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

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

There is a lack of carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotic for Staphylococcus aureus deep bone infections (DBIs). RIF is also associated with systemic side effects, and known for causing rapid development of antibiotic resistance when given as monotherapy. We evaluated a clinically usedbi-phasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). It was hypothesized that this combined approach could provide improved biofilm eradication and prevent the development of RIF resistance. Methods: 1) Biofilm eradication: Using a modified crystal violet staining biofilm quantification method, the antibiotics released at different time points (Day 1, 3, 7, 14, 21, 28 and 35) from the hemispherical pellets of CaS/HA(500 mg)-VAN (24.57 mg) / GEN (10.35 mg) composites with or without RIF (8.11 mg) were tested for their ability to disrupt the preformed 48-h old biofilms of S. aureus ATCC 25923, and S. aureus clinical strain P-3 in 96-well microtitre plate. For each tested group of antibiotic fractions, five separate wells were used (n=5). 2) Testing for resistance development: Similar to the method mentioned above the 48-h biofilm embeded bacteria exposed to antibiotic fractions from different time points continuously for 7 days. The biofilms remained were then tested for RIF resistant strains of bacteria. Overall, there was clear antibiofilm biofilm activity observed with CaS/HA-VAN/GEN+RIF combinations compared with CaS/HA-VAN/GEN alone. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with combinations of CaS/HA-VAN/GEN+RIF. Enhanced antibiofilm effects without development of RIF resistance indicates that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for additional local delivery in clinically demanding DBIs. Acknowledgement: We deeply acknowledge the Royal Fysiographic Society of Lund, Landshövding Per Westlings Minnesfond and the Stina and Gunnar Wiberg fond for financial support

Background. The different biodegradable local antibiotic delivery systems are widely used in recent years. The aim of this study was to evaluate the bactericidal activity antibiotic loaded PerOssal pellet in vitro and its effectiveness in the treatment of Staphylococcus aureus induced chronic osteomyelitis. Material and methods. MALDI-TOF have been applied to microbiological diagnosis in patient with osteomyelitis. In most cases, Staphylococcus aureus was isolated. In vitro Ceftriaxone-Loaded PerOssal pellet were placed in middle agar plate containing a stock strain of Staphylococcus aureus. Plates were incubated at 37ºC for 24 hours. The zones of bacterial inhibition were recorded after 24, 48 and 72 hours of incubation. In vivo evaluation was performed by prospectively studying of 21 patients with a clinically and bacteriologically diagnosed Staphylococcus aureus induced osteomyelitis. Mean age was 38±4,2(26 to 53)). After radical surgical debridement and ultrasound cavitation, the bone cavity was full filled with Perosal pellets loaded with different antibiotics depending from the antibiotic sensitivity test. Endpoints were the absence of clinical manifestation of infection or disease recurrence, no need for further surgery. Results. In vitro showed after 24 hrs inhibition zone was 4,2 х 4,9 cm, after 72 hrs the inhibition zone was increased till 7,6 х 8,4 cm. During the subsequent time, there were no changes. Results of the clinical study evidenced no signs of infection in 18 patients (86% (CI 69,8;100)) (p<0,05) at the follow up, while 3 (14%(CI 0;30,2)) (p<0,05) subjects showed infection recurrence at 6 months from operation and 2 of them needed further surgical procedures. Conclusion. PerOssal as an antibiotic carrier stabilizes the action of the antibiotic. This antibiotic carrier system allows to choose an antibiotic individually for each patient according to the antibiotic sensitivity test and can be successfully used in clinical cases of osteomyelitis

