Bacteriophage therapy in musculoskeletal infections: from basic science to clinical application.

The treatment of musculoskeletal infections (MSIs), including periprosthetic joint infection (PJI) and fracture-related infection (FRI), is often complicated by biofilm-related challenges necessitating multiple revision surgeries and incurring substantial costs. The emergence of antimicrobial resistance (AMR) adds to the complexity of the problem, leading to increased morbidity and healthcare expenses. There is an urgent need for novel antibacterial strategies, with the World Health Organization endorsing non-traditional approaches like bacteriophage (phage) therapy. Phage therapy, involving the targeted application of lytic potent phages, shows promise in the treatment of MSIs. Although historical clinical trials and recent case studies present significant milestones in the evolution of phage therapy over the past century, challenges persist, including variability in study designs, administration protocols and phage selection. Efforts to enhance treatment efficacy consist of personalized phage therapy and combination with antibiotics. Future perspectives entail addressing regulatory barriers, standardizing treatment protocols, and conducting high-quality clinical trials to establish phage therapy's efficacy for the treatment of MSIs. Initiatives like the PHAGEFORCE study and the PHAGEinLYON Clinic programme aim to streamline phage therapy, facilitating personalized treatment approaches and systematic data collection to advance its clinical utility in these challenging infections.

The global burden of fracture-related infection: can we do better?

Fracture-related infection is a major complication related to musculoskeletal injuries that not only has important clinical consequences, but also a substantial socioeconomic impact. Although fracture-related infection is one of the oldest disease entities known to mankind, it has only recently been defined and, therefore, its global burden is still largely unknown. In this Personal View, we describe the origin of the term fracture-related infection, present the available data on its global impact, and discuss important aspects regarding its prevention and management that could lead to improved outcomes in both high-resource and low-resource settings. We also highlight the need for health-care systems to be adequately compensated for the high cost of human resources (trained staff) and well-equipped facilities required to adequately care for these complex patients. Our aim is to increase awareness among clinicians and policy makers that fracture-related infection is a disease entity that deserves prioritisation in terms of research, with the goal to standardise treatment and improve patient outcomes on a global scale.

Alginate microbeads and hydrogels delivering meropenem and bacteriophages to treat Pseudomonas aeruginosa fracture-related infections.

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9-30 and MK.R3-15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future.

Stability of magistral phage preparations before therapeutic application in patients with chronic rhinosinusitis, sepsis, pulmonary, and musculoskeletal infections.

IMPORTANCE: As antimicrobial resistance becomes more prevalent, the application of (bacterio)phage therapy as an alternative treatment for difficult-to-treat infections is (re)gaining popularity. Over the past decade, numerous promising case reports and series have been published demonstrating the therapeutic potential of phage therapy. However, important questions remain regarding the optimal treatment protocol and, unlike for medicinal products, there are currently no predefined quality standards for the stability of phage preparations. Phage titers can be influenced by several factors which could lead to reduced titers after preparation and storage and, ultimately, subtherapeutic applications. Determining the stability of different phages in different recipients according to the route of administration is therefore one of the first important steps in establishing a standardized protocol for phage therapy.

Treatment and outcome of fracture-related infection of the clavicle.

INTRODUCTION: The number of operatively treated clavicle fractures has increased over the past decades. Consequently, this has led to an increase in secondary procedures required to treat complications such as fracture-related infection (FRI). The primary objective of this study was to assess the clinical and functional outcome of patients treated for FRI of the clavicle. The secondary objectives were to evaluate the healthcare costs and propose a standardized protocol for the surgical management of this complication. METHODS: All patients with a clavicle fracture who underwent open reduction and internal fixation (ORIF) between 1 January 2015 and 1 March 2022 were retrospectively evaluated. This study included patients with an FRI who were diagnosed and treated according to the recommendations of a multidisciplinary team at the University Hospitals Leuven, Belgium. RESULTS: We evaluated 626 patients with 630 clavicle fractures who underwent ORIF. In total, 28 patients were diagnosed with an FRI. Of these, eight (29%) underwent definitive implant removal, five (18%) underwent debridement, antimicrobial treatment and implant retention, and fourteen patients (50%) had their implant exchanged in either a single-stage procedure, a two-stage procedure or after multiple revisions. One patient (3.6%) underwent resection of the clavicle. Twelve patients (43%) underwent autologous bone grafting (tricortical iliac crest bone graft (n = 6), free vascularized fibular graft (n = 5), cancellous bone graft (n = 1)) to reconstruct the bone defect. The median follow-up was 32.3 (P25-P75: 23.9-51.1) months. Two patients (7.1%) experienced a recurrence of infection. The functional outcome was satisfactory, with 26 out of 28 patients (93%) having full range of motion. The median healthcare cost was € 11.506 (P25-P75: € 7.953-23.798) per patient. CONCLUSION: FRI is a serious complication that can occur after the surgical treatment of clavicle fractures. In our opinion, when treated adequately using a multidisciplinary patient-specific approach, the outcome of patients with an FRI of the clavicle is good. The median healthcare costs of these patients are up to 3.5 times higher compared to non-infected operatively treated clavicle fractures. Although not studied individually, we consider factors such as the size of the bone defect, condition of the soft tissue, and patient demand important when it comes to guiding our surgical decision making in cases of osseous defects.

