Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics.

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged by a low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 20 minutes and gentamicin. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L. Further studies confirmed that the mechanism of action was due to incomplete disruption of the extracellular matrix with subsequent metabolic stimulation of the dormant biofilm-associated bacteria due to increased nutrient availability and oxygen tension.

Low intensity pulsed ultrasound was associated with a 4-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations.

Antimicrobial resistance (AMR) is projected to result in 10 million deaths every year globally by 2050. Without urgent action, routine orthopaedic operations could become high risk and musculoskeletal infections incurable in a “post-antibiotic era.” However, current methods of studying AMR processes including bacterial biofilm formation are 2D in nature, and therefore unable to recapitulate the 3D processes within in vivo infection.

Within this study, 3D printing was applied for the first time alongside a custom-developed bioink to bioprint 3D bacterial biofilm constructs from clinically relevant species including Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa. Bacterial viability and biofilm formation in bioprinted constructs was excellent, with confocal laser scanning microscopy (CSLM) used to demonstrate biofilm production and maturation over 28 days. Bioprinted 3D MRSA and MSSA biofilm constructs had greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Raman spectroscopy was also adapted in a novel approach to non-invasively diagnose 3D bioprinted biofilm constructs located within a joint replacement model.

In conclusion, mature bacterial biofilm constructs were reproducibly 3D bioprinted for the first time using clinically relevant bacteria. This methodology allows the study of antimicrobial biofilm penetration in 3D, and potentially aids future antimicrobial research, replicating joint infection more closely than current 2D culture models. Furthermore, by deploying Raman spectroscopy in a novel fashion, it was possible to diagnose 3D bioprinted biofilm infections within a joint replacement model.

Introduction. Despite the routine use of irrigation, debridement and systemic antibiotics, there is a high incidence of infection in severe open fractures. The synergistic use of local and systemic antibiotics appreciably reduces infection rates although the time window within which this is effective is unknown. The aim was to determine if delaying treatment of wounds causes higher levels of infection. Methods. A defect was created in the femurs of 90 Sprague-Dawley rats and inoculated with 105CFUs Staphylococcus aureus. At 2, 6 and 24 hours following contamination, the defect was irrigated and debrided. The experimental groups had either vancomycin or tobramycin impregnated PMMA beads placed within the segmental defect. The controls received no further treatment. Two weeks after wound closure, the bacteria within the femur were quantified. Results. Delaying irrigation and debridement resulted in significantly more bacteria (p<0.01) within the control group (2 hr < 6 hr <24 hr). Both locally delivered tobramycin and vancomycin significantly reduced the bacteria (p<0.05) when administered at the earlier time points (2 and 6 hours). Locally-delivered antibiotics were ineffective when delivered at 24 hours. Conclusion. Delaying treatment of contaminated defects reduces its effectiveness to eradicate infection. This is presumably because of the biofilm formation by the bacteria. Biofilms begin to form within a couple of hours and are mature within 12 hours. Early treatment of the wound allows the surgeon to physically remove the bacteria or have antibiotics present before a mature biofilm protects the bacteria

Aims. Infections of bone usually require multiple surgery and prolonged periods of treatment. One reason for problems is found in the presence of stationary phase bacteria embedded in biofilms that show increased resistance against conventional antibiotic therapy (up to 1000x MIC). Biofilms adhere to surfaces of avital material making radical debridement a prerequisite for cure. Osseous defects are common in such conditions and need to be addressed. To avoid re-infection high local antbiotic concentrations are necessary. Allograft bone may be impregnated with high loads of antibiotics using a special incubation technique. The resulting antibiotic bone compound (ABC) provides high and long lasting concentrations at the site of infection and is likely to restore bone stock simultaneously. Based on this technology we have developed a new surgical technique. Methods. 42 patients (10–67yrs) with chronic osteitis were included into a prospective study using a standardized protocol. Infection was at the humerus (1x), femur (10x), tibia (29x) or femur+tibia (2x), respectively. Treatment consisted of removal of foreign material, radical sequestrectomy and soft-tissue debridement followed by pressurized lavage. Surfaces of sclerotic bone were trimmed down to vital areas. The remaining osseous defects were filled with ABC, using an impaction technique resulting in complete dead space management. The allograft was impregnated with vancomycin, in cases with mixed pathogens combinations with tobramycin were used. Internal fixation was performed the same time whenever applicable. Sites were drained and closed immediately; rehabilitation did not differ from uninfected procedures. Results. 1 patient died shortly after surgery from cardiac failure. 41 could be followed for a minimum of 2 and a maximum of 6years (mean 3,1years). In 2 patients wound healing was unsatisfactory requiring additional coverage with a muscle flap. 2 patients showed material failure after intramedullary nailing, requiring exchange of the implant. In those cases no sign of infection was present at the time of revision. There were 3 cases with recurrence of infection, all originating from foci not detected during the index operation and becoming apparent between 3 and 12 months after surgery. Two could successfully be revised using the same technique; one refused revision and shows continuing fistulation. Radiological incorporation of allografts appeared as after conventional bone grafting, union of pseudarthroses was achieved between 2 and 6 months after (re-) stabilization. 40 patients (95,2%) were fully weight bearing, painfree and without any sign of infection at the latest follow up. Conclusion. Using antibiotic impregnated allograft bone eradication of pathogens, grafting of defects, dead space management and insertion of osteosynthetic material may be accomplished in a one stage procedure. Since the graft gradually is replaced by healthy own bone improved long term results may be expected as well as improved conditions in the case of another revision. The new technique provides for quick rehabilitation, improved results and markedly reduced costs of treatment in cases of bone infection