Objectives. Vancomycin and fosfomycin are antibiotics commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. This study compares the in vitro inhibitory effects against MRSA of articulating cement spacers impregnated with either vancomycin or fosfomycin. Methods. Vancomycin-impregnated articulating cement spacers and fosfomycin-impregnated articulating cement spacers were immersed in sterile phosphate-buffered saline (PBS) solutions and then incubated. Samples were collected for bioactivity evaluation. The aliquots were tested for MRSA inhibition with the disc diffusion method, and the inhibition zone diameters were measured. The inhibition zone differences were evaluated using the Wilcoxon Rank Sum Test. Results. The vancomycin group had significantly larger inhibition zones than the fosfomycin group from day three through to completion of the fourth week of incubation (p < 0.001). The vancomycin group exhibited a MRSA inhibition zone up to four weeks but the fosfomycin group showed an inhibition zone for only three days and after that did not show the the potential to inhibit MRSA. Conclusion. This in vitro study found that the inhibitory effect of vancomycin-impregnated articulating cement spacers against MRSA outperformed fosfomycin-impregnated articulating cement spacers. Further comparing our results to other published reports suggests there might be a limitation of the disc diffusion bioassay to show a large inhibitory zone in a high concentration of a highly soluble antibiotic. Cite this article: V. Yuenyongviwat, N. Ingviya, P. Pathaburee, B. Tangtrakulwanich. Inhibitory effects of vancomycin and fosfomycin on methicillin-resistant Staphylococcus aureus from antibiotic-impregnated articulating cement spacers. Bone Joint Res 2017;6:132–136. DOI: 10.1302/2046-3758.63.2000639

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

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 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 explores, 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, bacterial biofilms were challenged by gentamicin +/− low-intensity ultrasound (1.5MHz, 30W/cm2, pulse duration 200µs/1KHz) for 20 minutes. 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. Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations

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/cm. 2. , 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 × 10. 9. 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. 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

Objectives. Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied. Methods. We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis. Results. Our results show that clinical strains adhere to the material surface at lower concentrations than collection strains. A destructive effect of bacteria on preosteoblastic cells was also detected, especially with higher concentrations of bacteria. Conclusions. The method described herein can be used to evaluate the effect of surface modifications on bacterial adherence more accurately than conventional monoculture studies. Clinical strains behave differently than collection strains with respect to bacterial adherence. Cite this article: M. Martinez-Perez, C. Perez-Jorge, D. Lozano, S. Portal-Nuñez, R. Perez-Tanoira, A. Conde, M. A. Arenas, J. M. Hernandez-Lopez, J. J. de Damborenea, E. Gomez-Barrena, P. Esbrit, J. Esteban. Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the “race for the surface” theory. Bone Joint Res 2017;6:315–322. DOI: 10.1302/2046-3758.65.BJR-2016-0226.R2

This longitudinal microCT study revealed the osteolytic response to a Staphylococcus epidermidis-infected implant in vivoand also demonstrates how antibiotics and/or a low bone mass state influence the morphological changes in bone and the course of the infection. Colonisation of orthopaedic implants with Staphylococcus aureusor S. epidermidisis a major clinical concern, since infection-induced osteolysis can drastically impair implant fixation or integration within bone. High fracture incidence in post-menopausal osteoporosis patients means that this patient group are at risk of implant infection. The low bone mass in these patients may exacerbate infection-induced osteolysis, or alter antibiotic efficacy. Therefore, the aims of this study were to examine the bone changes resulting from a S. epidermidisimplant infection in vivousing microCT imaging, and to determine if a low bone mass stateinfluences the course of the infection and the efficacy of antibiotic therapy. An in vivomodel system using microCT scanning [1], involving the implantation of either a sterile or a S. epidermidis-colonised PEEK screw into the proximal tibia of 24 week-old female Wistar rats, was used to investigate the morphological changes in bone following infection over a 28 day period. In addition, the efficacy of a combination antibiotic therapy (rifampin and cefazolin: administered twice daily from days 7–21 post-screw implantation) for affecting osteolysis was also assessed. A subgroup of animals was subjected to ovariectomy (OVX) at 12 weeks of age, allowing for a 12 week period for bone loss prior to screw implantation at 24 weeks. Bone resorption and formation rates, bone-implant contact and peri-implant bone volume in the proximity of the screw were assessed by microCT scanning at days 0, 3, 6, 9, 14, 20 and 28 days post-surgery. Following euthanasia at day 28, the implanted screw, bone and soft tissues were subjected to quantitative bacteriology as a measure of the efficacy of the antibiotic regimen. In non-OVX animals S. epidermidisinfection induced marked osteolysis, which peaked between 9 and 14 days post-screw implantation. Peak bone resorption was detected at day 6, before recovering to baseline levels at day 14. Infection also resulted in extensive deposition of mineralised tissue, initially within the periosteal region (day 9–14), then subsequently in the osteolytic region at day 20–28. Quantitative bacteriology indicated all non-OVX animals remained infected. Rifampin and cefazolin successfully cleared the infection in 5/6 non-OVX animals group although there was no difference observed in CT-derived bone parameters. OVX resulted in extensive loss of trabecular bone but this did not alter the temporal pattern of infection-induced osteolysis, or mineralised tissue deposition, which was similar to that observed in the non-OVX animals. Similarly, there was no difference in bacterial counts between non-OVX and OVX animals (39,005 colony-forming units (CFU) [range: 3,675–156,800] vs 37,665 CFU [range 3,250–84,000], respectively). Interestingly, antibiotic treatment was less effective in the OVX animals (3/5 remained infected), suggesting that antibiotics have reduced efficacy in OVX animals. This study demonstrates S. epidermidis-induced osteolysis displays a similar temporal pattern in both normal and low bone mass states, with comparable bacterial loads present within the localised infection site