Characterization of a Bacteriophage GEC_vB_Bfr_UZM3 Active against Bacteroides fragilis.

Bacteroides fragilis is a commensal gut bacterium that is associated with a number of blood and tissue infections. It has not yet been recognized as one of the drug-resistant human pathogens, but cases of the refractory infections, caused by strains that are not susceptible to the common antibiotic regimes established for B. fragilis, have been more frequently reported. Bacteriophages (phages) were found to be a successful antibacterial alternative to antibiotic therapy in many cases of multidrug-resistant (MDR) bacterial infections. We have characterized the bacteriophage GEC_vB_Bfr_UZM3 (UZM3), which was used for the treatment of a patient with a chronic osteomyelitis caused by a B. fragilis mixed infection. Studied biological and morphological properties of UZM3 showed that it seems to represent a strictly lytic phage belonging to a siphovirus morphotype. It is characterized by high stability at body temperature and in pH environments for about 6 h. Whole genome sequencing analysis of the phage UZM3 showed that it does not harbor any known virulence genes and can be considered as a potential therapeutic phage to be used against B. fragilis infections.

Foundation of the Belgian Society for Viruses of Microbes and Meeting Report of Its Inaugural Symposium.

The Belgian Society for Viruses of Microbes (BSVoM) was founded on 9 June 2022 to capture and enhance the collaborative spirit among the expanding community of microbial virus researchers in Belgium. The sixteen founders are affiliated to fourteen different research entities across academia, industry and government. Its inaugural symposium was held on 23 September 2022 in the Thermotechnical Institute at KU Leuven. The meeting program covered three thematic sessions launched by international keynote speakers: (1) virus-host interactions, (2) viral ecology, evolution and diversity and (3) present and future applications. During the one-day symposium, four invited keynote lectures, ten selected talks and eight student pitches were given along with 41 presented posters. The meeting hosted 155 participants from twelve countries.

Diagnosis of fracture-related infection in patients without clinical confirmatory criteria: an international retrospective cohort study.

Background: fracture-related infection (FRI) remains a serious complication in orthopedic trauma. To standardize daily clinical practice, a consensus definition was established, based on confirmatory and suggestive criteria. In the presence of clinical confirmatory criteria, the diagnosis of an FRI is evident, and treatment can be started. However, if these criteria are absent, the decision to surgically collect deep tissue cultures can only be based on suggestive criteria. The primary study aim was to characterize the subpopulation of FRI patients presenting without clinical confirmatory criteria (fistula, sinus, wound breakdown, purulent wound drainage or presence of pus during surgery). The secondary aims were to describe the prevalence of the diagnostic criteria for FRI and present the microbiological characteristics, both for the entire FRI population. Methods: a multicenter, retrospective cohort study was performed, reporting the demographic, clinical and microbiological characteristics of 609 patients (with 613 fractures) who were treated for FRI based on the recommendations of a multidisciplinary team. Patients were divided in three groups, including the total population and two subgroups of patients presenting with or without clinical confirmatory criteria. Results: clinical and microbiological confirmatory criteria were present in 77 % and 87 % of the included fractures, respectively. Of patients, 23 % presented without clinical confirmatory criteria, and they mostly displayed one (31 %) or two (23 %) suggestive clinical criteria (redness, swelling, warmth, pain, fever, new-onset joint effusion, persisting/increasing/new-onset wound drainage). The prevalence of any suggestive clinical, radiological or laboratory criteria in this subgroup was 85 %, 55 % and 97 %, respectively. Most infections were monomicrobial (64 %) and caused by Staphylococcus aureus. Conclusion: clinical confirmatory criteria were absent in 23 % of the FRIs. In these cases, the decision to operatively collect deep tissue cultures was based on clinical, radiological and laboratory suggestive criteria. The combined use of these criteria should guide physicians in the management pathway of FRI. Further research is needed to provide guidelines on the decision to proceed with surgery when only these suggestive criteria are present.

