Orthopedic device-related infection (ODRI) preclinical models are widely used in translational research. Most models require induction of general anesthesia, which frequently results in hypothermia in rodents. This study aimed to evaluate the impact of peri anesthetic hypothermia in rodents on outcomes in preclinical orthopedic device-related infection studies. A retrospective analysis of all rodents that underwent surgery under general anesthesia to induce an ODRI model with inoculation of Staphylococcus epidermidis between 2016 and 2020 was conducted. A one-way multivariate analysis of covariance was used to determine the fixed effect of peri anesthetic hypothermia (hypothermic defined as rectal temperature <35°C) on the combined harvested tissue and implant colonies forming unit counts, and having controlled for the study groups including treatments received duration of surgery and anesthesia and study period. All animal experiments were approved by relevant ethical committee. A total of 127 rodents (102 rats and 25 mice) were enrolled in an ODRI and met the inclusion criteria. The mean lowest peri-anesthetic temperature was 35.3 ± 1.5 °C. The overall incidence of peri-anesthetic hypothermia was 41% and was less frequently reported in rats (34% in rats versus 68% in mice). Statistical analysis showed a significant effect of peri anesthetic hypothermia on the post-mortem combined colonies forming unit counts from the harvested tissue and implant(s) (p=0.01) when comparing normo- versus hypothermic rodents. Using Wilks’ Λ as a criterion to determine the contribution of independent variables to the model, peri-anesthetic hypothermia was the most significant, though still a weak predictor, of increased harvested colonies forming unit counts. Altogether, the data corroborate the concept that bacterial colonization is affected by abnormal body temperature during general anesthesia at the time of bacterial inoculation in rodents, which needs to be taken into consideration to decrease infection data variability and improve experimental reproducibility

One of the most common bacteria in orthopaedic prosthetic infections is Staphylococcus Aureus. Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion on nanofabricated materials. We hypothesise that surface nanotopography impacts the differential ability of staphylococci species to adhere via altered metabolomics and may reduce orthopaedic implant infection rate. Bacteria were grown and growth conditions optimised. Polystyrene and titanium (Ti) nanosurfaces were studied. The polystyrene surfaces had different nanopit arrays, while the Ti surfaces expressed different nanowire structures. Adhesion analysis was performed using fluorescence imaging, quantitative PCR and bacterial percentage coverage calculations. Further substitution with ‘heavy’ labelled glucose into growth medium allowed for bacterial metabolomic analysis and identification of any up-regulated metabolites and pathways. Our data demonstrates reduced bacterial adhesion on specific nanopit polystyrene arrays, while nanowired titanium showed increased bacterial adhesion following qPCR (P<0.05) and percentage coverage calculations (P<0.001). Further metabolomic analysis identified significantly increased intensity counts of specific metabolites (Pyruvate, Aspartate, Alanine and Carbamoyl aspartate). Our study shows that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. The identification of metabolic pathways involved in adhesion may allow for a targeted approach to biofilm eradication in S. aureus. This is of significant benefit to both the patient and the surgeon, and may well extend far beyond the realms of orthopaedics

The most common bacteria in orthopaedic prosthetic infections are Staphylococcus, namely Staphylococcus Epidermidis (SE) and Staphylococcus Aureus (SA). Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion and biofilm formation on nanofabricated materials. Bacteria studied were clinically relevant from an orthopaedic perspective, SA and SE. We hypothesise that that nanosurfaces can modulate bacterial adherence and biofilm formation and may reduce orthopaedic implant infection rate. Isolated bacteria were grown and growth conditions optimised. Bacterial concentrations were calculated by using qPCR. Statistical analysis allowed identification of optimal biofilm growth conditions. These were refined on standard, non-nanopatterned surfaces, and then control and nanopatterned polystyrene (nanopits) and titanium plates (nanowires). Adhesion analysis was performed using fluorescence imaging and quantitative PCR. 4 bacterial strains were isolated and cultured. Growth kinetics based on 24hr cultures allowed isolation of optimal media for biofilm conditions (Dulbecco's Modified Eagle Medium with additional supplements). Highest bacterial concentrations were found following 2hrs incubation with Lysozyme during qPCR. Bacterial concentration significantly increased between 30, 60 and 90 minutes incubation. Differences in percentage coverage on different polysyrene nanosurfaces (nanopits) were noted varying. This was confirmed by qPCR extractions that showed different bacterial concentrations on different nanopatterns. Titanium nanowire surfaces significantly increased bacterial adhesion (P<0.05). Our study cultured and quantified bacterial biofilm and suggests that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. Clearly this is of significant benefit to the patient, the surgeon and the NHS, and may well extend far beyond the realms of orthopaedics