Introduction. Carriers of Staphylococcus aureus, both methicillin sensitive (MSSA) and methicillin resistant (MRSA), have an increased risk for health-care associated infections. Despite WHO recommendations there is currently no national screening and eradication policy for the detection of MSSA in the UK or USA. This study aimed to evaluate the effectiveness of current standard MRSA eradication therapies in the context of S. aureus decolonisation prior to joint replacement surgery. Methods. Pre-operative PCR nasal screening was performed in 273 Orthopaedic patients awaiting joint replacement surgery. In all 100 patients were positive for S. aureus and enrolled into the study. All enrolled patients received and were instructed to administer the decolonisation regimen for five days. Prior to commencement of the eradication therapy swabs of the anterior nares, throat, and perineum were taken for culture. Further culture swabs were taken at; 48–96 hours after completion of the five-day eradication regimen, at hospital admission for surgery, and at hospital discharge. Patients were followed up for six weeks post-surgery. Following completion of the five-day course patients were asked to provide feedback on their experience using Likert rating scales. The primary outcome of this study was S. aureus clearance 48–96 hours post-completion of eradication therapy. Results. At 48–96 hours post-completion there was S. aureus clearance from: the anterior nares 93.8% (95% Confidence Interval (CI) 79.2–99.2%), throat 65.6% (95% CI 46.8–81.4%), and groin 87.5% (95%CI 71–96.5). Mean compliance with nasal mupirocin was 98.2% (standard deviation ±5.2). There was no statistically significant recolonisation effect between completion of eradication therapy and the day of surgery (P>0.05) at a median time of 9.5 days (Interquartile range 6–13 days) at all sites. Discussion and Conclusion. Current MRSA decolonisation regimens are well-tolerated and effective for S. aureus decolonisation for the anterior nares and groin. The decolonisation effect is preserved for up to 10 days following completion

