters in a methicillin-resistant Staphylococcus aureus (MRSA) biofilm after treatment with high concentrations of vancomycin (VAN) and their ability to revert to a normal-growing phenotype during incubation in fresh medium without antibiotic. Moreover, the ability of daptomycin to eradicate the infection by killing persisters was also investigated. Method. A 24h-old MRSA ATCC 43300 biofilm was exposed to 1024 µg/ml VAN for 24h. Metabolism-related heat of biofilm-embedded cells, either during or after VAN-treatment, was monitored in real-time by IMC for 24 or 48h, respectively. To evaluate the presence of VAN-derived “persisters” after antibiotic treatment, beads were sonicated and detached free-floating bacteria were further challenged with 100xMIC VAN (100 µg/ml) in PBS+1% Cation Adjusted Mueller Hinton Broth (CAMHB).. Suspensions were plated for colony counting. The resumption of persister cells' normal growth was analysed by IMC on dislodged trated cells for 15h in CAMHB. Activity of 16 µg/ml daptomycin was assessed against persister cells by colony counting. Results. When incubated with 1024 µg/ml VAN, MRSA biofilm produced undetectable heat, suggesting a strong reduction of cell viability and/or cellular metabolism. However, the same samples re-inoculated in fresh medium produced a detectable and delayed metabolism-related heat signal, similarly to that generated by persister cells. The following exposure to 100xMIC VAN resulted in neither complete killing nor bacterial growth, strongly supporting the hypothesis of a persistent phenotype. IMC analysis indicated that VAN-treated biofilm cells resumed normal growth with a ∼3h-delay, as compared to the untreated growth control. Daptomycin treatment yielded a complete eradication of persister cells selected after VAN treatment. Conclusions. Hostile environmental conditions (e.g. high antibiotic bactericidal concentrations) select for persister cells in MRSA biofilm after 24h-treatment in vitro. A staggered treatment vancomycin/daptomycin allows complete biofilm eradication. These results support the use in clinical practice of a therapeutic regimen based on the combined use of antibiotics to kill persisters and eradicate MRSA biofilms. IMC represents a suitable technique to detect persisters and characterize in real-time their reversion to a metabolically-active phenotype

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)

Aim. bone and joint infection (BJI) in aging population, continues to be associated with significant morbi-mortality. In western Europeans countries, the Gram positive BJI are preponderant. Vancomycin was the “gold standard” and the full treatment requires prolonged antibiotic therapy. Dalbavancin is a semi-synthetic lipoglycopeptideanalog of teicoplanin class of antibiotics with bactericidal activity and a long half-life. The use of dalbavancin in BJI could be an option. Methods. during November 2017 and April 2019, Dalbavancin was used in monotherapy as salvage option in BJI: 1500 mg, 1. st. (D1) and 8. th. day (D8), repeated if needed. The clinical and biological follow up was for 6 months if osteomyelitis or BJI without prosthesis and 1 year if prosthesis (PJI). Results. the demographics of 16 patients are: 75.0% men (n=12), mean age 77.8 years [64–90], 37.5% (n=6) diabetes, 68.8% (n=11) renal failure, 37.5% (n=6) atrial fibrillation, 18.8% (n=3) cardiac bioprosthesis, 31.2% (n=5) lower limb arteriopathy, and one patient with active neoplasia. The BJI characteristic's: 50% (n=8) secondary to health care;5 vertebral osteomyelitis; 12 lower limb BJI : 8 joint infection of witch 6 PJI (4 knee, 2 hip) and 4 foot osteomyelitis; 2 shoulder PJI; 3 patients had 2 or more localisations of BJI. In 68.8% (11/16) BJI, bacteraemia occurred with 68.8% (n=11) of possible or certain infective endocarditis (Duke criteria) and 37.5% (n=6) of deep abscess. The DAIR was of 83.4% (5/6). Monobacterian biopsy in 75.0% (n=12). Out of 32 micro-organisms, 25 were Dalbavancin susceptible:56.0% (14/25) Staphylococcus aureus (10 methicillin susceptible), 3 Streptococcus, 5 Enterococcus faecalis, 2 Corynebacterium, 1 coagulase negative staphylococcus. Mean of 1. st. antibiotherapy: 18.3 days [0–49], with 2 patients who had dalbavancine as only antibiotic. Number of dalbavancine doses: 75% (n=12) patients had 2 injection (D1, D8), 18.8% (n=3), 4 injections D1, D8, D28 and D35 and 1 patient had one dose. Principal reason of changing by dalbavancine: 50% (8/16) poor tolerance of antibiotics, 12.5% (2/16) poor compliance of patient, 18.8% (3/16) poor efficacy of 1. st. antibiotherapy, 18.8 %(3/16) only for the patient's comfort. Clinically success: 75% (12/16) with 5 patients in follow up today. Three patients died and one is cured with teicoplanin and rifampicin. Three patients presented side effects: one diarrhea, one headache and one transient asthenia. No renal damage was found and no allergy. Conclusion. This report highlights the potential role of dalbavancin in treating unstable and weak patients who require long-term antimicrobial therapy with fewer antibiotic choices