Bacteriophage therapy for human musculoskeletal and skin/soft tissue infections.

BACKGROUND: Bacteriophage therapy has a long history in the treatment of musculoskeletal and skin/soft tissue infections, particularly in the former Soviet Union. Due to the global rise in antimicrobial resistance, phage application has experienced a resurgence of interest and expanded to many countries. OBJECTIVES: This narrative review aims to provide clinical microbiologists, infectious disease specialists and surgeons a brief history of bacteriophage therapy for human musculoskeletal and soft tissue infections, as well as data on current practices and ongoing clinical studies. SOURCES: A search of PubMed and Clinicaltrials.gov was performed to identify relevant studies. Search terms were 'bacteriophage therapy', 'musculoskeletal infection' and 'soft tissue infection'. The bibliography of all retrieved articles was checked for additional relevant references. CONTENT: Past and current data on the use of bacteriophage therapy for human musculoskeletal, skin and soft tissue infections are evaluated. Moreover, we present the clinical trials registered in public databases. Based on current clinical experience and data, several scenarios of bacteriophage application for human therapy are examined. Finally, we discuss legislative hurdles in the regulatory approval process and present future perspectives for bacteriophage therapy. IMPLICATIONS: Antimicrobial resistance is one of the most important global public health challenges. Several different alternatives to conventional antibiotics are under development; bacteriophage therapy is one of them. Currently, therapeutic use of phages is restrained by regulatory hurdles and largely limited to sporadic authorization in compassionate use or under temporary approval as new drugs in Europe and the US. Although bacteriophage therapy seems to be safe and clinical results of phage treatment are promising, future data from high-quality (randomized controlled) trials could provide a better understanding of the reasonable minimal criteria required for expansion of bacteriophage therapy.

The Microbiological Etiology of Fracture-Related Infection.

Purpose: Fracture-related infection (FRI) is an important complication related to orthopaedic trauma. Although the scientific interest with respect to the diagnosis and treatment of FRI is increasing, data on the microbiological epidemiology remains limited. Therefore, the primary aim of this study was to evaluate the microbiological epidemiology related to FRI, including the association with clinical symptoms and antimicrobial susceptibility data. The secondary aim was to analyze whether there was a relationship between the time to onset of infection and the microbiological etiology of FRI. Methods: FRI patients treated at the University Hospitals of Leuven, Belgium, between January 1st 2015 and November 24th 2019 were evaluated retrospectively. The microbiological etiology and antimicrobial susceptibility data were analyzed. Patients were classified as having an early (<2 weeks after implantation), delayed (2-10 weeks) or late-onset (> 10 weeks) FRI. Results: One hundred ninety-one patients with 194 FRIs, most frequently involving the tibia (23.7%) and femur (18.6%), were included. Staphylococcus aureus was the most frequently isolated pathogen, regardless of time to onset (n=61; 31.4%), followed by S. epidermidis (n=50; 25.8%) and non-epidermidis coagulase-negative staphylococci (n=35; 18.0%). Polymicrobial infections (n=49; 25.3%), mainly involving Gram negative bacilli (GNB) (n=32; 65.3%), were less common than monomicrobial infections (n=138; 71.1%). Virulent pathogens in monomicrobial FRIs were more likely to cause pus or purulent discharge (n=45;54.9%; p=0.002) and fistulas (n=21;25.6%; p=0.030). Susceptibility to piperacillin/tazobactam for GNB was 75.9%. Vancomycin covered 100% of Gram positive cocci. Conclusion: This study revealed that in early FRIs, polymicrobial infections and infections including Enterobacterales and enterococcal species were more frequent. A time-based FRI classification is not meaningful to estimate the microbiological epidemiology and cannot be used to guide empiric antibiotic therapy. Large multicenter prospective studies are necessary to gain more insight into the added value of (broad) empirical antibiotic therapy.