INTRODUCTION. Staphylococci species account for ∼80 % of osteomyelitis cases. While the most severe infections are caused by Staphylococcus aureus (S. aureus), the clinical significance of coagulase negative Staphylococcus epidermidis (S. epidermidis) infections remain controversial. In general, S. epidermidis was known to be a protective commensal bacterium. However, recent studies have shown that intra-operative low-grade S. epidermidis contamination prevents bone healing. Thus, the purpose of this study is to compare the pathogenic features of S. aureus and S. epidermidis in an established murine model of implant-associated osteomyelitis. METHODS. All animal experiments were performed on IACUC approved protocols. USA300LAC (MRSA) and RP62A(S. epidermidis) were used as prototypic bacterial strains. After sterilization, stainless steel pins were implanted into the tibiae of BALB/c mice (n=5 each) with or without Staphylococci. Mice were euthanized on day 14, and the implants were removed for scanning electron microscopy (SEM). Tibiae were fixed for mCT prior to decalcification for histology. RESULTS. The histology of S. aureus infected tibiae demonstrated massive osteolysis and abscesses formation. In contrast, the histology from S. epidermidis infected tibiae was indistinguishable from uninfected controls. Gross mCT analyses revealed massive bone defects around the infected implant with reactive bone formation only in the S. aureus group. The osteolysis findings were confirmed by quantitative analysis, as the medial hole area of S. aureus infected tibiae (1.67 ± 0.37 mm2) was larger than uninfected (0.15 ± 0.10 mm2) (p < 0.001) and S. epidermidis (0.19 ± 0.14 mm2) (p < 0.001) groups. Consistently, the %biofilm area on the implants of the S. aureus group (39.0 ± 13.7 %) was significantly larger than uninfected (6.3 ± 2.3 %) (p < 0.001) and S. epidermidis (12.9 ± 7.4 %) (p < 0.001). Although the amount of biofilm of S. epidermidis was much smaller than S. aureus, the presence of bacteria on the implant were confirmed by SEM. In addition, the empty lacunae, which is a feature of mature biofilm and evidence of bacterial emigration, were also present on both S. epidermidis and S. aureus infected implants. DISCUSSION. In this study, we confirmed the aggressive pathologic features S. aureus on host bone, soft tissues and biofilm formation. In contrast, we show that S. epidermidis is incapable of inducing osteolysis, reactive bone formation or soft tissue abscesses, even though it colonizes the implant in small biofilms. Collectively, the results support a potential role for S. epidermidis in implant loosening and fracture non-unions, as the bacteria can form small biofilms that could interfere with osseous integration and bone healing. However, future studies are warranted to assess the effects of S. epidermidis biofilm on implant loosening

Background. Staphylococcus aureus is a human pathogen involved in implant-related infections. In these diseases, biofilm production is the key pathogenic event, and it increases antibiotic resistance of the organism. Because this phenomenon, local delivery of antibiotics could allows reaching high concentrations in the infected tissue without the secondary effects linked to systemic administration. Here we report the use of a ceramic biomaterial (SBA-15) as a carrier of antibiotics in order to deliver them directly in the infected tissue. Material and methods. SBA-15 discs were loaded with vancomycin, rifampin and a combination of both according to the protocol described by Molina-Manso et al. Loaded discs were introduced in a 0.5 McFarland suspension of S. aureus 15981 and incubated during 6 and 24 hours in order to develop a biofilm. After incubation, samples were sonicated during 5 minutes and 1:10 serial dilutions were performed in order to count viable bacteria. All experiments were performed in triplicate. Results. A statistically significant decrease in the number of viable bacteria was detected for all antibiotics at 6 hours, and also for vancomycin and the combination. Rifampin showed an increase in the number of viable bacteria at 24 hours. No differences were detected between vancomycin and the combination of antibiotics. Conclusion. SBA-15 can carry antibiotics that have effect on bacterial biofilm. The use of rifampin alone showed a loss of the effect after 24 hours of incubation, probably due to the selection of resistant mutants that nullify the effect of the antibiotic. No differences have been detected between vancomycin alone and its combination with rifampin in this experiment

Serial section electron microscopy (SSEM) was initially developed to map the neural connections in the brain. SSEM eventually led to the term ‘Connectomics’ to be coined to describe process of following a cell or structure through a volume of tissue. This permits the true three-dimensionality to be appreciated and relationships between cells and structures. The purpose of this study was to utilize this methodology to interrogate S. aureus infected bone.