Orthopedic device-related bone infection is one of the most distressing complications of the surgical fixation of fractures. Despite best practice in medical and surgical interventions, the rate of infection remains stubbornly persistent, and current estimates indicate that treatment failure rates are also significant. As we approach the limit of the effectiveness of current anti-infective preventative and therapeutic strategies, novel approaches to infection management assume great importance. This presentation will describe our efforts to develop and test various hydrogels to serve as customized antibiotic delivery vehicles for infection prevention and treatment. Hydrogels offer solutions for many of the challenges faced by complex trauma wounds as they are not restricted spatially within a poorly defined surgical field, they often degrade rapidly with no compatibility issues, and releases 100% of the loaded antibiotic. The preliminary data set proving efficacy in preventing and treating infection in both rabbit and sheep studies will be described, including local antibiotic concentrations in the intramedullary canal over time, compared to that of the more conventional antibiotic loaded bone cement. These two technologies show potential for the prevention and treatment of infection in trauma patients, with a clear focus on optimized antibiotic delivery tailored for complex wounds.

Objectives. Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro?. Methods. Titanium alloy cylinders (Ti6Al4V) were exposed to PEMF from an induction heater with maximum 2000 watts at 27 kHz after being contaminated with five different types of micro-organisms: Staphylococcus epidermidis; Staphylococcus aureus; Pseudomonas aeruginosa; spore-forming Bacillus cereus; and yeast Candida albicans. The cylinders were exposed to incremental target temperatures (35°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C) for up to 3.5 minutes. Results. There was an average linear heating rate of 0.39°C per second up to the target temperature, and thereafter the target temperature was maintained until the end of the experiment. At 60°C and higher (duration 3.5 minutes), there was a 6-log reduction or higher for every micro-organism tested. At 60°C, we found that the shortest duration of effective induction heating was 1.5 minutes. This resulted in a 5-log reduction or higher for every micro-organism tested. Conclusion. Non-contact induction heating of a titanium disk is effective in reducing bacterial load in vitro. These promising results can be further explored as a new treatment modality for infections of metal orthopaedic implants. Cite this article: B. G. Pijls, I. M. J. G. Sanders, E. J. Kuijper, R. G. H. H. Nelissen. Non-contact electromagnetic induction heating for eradicating bacteria and yeasts on biomaterials and possible relevance to orthopaedic implant infections: In vitro findings. Bone Joint Res 2017;6:323–330. DOI: 10.1302/2046-3758.65.BJR-2016-0308.R1