Infected mega-endoprostheses are difficult to treat with systemic antibiotics due to encapsulation of the implant by fibrous tissue, formation of biofilms and antibiotic resistant bacteria. Modifying the implant surface by incorporating a bactericidal agent may reduce infection. Infection rates are typically in the range of 8% to 30%. This study describes a novel process method of “stitching-in” ionic silver into the implant surface, in vitro testing and its early clinical usage. A novel process has been developed to “stitch in” ionic silver into the upper surface of titanium alloy (Ti6Al4V). The process produces a modification by anodisation of the titanium alloy in dilute phosphoric acid, followed by absorption of silver from an aqueous solution. The engineered surface modification is therefore integral with the substrate and loaded with silver by an ion exchange reaction. Using this technique the maximum inventory of silver for typical a mega-prosthesis is 6mg and this is greater than 300 times lower than the No Observable Adverse Affects Level (NOAEL). Scanning electron microscopy revealed that the silver was concentrated in pits and forming reservoirs of ionic silver exposed to the body tissues. Laboratory-based studies focusing on the safety and efficacy of silver as a bactericidal agent have included investigation into cytotoxicity using fibroblast and osteoblast cell lines, the impact of silver in reducing corrosion and laboratory testing to establish if the modified surface has an effect on the wear and mechanical characteristics. A range of fatigue, static, tensile pull off tests were performed. The silver elution profiles for both silver loaded and HA coated over a silver loaded surface have been examined. Histological studies were also performed to examine the impact of the silver on osseointegration. The in vitro results confirm that silver is an effective antimicrobial agent. The mechanical characterization studies have identified that the surface treatment has no or minimal impact on the implant surface. Early results of the elution studies are encouraging showing that the HA coating of a silver loaded surface does not “seal” in the silver. To date (May 10) 147 silver treated mega-prostheses have been implanted since March 2006. The majority of implants were distal femoral (29%), proximal tibial (23%) or hemiplevic (10%). The most common indication was revision of a failed limb salvage reconstruction (58%), with the dominant cause of failure being infection. The next most common indication was bone tumour (31%) and the large majority were used in the high risk skeletal locations of the tibia (44%) and the pelvis (27%). Early clinical results are encouraging indicating a significant reduction in the incidence of infection. Three implants have been retrieved. An analysis of a proximal humeral replacement that had been in situ for 6 mths identified that there was 10-20% remaining on the implant surface. This novel process of “stitching-in” silver appears to be a safe and effective surface treatment in helping to control infections of mega-prostheses. This technology has the potential to be transferred to other arthroplasty joints

Aim. Virulent bacteriophages are known to be an effective therapy against various human bacterial infections. The aims of the study are to evaluate i) the killing activity of an antistaphylococcal phage lysate (ASPL), available in the Czech Republic for topical application, against Staphylococcal aureus (Sa) strains isolated in orthopedic infections; ii) the antimicrobial activity of ASPL against biofilm-embedded cells of a methicillin-resistant Sa (MRSA) standard strain. Method. The susceptibility of 25 MRSA and 18 methicillin-sensitive Sa (MSSA) strains to the ASPL was evaluated by spot assay. In addition, susceptibility of four laboratory MRSA strains, including ATCC 43300, ATCC 33591, Mu3 (MRSA/hetero vancomycin intermediate resistant Sa) and Mu50 (MRSA/vancomycin-resistant Sa) was also tested. The activity of ASPL against planktonic and biofilm-embedded MRSA ATCC 43300 was evaluated in real-time by isothermal microcalorimetry. The minimum heat inhibitory concentrations (MHIC) was defined as the lowest antimicrobial concentration leading to the lack of heat flow production after 24h for both planktonic and biofilm-embedded cells. The viability of bacterial cells was assessed by plating and colony counting. The minimum bactericidal concentration (MBC) was defined as the lowest antimicrobial concentration leading the reduction of 3 log CFU compared to the untreated control. Results. Around 34 out of 43 (79%) Sa strains were susceptible to the ASPL, including 17 MRSA and 17 MSSA strains. Both Mu3 and Mu50 (vancomycin intermediate and resistant MRSA, respectively) strains were also susceptible. Microcalorimetric evaluation of the activity of ASPL against planktonic cells of MRSA ATCC 43300 revealed the MHIC and the MBC were 10. 4. PFU/ml and 10. 5. PFU/ml, respectively. ASPL tested at 10. 5. PFU/ml was able to suppress the heat produced by biofilm bacterial cells, although this titer was not able to completely eradicate MRSA biofilm. Conclusions. ASPL showed a broad host spectrum among MRSA and MSSA strains associated with infections on implants, including strains that are resistant to vancomycin as well. ASPL exhibits a lytic activity against planktonic and biofilm MRSA and a titer of phage higher than 10. 5. PFU/ml is needed in order to achieve a complete eradication of MRSA biofilm. In conclusion, the antistaphylococcal phage lysate shows an excellent potential treatment of implant-related infections caused by Sa strains