Bone samples were harvested from mice tibia infected with S. aureus and were fixed, decalcified, and osmicated. The samples were paraffin embedded and 5-micron sections were cut to identify regions of bacterial invasion into the osteocyte-lacuna-canalicular-network (OLCN). This area was cut from the paraffin block, deparaffinized, post-fixed and reprocessed into epoxy resin. Serial sections were cut at 60nm and collected onto Kapton tape utilizing the Automated Tape-collecting Ultramicrotome (ATUMtome) system. Samples were mounted onto 4” silicon wafers and post-stained with 2% uranyl acetate followed by 0.3% lead citrate and carbon coated. A ZEISS GeminiSEM 450 scanning electron microscope fitted with an electron backscatter diffusion detector was used to image the sections. The image stack was aligned and segmented using the open-source software, VASTlite.

264 serial sections were imaged, representing approximately 40 × 45 × 15-micron (x, y, z) volume of tissue. 70% of the canaliculi demonstrated infiltration by S. aureus.

This study demonstrates that SSEM can be applied to the skeletal system and provide a new solution to investigate the OLCN system. It is feasible that this methodology could be implemented to investigate why some canaliculi are resistant to colonization and potentially opens up a new direction for the prevention of chronic osteomyelitis. In order to make this a realistic target, automated segmentation methodologies utilizing machine learning must be developed and applied to the bone tissue datasets.

Failure of osseointegration and periprosthetic joint infection (PJI) are the two main reasons of implant failure after total joint replacement (TJR). Nanofiber (NF) implant surface coating represents an alternative local drug eluting device that improves osseointegration and decreases the risk of PJI. The purpose of this study was to investigate the therapeutic efficacies of erythromycin (EM)-loaded coaxial PLGA/PCL-PVA NF coating in a rat S. aureus-infected tibia model.

NF coatings with 100mg and 1000mg EM were prepared. NF without EM was included as positive control. 56 Sprague Dawley rats were divided into 4 groups. A titanium pin (1.0-mm x 8 mm) was placed into the tibia through the intercondylar notch. S. aureus (SA) was introduced by both direct injection of 10 μl broth (1 × 10⁴ CFU) into the medullary cavity and single dip of Ti pins into a similar solution prior to insertion. Rats were sacrificed at 8 and 16 weeks after surgery. The outcome measurements include μCT based quantitative osteolysis evaluation and hard tissue histology.

Results: EM-NF coating (EM100 and EM1000) reduced osteolysis at 8 and 16 weeks, compared to EM0 and negative control. The effective infection control by EM-NFs was further confirmed by hard tissue section analysis. The Bone implant contact (BIC) and bone area fraction Occupancy (BAFO) within 200 µm of the surface of the pins were used to evaluate the osseointegration and new bone formation around the implants. At 16 weeks, the bone implant contact (BIC) of EM 100 (35.08%) was higher than that of negative control (3.43%) and EM0 (0%). The bone area fraction occupancy within 200 µm (BAFO) of EM100 (0.63 mm2) was higher than that of negative control (0.390 mm2) and EM0 (0.0 mm²). The BAFO of EM100 was also higher than that of EM1000 (0.3mm²).

There was much less osteolysis observed with EM100 and EM1000 NF coatings at 16 weeks, as compared to EM0 positive control, p=0.08 and p=0.1, respectively. Osseointegration and periprosthetic bone formation was enhanced by EM-NFs, especially EM100. Data from this pilot study is promising for improving implant surface fabrication strategies.