Validation of the diagnostic criteria of the consensus definition of fracture-related infection.

BACKGROUND: The recently developed fracture-related infection (FRI) consensus definition, which is based on specific diagnostic criteria, has not been fully validated in clinical studies. We aimed to determine the diagnostic performance of the criteria of the FRI consensus definition and evaluated the effect of the combination of certain suggestive and confirmatory criteria on the diagnostic performance. METHODS: A multicenter, multi-national, retrospective cohort study was performed. Patients were subdivided into an FRI or a control group, according to the treatment they received and the recommendations from a multidisciplinary team ('intention to treat'). Exclusion criteria were patients with an FRI diagnosed outside the study period, patients younger than 18 years of age, patients with pathological fractures or patients with fractures of the skull, cervical, thoracic and lumbar spine. Minimum follow up for all patients was 18 months. RESULTS: Overall, 637 patients underwent revision surgery for suspicion of FRI. Of these, 480 patients were diagnosed with FRI, treated accordingly, and included in the FRI group. The other 157 patients were included in the control group. The presence of at least one confirmatory sign was associated with a sensitivity of 97.5%, a specificity of 100% and a high discriminatory value (AUROC 0.99, p < 0.001). The presence of a clinical confirmatory criterion or, if not present, at least one positive culture was associated with the highest diagnostic performance (sensitivity: 98.6%, specificity: 100%, AUROC: 0.99 (p < 0.001)). In the subgroup of patients without clinical confirmatory signs at presentation, specificities of at least 95% were found for the clinical suggestive signs of fever, wound drainage, local warmth and redness. CONCLUSIONS: The presence of at least one confirmatory criterion identifies the vast majority of patients with an FRI and was associated with an excellent diagnostic discriminatory value. Therefore, our study validates the confirmatory criteria of the FRI consensus definition. Infection is highly likely in case of the presence of a single positive culture with a virulent pathogen. When certain clinical suggestive signs (e.g., wound drainage) are observed (individually or in combination and even without a confirmatory criterion), it is more likely than not, that an infection is present.

Safety and efficacy of phage therapy in difficult-to-treat infections: a systematic review.

According to the latest reports from WHO, the incidence of antibiotic-resistant bacterial infections is increasing worldwide, resulting in increased morbidity and mortality and a rising pressure on health-care systems. However, the development of new antibiotics is an expensive and time-consuming process, urging scientists to seek alternative antimicrobial strategies. Over the past few decades, the concept of therapeutic administration of bacteriophages (also known as phages) has gained popularity worldwide. Although conceptually promising, the widespread implementation of phage therapy in routine clinical practice is restricted by the scarcity of safety and efficacy data obtained according to the strict standards of the applicable clinical trial regulations. In this systematic review, we list clinical data published between Jan 1, 2000 and Aug 14, 2021 on the safety and efficacy of phage therapy for difficult-to-treat bacterial infections, and provide an overview of trials and case studies on the use of phage therapy in several medical disciplines.

Bacteriophage Therapy for the Prevention and Treatment of Fracture-Related Infection Caused by Staphylococcus aureus: a Preclinical Study.