Abstract. Objectives. The objective of this study is to investigate if genomic sequencing is a useful method to diagnose orthopaedic infections. Current methods used to identify the species of bacteria causing orthopaedic infections take considerable time and the results are frequently insufficient for guiding antibiotic treatment. The aim here is to investigate if genomic sequencing is a faster and more reliable method to identify the species of bacteria causing infections. Current methods include a combination of biochemical markers and microbiological cultures which frequently produce false positive results and false negative results. Methods. Samples of prosthetic fluid were obtained from surgical interventions to treat orthopaedic infections. DNA is extracted from these samples lab and nanopore genomic sequencing is performed. Initial investigations informed that a sequencing time of 15 minutes was sufficient. The resulting genomic sequence data was classified using Basic Local Alignment Tool (BLAST) against the NCBI bacterial database and filtered by only including reads with an identity score of 90 and E-value of 1e-50. An E-value of 1e-50 suggests a high-quality result and is commonly used when analysing genomic data. This data was then filtered in R Studio to identify if any species were associated with orthopaedic infections. The results from genomic sequencing were compared to microbiology results from the hospital to see if the same species had been identified. The whole process from DNA extraction to output took approximately 2 hours, which was faster than parallel microbiological cultures. Results. In these preliminary analyses, 15 samples have been collected from patients with confirmed/suspected orthopaedic infections. To date, 11 samples from confirmed infected patients have been sequenced and a summary of the findings are presented in the table attached. As well as finding bacteria species to match microbiological cultures, genomic sequencing has also identified bacteria when culture results have been negative, but the patient is known to have an infection due to clinical indication and previous culture results. This example suggests genomic sequencing may have higher sensitivity than microbiological cultures at detecting bacteria causing orthopaedic infections. Results in table indicate the identification of bacteria from genomic sequencing that match microbiological cultures are high quality. Conclusions. Preliminary data presented using genomic sequencing suggests that the technique may be useful to identify bacterial species causing orthopaedic infections and can do so in a shorter time frame than current microbial methods. The results from genomic sequencing all produced a number of false positive results which hopefully can be reduced by improving the bioinformatic techniques used and increasing the sample number to include individuals without an infection. Further analysis will also look at identifying antibiotic resistance genes in the sequencing data and seeing if this ca be used to predict which patients will and will not respond to antibiotic treatment. The aim at the end of this project is to demonstrate if genomic sequencing is a more sensitive method to identify bacteria causing orthopaedic infections that current methods and if it can be used to guide antibiotic treatment. Include limitations, next steps and bigger picture. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

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

Thermostability is a key property in determining the suitability of local delivery of antibiotics in the treatment of orthopaedic infections. Herein, we aimed to assess the thermal stability and antibacterial activity of ciprofloxacin, ceftriaxone, gentamycine and vancomycine in high temperature conditions. Using a standardized E-test method, minimally inhibited concentration of each antibiotic substance against Staphylococcus aureus cultures were determined. The solutions of antimicrobial drugs ciprofloxacin 2 mg/ml, ceftriaxone 200 mg/ml, gentamycine 40 mg/ml and vancomycine 200 mg/ml were diluted twofold in deionised water. Acquired solutions were divided into three aliquots. The first aliquot was held at 40°C for 30 min in a waterbath, the second and the third aliquots were exposed to 80 and 100°C for 30 min in hot-air sterilizer, respectively. The treated solutions were tested for residual activity against S. aureus using a standardized disk diffusion method. Mediums with untreated antibiotic solutions and S. aureus were used as control. Plates were incubated at 37°C, at which time zones of inhibition (ZoI) were measured to the nearest whole millimeter for 14 days. The investigation indicated that the temperature elevation impacted considerably on antimicrobial activity and antibiotic stability overall. The in vitro temperature-response curves showed that ZoI diameter decreases logarithmically with elevated temperatures. Gentamicin was the only drug that was found to be affected to some extent. Results from the study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics and methods of antibiotic impregnation

Orthopaedic infection with bacteria leads to high societal cost and is detrimental to the life quality. Particularly, deep bone infection leading to osteomyelitis results in an inflammatory response whereby localized bone destruction occurs. Current treatments like antibiotic-containing polymethymethacrylate (PMMA) still has the high risk of bacterial resistance. Taking advantages of silver which has antibacterial and anti-inflammatory effect and bioactive collagen, we fabricated a silver nanoparticle (AgNP)-coated collagen membrane by sonication and sputtering. SEM showed good deposition of AgNPs on collagen membrane by both coating methods. The optimal coating concentration was finalized by assessing optimal antibacterial effect against cytotoxicity and finally collagen membrane coated with 1mg/mL AgNPs solution was selected. We also found that the coated collagen membrane demonstrating short-term cytotoxicity within 24 hours with damage to the cell membrane, which was evidenced by MTS and LDH release test, but had no significant influence (p > 0.05) thereafter. The amount of released AgNPs from coated collagen membrane had negligible cytotoxicity (p > 0.05). Confocal laser scanning microscope displayed similar cell morphology in both coated and uncoated collagen membrane. ELISA and qPCR presented the decreased secretion and expression (p < 0.001) of IL-6 and TNF-alpha. Upregulated expression (p < 0.001) of osteogenesis markers (RUNX2, ALP and OPN) could be found and this might be attributed to the modified collagen fibre surface coated by AgNPs. Collectively, the osteogenesis induced by AgNPs demonstrates a promising application in orthopaedic surgery for its use both as an antimicrobial agent, and to enhance bone regeneration