The main problem of infected orthopaedic implants is that the presence of microorganisms in an organized biofilm making them difficult accessible for antibiotics. This biofilm consists of a complex community of microorganisms embedded in an extracellular matrix that forms on surfaces such as an implant. Non-contact induction heating uses pulsed electromagnetic fields to induce so-called ‘eddy currents’ within metal objects which causes them to heat up. This heat causes thermal damage to the bacterial biofilm hence killing the bacteria on the metal implant. The purpose of this study is to determine the effectiveness of induction heating on killing Staphylococcus epidermidis in a biofilm. S. epidermidis biofilms were grown on Titanium alloy (Ti6Al4V) coupons and subsequently were heated with a custom-built induction heater to temperatures of 60°C, 70°C, 80°C and 90°C for 3.5 minutes. Temperature was controlled with an infra-red thermal sensor and micro-controller. We also included two control conditions without induction heating: C1 without induction heating and C2 with chlorhexidine 0.5% in 70% alcohol without induction heating. Experiments were repeated 5 times. In the C1 group (no induction heating), 1.3 * 10(7) colony forming units (CFU)/cm(−2) of S. epidermidis were observed. For 60°C, 70C, 80 C and 90C, a 3.9-log reduction, 5.3-log reduction, 5.5-log reduction and 6.1-log reduction in CFU/cm(−2) were observed, respectively. For the C2 (chlorhexidine) there was a 6.7-log reduction CFU/cm(-2). We concluded that induction heating of Titanium coupons is effective in reducing bacterial load in vitro for S. epidermidis biofilms

We have compared the rates of infection and resistance in an animal model of an orthopaedic procedure which was contaminated with a low-dose inoculum of Staphylococcus epidermidis. We randomised 44 Sprague-Dawley rats to have bone cement implanted subcutaneously containing either gentamicin or saline (control). The wound was inoculated with a dilute solution of gentamicin-sensitive Staphylococcus epidermidis. At two weeks the cement was retrieved and microbiologically tested. A lower overall rate of infection was seen in the gentamicin-loaded cement group, but there was a significantly higher rate of gentamicin-resistant infection in this group (Fisher’s exact test, p < 0.01). Antibiotic-impregnated cement has an optimum surface for colonisation and prolonged exposure to antibiotic allows mutational resistance to occur. Gentamicin-loaded cement may not be appropriate for revision surgery if it has been used already in previous surgery

Prosthetic joint infections (PJI) occur infrequently, but they represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus are the most common infecting agents associated with acute PJI, and also appear in some cases of delayed PJI. 1. S. aureus biofilm development can be divided in two stages: adhesion and proliferation. 2. To avoid PJI bacterial adhesion has to be decreased. Hybrid organo-inorganic sol-gel coatings are proposed as a promising biomaterial improvement. 3. One of these compounds is a mixture of two organopolisiloxanes: 3-methacryloxypropyltrimethoxysilane (MAPTMS) and tetramethylorthosilicate (TMOS). The aim of this work was to evaluate bacterial adhesion on MAPTMS-TMOS coating compared to titanium parts made by powder metallurgy. MAPTMS-TMOS sol-gel coating was produced using a molar ratio of 1:2 (MAPTMS:TMOS) and dispersed in ethanol. The sol-gel was deposited by dip-coating on titanium parts made by powder metallurgy followed by a thermal treatment at 120 ºC for 30 minutes. 4. Titanium parts without sol-gel coating were used as control. S. aureus 15981 strain adherence study was performed using the protocol described by Kinnari et al. 5. with 90 min incubation. After incubation, the samples were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Proportion of total adhered, live and dead bacteria was calculated and studied by using ImageJ software. The experiments were performed in triplicate. The statistical data were analyzed by pairwise comparisons using the nonparametric Mann-Whitney test with a level of statistical significance of p<0.05. Values are cited and represented as medians. S. aureus 15981 adherence was 942-fold lower on MAPTMS-TMOS coating than on uncoated titanium. According with our results, MAPTMS-TMOS sol-gel coating is a promising antiadherent surface for S. aureus. More studies are necessary in order to evaluate this property with other species and strains