Although several studies have shown promising clinical outcomes of phage therapy in patients with orthopedic device-related infections, questions remain regarding the optimal application protocol, systemic effects, and the impact of the immune response. This study provides a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection (FRI) caused by Staphylococcus aureus. In a prevention setting, phage in saline (without any biomaterial-based carrier) was highly effective in the prevention of FRI, compared to systemic antibiotic prophylaxis alone. In the subsequent study involving treatment of established infection, daily administration of phage in saline through a subcutaneous access tube was compared to a single intraoperative application of a phage-loaded hydrogel and a control group receiving antibiotics only. In this setting, although a possible trend of bacterial load reduction on the implant was observed with the phage-loaded hydrogel, no superior effect of phage therapy was found compared to antibiotic treatment alone. The application of phage in saline through a subcutaneous access tube was, however, complicated by superinfection and the development of neutralizing antibodies. The latter was not found in the animals that received the phage-loaded hydrogel, which may indicate that encapsulation of phages into a carrier such as a hydrogel limits their exposure to the adaptive immune system. These studies show phage therapy can be useful in targeting orthopedic device-related infection, however, further research and improvements of these application methods are required for this complex clinical setting. IMPORTANCE Because of the growing spread of antimicrobial resistance, the use of alternative prevention and treatment strategies is gaining interest. Although the therapeutic potential of bacteriophages has been demonstrated in a number of case reports and series over the past decade, many unanswered questions remain regarding the optimal application protocol. Furthermore, a major concern during phage therapy is the induction of phage neutralizing antibodies. This study aimed at providing a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection caused by Staphylococcus aureus. Phage therapy was applied as prophylaxis in a first phase, and as treatment of an established infection in a second phase. The development of phage neutralizing antibodies was evaluated in the treatment study. This study demonstrates that phage therapy can be useful in targeting orthopedic device-related infection, especially as prophylaxis; however, further research and improvements of these application methods are required.

Fracture-related infection in long bone fractures: A comprehensive analysis of the economic impact and influence on quality of life.

OBJECTIVES: Fracture-related infection (FRI) is a feared complication with substantial clinical and economic consequences. The main objective of this study was to compare direct and indirect healthcare costs related to long bone fractures in patients with and without FRI and to assess its impact on the patient's quality of life (QoL). PATIENTS AND METHODS: Between January 2015 and March 2019, 175 patients with FRI were treated at the University Hospitals Leuven (Belgium). Using a matched-pair analysis, patients with an FRI were matched by age, sex, and fracture location (humeral, femoral, or tibial shaft) to a non-FRI cohort treated during the same time period. Clinical and process-related variables, direct hospital-related healthcare costs, and indirect costs due to absenteeism were compared between the two groups. Furthermore, the patient's QoL was evaluated using Patient-Reported Outcomes Measurement Information System (PROMIS) physical function and pain interference. RESULTS: After matched-pair analysis, 15 patients in both the FRI and non-FRI group were included. FRI was associated with direct hospital-related costs being eight times that of non-FRI patients (€ 47,845 [€ 43,072-€ 82,548] vs. € 5,983 [€ 4,519-€ 8,428], p < 0.001). Furthermore, FRI was associated with prolonged absenteeism (340 [340-676] vs. 86 [65-216] days, p = 0.007) and a median indirect cost that was nearly four times that of patients without FRI (€ 77,909 vs. € 19,706). Lastly, FRI patients showed significantly poorer outcomes on both physical function (35.6 vs. 48.4, p < 0.001) and pain interference (60.4 vs. 46.3, p < 0.001) PROMIS scales. CONCLUSION: Direct hospital-related healthcare costs of FRI are eight times that of non-FRI long bone fractures. Total healthcare costs are mainly driven by hospitalisation costs, wherein FRI is associated with prolonged length of stay. To the best of our knowledge, this study is the first to demonstrate that FRI is also associated with substantial absenteeism, which is almost four times higher compared to non-FRI patients. In addition to this economic impact, FRI significantly deteriorates QoL. Generalizing the outcome of this study should be done cautiously due to the small sample size of 15 patients in both the FRI and non-FRI group after matched-pair analysis.

What is the diagnostic value of the Centers for Disease Control and Prevention criteria for surgical site infection in fracture-related infection?

BACKGROUND: Fracture-related infection (FRI) remains one of the most challenging complications in orthopaedic trauma surgery. An early diagnosis is of paramount importance to guide treatment. The primary aim of this study was to compare the Centers for Disease Control and Prevention (CDC) criteria for the diagnosis of organ/space surgical site infection (SSI) to the recently developed diagnostic criteria of the FRI consensus definition in operatively treated fracture patients. METHODS: This international multicenter retrospective cohort study evaluated 257 patients with 261 infections after operative fracture treatment. All patients included in this study were considered to have an FRI and treated accordingly ('intention to treat'). The minimum follow-up was one year. Infections were scored according to the CDC criteria for organ/space SSI and the diagnostic criteria of the FRI consensus definition. RESULTS: Overall, 130 (49.8%) FRIs were captured when applying the CDC criteria for organ/space SSI, whereas 258 (98.9%) FRIs were captured when applying the FRI consensus criteria. Patients could not be classified as having an infection according to the CDC criteria mainly due to a lack of symptoms within 90 days after the surgical procedure (n = 96; 36.8%) and due to the fact that the surgery was performed at an anatomical localization not listed in the National Healthcare Safety Network (NHSN) operative procedure code mapping (n = 37; 14.2%). CONCLUSION: This study confirms the importance of standardization with respect to the diagnosis of FRI. The results endorse the recently developed FRI consensus definition. When applying these diagnostic criteria, 98.9% of the infections that occured after operative fracture treatment could be captured. The CDC criteria for organ/space SSI captured less than half of the patients with an FRI requiring treatment, and seemed to have less diagnostic value in this patient population.