Objectives. Thermal stability is a key property in determining the suitability of an antibiotic agent for local application in the treatment of orthopaedic infections. Despite the fact that long-term therapy is a stated goal of novel local delivery carriers, data describing thermal stability over a long period are scarce, and studies that avoid interference from specific carrier materials are absent from the orthopaedic literature. Methods. In this study, a total of 38 frequently used antibiotic agents were maintained at 37°C in saline solution, and degradation and antibacterial activity assessed over six weeks. The impact of an initial supplementary heat exposure mimicking exothermically curing bone cement was also tested as this material is commonly used as a local delivery vehicle. Antibiotic degradation was assessed by liquid chromatography coupled to mass spectrometry, or by immunoassays, as appropriate. Antibacterial activity over time was determined by the Kirby-Bauer disk diffusion assay. Results. The heat exposure mimicking curing bone cement had minimal effect on stability for most antibiotics, except for gentamicin which experienced approximately 25% degradation as measured by immunoassay. Beta-lactam antibiotics were found to degrade quite rapidly at 37°C regardless of whether there was an initial heat exposure. Excellent long-term stability was observed for aminoglycosides, glycopeptides, tetracyclines and quinolones under both conditions. Conclusions. This study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics, and for material scientists looking to develop next-generation controlled or extended-release antibiotic carriers. Cite this article: E. Samara, T. F. Moriarty, L. A. Decosterd, R. G. Richards, E. Gautier, P. Wahl. Antibiotic stability over six weeks in aqueous solution at body temperature with and without heat treatment that mimics the curing of bone cement. Bone Joint J 2017;6:296–306. DOI: 10.1302/2046-3758.65.BJR-2017-0276.R1