Aim. Staphylococcus aureus and Pseudomonas aeruginosa are ubiquitous pathogens often found together in polymicrobial, biofilm-associated infections. The mixed-species biofilm are significantly more resistant to antimicrobial treatment and are associated with failures. Bacteriophages present a promising alternative to treat biofilm-related infections due to their rapid bactericidal activity on multi-drug resistant bacteria. In this study, we assess the simultaneous or sequential application of phages and ciprofloxacin on the mixed-species biofilm in vitro. Method. Ciprofloxacin was tested alone and in combination with Pyo-bacteriophage cocktail against P.aeurginosa ATCC 27853 and MRSA ATCC 43300 mixed-species biofilm. In order to evaluate the effect of combined treatment on biofilm-embedded cells, mature biofilms were grown on porous glass beads with MRSA (10. 6. CFU/ml) and P.aeruginosa (10. 3. CFU/ml) and incubated for 24h at 37° C in LB broth. The beads were then washed and placed in fresh LB in the presence of sub-eradicating titers/concentrations of phages and ciprofloxacin (corresponding to 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 × MBEC. biofilm. ), respectively, simultaneous or in order (pretreated with phages for 3-6-12-24 hours) at 37°C. In all cases, heat flow produced by the viable cells still embedded in the biofilm was measured for 48 hours by isothermal microcalorimetry. Results. Simultaneous or sequential treatment with pyo-bacteriophage (10. 5. and 10. 6. PFU/ml) and ciprofloxacin, producing a synergistic effect resulting in the complete eradication of the biofilm was evaluated. When sub-eradicating concentrations of ciprofloxacin together with sub-eradicating titers of phages simultaneously used to treat mixed-species biofilm, a delay and/or reduction of heat flow produced by bacteria was observed. The same effect was seen when mix-biofilm was pre-treated with phages for 3 hours and 24 hours, respectively. However, antibiotic introduction after 6 and 12 hours resulted in a high synergistic eradicating effect with pyo-bacteriophage. The concentration of ciprofloxacin decreased dramatically from >512 μg/ml to < 16 μg/ml. Conclusions. While MBEC of ciprofloxacin against mixed-species biofilm of Pseudomonas aeruginosa and Staphylococcus aureus was above drug concentrations reachable in clinical practice, the co-administration with bacteriophage strongly reduced the antibiotic doses needed to eradicate biofilm. There is a specific time delay in antibiotic introduction to reach the eradication of mix-species biofilm. These results have implications for optimal combined treatment approaches

Titanium knee, shoulder and hip implants are typically grit-blasted, thermal plasma spray coated, or sintered to provide ingrowth surface features having texture with pore sizes on the order of hundreds of micrometers. This provides macro and micro-mechanical locking upon bone remodeling. However, at the nanoscale and cellular level, these surfaces appear smooth. In vitro and in vivo research shows surfaces with nanoscale features result in enhanced osseointegration, greater bone-implant contact area and pullout force, and the potential to be bactericidal via a simple hybrid anodization surface modification process. Prior processes for creating nanotube nano-textured surfaces via electrochemical anodization relied on hydrofluoric acid electrolyte and platinum cathodes. This novel process uses ammonium fluoride electrolytes and graphite cathodes which are more cost effective and easier to handle during processing. Hybrid electrolytes with differing concentrations of ethylene glycol, water, and ammonium fluoride provide a variety of nanotube morphologies and sizes. Nano-tubular surfaces on knee tibial and femoral implants, hip stems and acetabular cups, bone screws and other 3D printed parts have been enhanced by this method of nano-texturing in as little as 30 minutes. In vivo work in a Sprague Dawley rat model showed bone-implant contact area up to 2.9-times greater, and uniaxial pullout forces up to 6.9-times greater, than implanted smooth titanium controls at 4 and 12-week time points. In these tests, 1.25mm Kirschner wires were implanted in the rat femora to simulate an intramedullary nail. Histomorphometry in the mid-shaft and distal regions showed greater trabecular thickness and bone tissue mineral density than controls. Axial pullout tests often resulted in bone failure before the bone-implant interface. In vitro evidence suggests that nanoscale surfaces may have an antibacterial effect due to surface energy changes that reduce the ability of bacteria to adhere. However, it is recognized that silver is highly antibacterial in appropriate concentrations. It is also recognized that nanosilver, approximately 10–20nm, is especially effective. Ammonium fluoride anodization is modified using a hybrid electrolyte that includes silver fluoride. By substituting some of the ammonium fluoride with silver fluoride, to maintain a constant total fluorine mass, nanosilver is integrated within and among the nanotubes in the same single process that forms the nanotubes. This hybrid process in nano-texturing titanium implants can be integrated into current manufacturing production at low cost