Bacteriophage Therapy for Difficult-to-Treat Infections: The Implementation of a Multidisciplinary Phage Task Force (The PHAGEFORCE Study Protocol).

In times where only a few novel antibiotics are to be expected, antimicrobial resistance remains an expanding global health threat. In case of chronic infections caused by therapy-resistant pathogens, physicians have limited therapeutic options, which are often associated with detrimental consequences for the patient. This has resulted in a renewed interest in alternative strategies, such as bacteriophage (phage) therapy. However, there are still important hurdles that currently impede the more widespread implementation of phage therapy in clinical practice. First, the limited number of good-quality case series and clinical trials have failed to show the optimal application protocol in terms of route of administration, frequency of administration, treatment duration and phage titer. Second, there is limited information on the systemic effects of phage therapy. Finally, in the past, phage therapy has been applied intuitively in terms of the selection of phages and their combination as parts of phage cocktails. This has led to an enormous heterogeneity in previously published studies, resulting in a lack of reliable safety and efficacy data for phage therapy. We hereby present a study protocol that addresses these scientific hurdles using a multidisciplinary approach, bringing together the experience of clinical, pharmaceutical and molecular microbiology experts.

The Potential Role of Bacteriophages in the Treatment of Recalcitrant Chronic Rhinosinusitis.

Chronic rhinosinusitis is a common condition affecting 5-12% of the general population worldwide. In a limited number of cases, the disease is recalcitrant to medical and surgical interventions, causing a major impact on physical, social and emotional well-being and increasing pressure on healthcare systems. Biofilm formation and dysbiosis caused by Staphylococcus aureus and Pseudomonas aeruginosa play a role in the pathogenesis of recalcitrant chronic rhinosinusitis. In these cases, a promising treatment alternative is the application of bacteriophages, which are viruses that infect and lyse bacteria. In this review, we appraise the evidence for the use of bacteriophages in the treatment of recalcitrant chronic rhinosinusitis. Additionally, (dis)advantages of bacteriophages and considerations for implementation of phage therapy in otorhinolaryngology practice will be discussed.

An Improved 2-Aminoimidazole Based Anti-Biofilm Coating for Orthopedic Implants: Activity, Stability, and in vivo Biocompatibility.

Orthopedic device-related infections remain a serious challenge to treat. Central to these infections are bacterial biofilms that form on the orthopedic implant itself. These biofilms shield the bacteria from the host immune system and most common antibiotic drugs, which renders them essentially antibiotic-tolerant. There is an urgent clinical need for novel strategies to prevent these serious infections that do not involve conventional antibiotics. Recently, a novel antibiofilm coating for titanium surfaces was developed based on 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine as an active biofilm inhibitor. In the current study we present an optimized coating protocol that allowed for a 5-fold higher load of this active compound, whilst shortening the manufacturing process. When applied to titanium disks, the newly optimized coating was resilient to the most common sterilization procedures and it induced a 1 log reduction in biofilm cells of a clinical Staphylococcus aureus isolate (JAR060131) in vitro, without affecting the planktonic phase. Moreover, the antibiofilm effect of the coating in combination with the antibiotic cefuroxime was higher than cefuroxime treatment alone. Furthermore, the coating was successfully applied to a human-scale fracture fixation device resulting in a loading that was comparable to the titanium disk model. Finally, an in vivo biocompatibility and healing study in a rabbit osteotomy model indicated that these coated implants did not negatively affect fracture healing or osteointegration. These findings put our technology one step closer to clinical trials, confirming its potential in fighting orthopedic infections without compromising healing.