Abstract. OBJECTIVES. Staphylococcus aureus is one of the most common pathogens in orthopaedic biomaterial-associated infections. The transition of planktonic S. aureus to its biofilm phenotype is critical in the pathogenesis of biomaterial-associated infections and the development of antimicrobial tolerance, which leads to ineffective eradication in clinical practice. This study sought to elucidate the effect of non-lethal dispersion on antimicrobial tolerance in S. aureus biofilms. METHODS. Using a methicillin-sensitive S. aureus reference strain, the effect of non-lethal dispersion on gentamicin tolerance, cellular activity, and the intracellular metabolome of biofilm-associated bacteria were examined. Gentamicin tolerance was estimated using the dissolvable bead biofilm assay. Cellular activity was estimated using the triphenyltetrazolium chloride assay. Metabolome analysis was performed using tandem high-performance liquid chromatography and mass spectrometry. RESULTS. Non-lethal dispersion of biofilm-associated S. aureus was associated with a four-fold reduction in gentamicin tolerance and a 25% increase in cellular respiration of both dispersed and adherent cells. Metabolome analysis found non-lethal dispersion reduced intracellular levels of L-ornithine and L-proline, with increased levels of cyclic nucleotides (p<0.05) in both liberated cells and the remaining biofilm-associated bacteria. These metabolomic changes have previously been shown to be associated with inactivation of the carbon catabolite repression mechanism, which is a key regulatory gatekeeper in the cellular resuscitation of dormant S. aureus cells. CONCLUSION. The metabolomic pipeline described in this study presents a valuable tool in the elucidation of molecular mechanistic pathways in biofilm pathogenesis. Kreb's cycle reactivation, through the carbon catabolite repression regulatory mechanism, has been shown to be associated with the reversal of biofilm-associated gentamicin tolerance. Understanding of the biosynthetic changes associated with the biofilm state will assist in the discovery of novel therapeutic targets in the management of biomaterial-related infections. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction and Objective. Found in bone-associated prosthesis, Cutibacterium acnes (C. acnes) is isolated in more than 50% of osteoarticular prosthesis infections, particularly those involving shoulder prostheses. Ongoing controversies exist concerning the origin of C. acnes infection. Few reports construct a reasonable hypothesis about probable contaminant displaced from the superficial skin into the surgical wound. Indeed, despite strict aseptic procedures, transecting the sebaceous glands after incision might result in C. acnes leakage into the surgical wound. More recently, the presence of commensal C. acnes in deep intra-articular tissues was reported. C. acnes was thus detected in the intracellular compartment of macrophages and stromal cells in 62.5% of the tested patients who did not undergo skin penetration. Among bone stromal cells, mesenchymal stem cells (MSCs) are predominantly found in bone marrow and periosteum. MSCs are the source of osteogenic lines of cells capable of forming bone matter. In this study, the pathogenicity of C. acnes in bone repair context was investigated. Materials and Methods. Human bone marrow derived MSCs were challenged with C. acnes clinical strains harvested from non-infected bone site (Cb). The behaviour of Cb strain was compared to C. acnes took from orthopaedic implant-associated infection (Ci). The infective capabilities of both strains was determined following gentamicin-based antibiotic protection assay. The morphology and ultrastructural analysis of infected MSCs was performed respectively through CLSM pictures of Phalloidin. ®. stained MSCs cytoskeleton and DAPI labelled Cb, and transmission and scanning electron microscopies. The virulence of intracellular Ci and Cb (Ci-MSCs and Cb-MSCs) was investigated by biofilm formation on non-living bone materials; and the immunomodulatory response of infected MSCs was investigated (PGE-2 and IDO secretion detected by ELISA). Bone cells (osteoblasts and PMA differentiated macrophages) were then challenged with Cb-MSCs and Ci-MSCs. Intracellular accumulation of ROS within infected macrophages was assessed by flow cytometry after 2 h of infection and the catalase production by Cb-MSC and Ci-MSC was evaluated. Statistical analyses were performed using Mann & Whitney test. Results. Following MSCs infection by C. acnes, the rate of viable bacteria inside MSCs was about 4% and 6% for Cb and Ci, respectively. Cb showed however a lower invasiveness in comparison to Ci (0.6-fold, p=0.01), confirming the higher pathogenicity of Ci. The ultrastructural and morphology analysis of infected MSCs confirmed the presence of bacteria free in MSCs cytoplasm, localized between F-actin fibers of MSCs, which preserved their elongated morphology. Considering the high level of secreted immunomodulatory mediators (PGE-2 and IDO), our results suggest that Cb-infected MSCs could promote a transition of macrophages from a primarily pro-inflammatory M1 to a more anti-inflammatory M2 phenotype. In comparison with Cb, Cb-MSCs increased significantly the formation of biofilm on TA6V and PEEK but reduced the biofilm formation on 316L SS. Ci-MSCs showed a significant increase in biofilm formation on PEEK vs Ci, while no difference in biofilm formation was noticed on TA6V and 316L SS. Regarding the ability of MSCs bacteria to infect osteoblasts, our results showed a higher infective capabilities of Cb-MSCs versus Cb (>2-fold, p=0.02), while no difference was noticed between Ci and Ci-MSCs. Along with an increase in catalase production by Cb-MSCs, we noticed its higher persistence to macrophage degradation. Conclusions. Taken together, our results demonstrate a shift in commensal Cb to pathogenic following infection. Indeed, Cb- MSCs acquires features that (i) increase biofilm formation on orthopedic based materials, (ii) increase the osteoblast infection and (iii) develop resistance to the macrophage degradation, through the increase of catalase production. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections

Introduction. In 2011 the Scottish Government published national MRSA screening requirements. A comparison of Orthopaedic and ENT elective surgery intended to juxtapose a specialty known to take MRSA screening seriously with one that has little clinical concern with regards MRSA infection. ENT surgery parallels Orthopaedics in using implants and there potentially being MRSA colonisation at or close to the site of surgery. In Orthopaedics MRSA infection is infrequent, but implant infection with antibiotic resistant bacteria has a particularly poor prognosis. In ENT MRSA infection is rare and colonisation does not influence patient care. Aims. An evaluation of MRSA screening practice for elective Orthopaedics and ENT surgery at Gartnavel General Hospital with regards strategy and implementation. Method. Review of 342 consecutive elective ENT patients and 325 Orthopaedic patients attending for inpatient or day case surgery. The reference standards were the regional and national guidelines on MRSA screening. Results. Overall screening rates were 145 (42%) of 342 ENT patients and 270 (83%) of 326 Orthopaedic patients. 100% of Orthopaedic patients admitted (154) were screened, in compliance with both regional and national policy. 91 (70%) of 130 ENT patients admitted were screened for MRSA, and no risk assessment was carried out, which was not in compliance with either regional or national policy. Discussion. Orthopaedic surgery has an established and reliable practice of screening elective inpatient cases, and when identified MRSA colonisation results in a change in patient management. ENT surgery should have established a similar practice according to existing local guidelines. The Government consider ENT a lower risk speciality for MRSA, but still require as a minimum a documented MRSA risk assessment process

Summary Statement. Conventional culture techniques have poor sensitivity for detecting bacteria growing in biofilms, which can result in under-diagnosis of infections. Sonication of biofilm colonised orthopaedic biomaterials can render bacteria in biofilm more culturable, thereby improving diagnosis of orthopaedic implant infections. Introduction. Prosthetic joint infection (PJI) is a potentially devastating complication in arthroplasty. Biofilm formation is central to PJI offering protection to the contained bacteria against host defence system and antimicrobials. Orthopaedic biomaterials generally have a proclivity to biofilm colonisation. Conventional culture technique has a low sensitivity for detecting bacteria in biofilm. Sonication can disrupt bacteria biofilms aggregations and dislodge them from colonised surfaces, rendering them culturable and consequently improve the diagnosis of otherwise culture-negative PJI. We investigated the effect of ultrasonication on biofilms adherent to poylmethylmethacrylate PMMA cement. Method. Identical PMMA cement beads were aseptically prepared using 7mm bead templates. Each sample comprised of two beads and with multiple replicates made for each sample. Two proficient biofilm forming strains of Staphylococcus epidermidis (5179-R1 and 1457) were used for the experiments. Each set of cement sample was immersed in Brain Heart Infusion broth inoculated with a pre-culture of the chosen bacteria strains (final concentration approximately 4 × 10. 6. CFU/ml). All samples were then incubated for 24 hours at 37°C to allow for biofilm growth and colonisation of the cement surfaces, as well as for biofilm maturity. After incubation, each sample was washed twice with sterile phosphate buffer saline (PBS) to remove non-adherent and loosely adherent bacteria. The cement beads were transferred to a fresh sterile bottle at each stage of the experiment, while ensuring the maintenance of asepsis. After the final wash, 10ml of sterile PBS was added to the cement beads and each sample was sonicated for varying periods: 0min, 5min, 10min, 20min and 40min. Sonicate fluid were collected after each period of sonication, with which culture plates were inoculated for the purpose of viable bacteria counting. Results. The optimum sonication period was between 5min and10 min. The mean pre-sonication CFU/ml were 4.7 × 10. 5. and 8.3 × 10. 5. for bacteria strains 5179-R1 and 1457 respectively, while the mean CFU/ml after 10min of sonication were 1.4 × 10. 7. and 0.74 × 10. 7. for bacteria strains the respective bacteria strains. Discussion / Conclusion. Our study showed a significant increase (almost 100 fold) in bacteria culture yield following sonication. We were also able to demonstrate that the optimum duration for sonication (using comparable sonicators) was approximately 10min. Sonication was able to completely remove adherent bacteria from the surfaces of our cement samples allowing them to be cultured. Our result suggests that sonication of bone cement can be instrumental in improving the diagnosis of biofilm associated PJI

Objectives

The objective of this study was to compare the elution characteristics, antimicrobial activity and mechanical properties of antibiotic-loaded bone cement (ALBC) loaded with powdered antibiotic, powdered antibiotic with inert filler (xylitol), or liquid antibiotic, particularly focusing on vancomycin and amphotericin B.