Summary Statement. A single, locally-delivered injection of a human placental product containing multipotent stromal cells reduced severity of infection in an immunosuppressed murine osteomyelitis model and eliminated infection in 25% of animals compared with 0% of controls without the use of antibiotics. Introduction. Implant–associated osteomyelitis is a serious orthopaedic condition and is particularly difficult to treat in immunosuppressed individuals. Despite great advancement in the field of biomaterials and pharmaceuticals, emerging patterns of antibiotic resistance, complex biofilm production and penetration of therapeutic concentrations of effective antibiotics into bone continue to represent unmet clinical challenges. The promise of adult multipotent stromal cells (MSCs) for tissue regeneration has been of intense interest in recent years. Among their many potential therapeutic uses, MSCs have also been shown to have direct antimicrobial properties. The objective of this study was to evaluate the efficacy of a locally–delivered human placental-based tissue product containing multipotent stromal cells (hAmSC) to reduce the severity of implant-associated Staphylococcus aureus osteomyelitis in an immunosuppressed murine model. We hypothesised that athymic mice with implant-associated osteomyelitis would have diminished infection following treatment with hAmSC as evidenced by decreased bioluminescence intensity and lower histologic scores for infection and bacterial load when compared to saline-treated controls. Methods. An athymic murine model of chronic implant-associated osteomyelitis was developed using luciferase-transfected Staphylococcus aureus to study the antimicrobial effects of a human placental-based product containing multi-potent stromal cells (hAmSC). Sixteen athymic mice had osteomyelitis established in the right femoral diaphysis. Fifteen days after inducing luc S. aureus osteomyelitis, the mice were randomised to receive a single 0.5 cc injection of hAmSC (n=8) or vehicle (0.9% saline) (n=8) into the soft tissues immediately adjacent to the infected bone. No antibiotics were administered throughout the duration of the study. Mice were imaged with an In Vivo Imaging System (IVIS 1000, PerkinElmer) twice weekly for 30 days to assess change in bioluminescence intensity from baseline immediately prior to treatment with either hAmSC or saline. Radiographs were obtained at days −10, 0, 10, 20 and 30 days post-injection and scored for bone changes secondary to osteomyelitis by a reviewer blinded to treatment group. Mice were sacrificed 30 days after treatment and femurs were examined histologically and scored for bacterial load and degree of inflammation by a pathologist blinded to treatment group. Results. Osteomyelitis was successfully established in all mice as evidenced by baseline bioluminescence imaging and radiographs. Mean bioluminescence intensity decreased from baseline in animals receiving hAmSC and remained below baseline for 28 days, whereas vehicle-treated animals showed an increase in mean bioluminescence intensity throughout the study period. Osteomyelitis resolved in 2/8 hAmSC-treated animals and 0/8 vehicle-treated animals as evidenced by bioluminescence imaging and histological examination for bacteria/inflammation at sacrifice. Radiograph scores for secondary bone changes were lower in mice treated with hAmSC than vehicle at 10, 20 and 30 days post injection. Median inflammatory score was lower in the hAmSC-treated mice than vehicle treated controls. Conclusions. A single injection of hAmSC was effective at reducing the severity of S. aureus infection without the use of antibiotics in this chronic implant associated osteomyelitis immunosuppressed murine model. In addition to reduced bioluminescence intensity below baseline for 28 days during the study period, infection was eliminated in 25% of animals in the hAmSC-treated group

Summary. Staphylococcus aureus isolates from Fracture fixation device related infections contained fewer isolates that form a strong biofilm in comparison with isolates from Prosthetic joint infections. Both orthopaedic implant related infection groups possessed fnbB and sdrE more frequently than the non-implant related infection groups. Introduction. One of the most common pathogen causing musculoskeletal infections is Staphylococcus aureus. The aim was to characterise S. aureus isolated from these infections and to look for differences between the isolates from orthopaedic implant related infections (OIRI) and those in non-implant related infections (NIRI). The OIRI are further differentiated in those associated with fracture fixation (FFI) devices and those found in prosthetic joint infections (PJI). Methods. Three-hundred and five S. aureus isolates were collected from different Swiss and French hospitals (FFI, n=112; PJI, n=105; NIRI, n=88). The cases of NIRI were composed of 27 osteomyelitis (OM), 23 diabetic foot infections (DFI), 27 soft tissue infections (STI) and 11 postoperative spinal infections (SI). Isolates were tested for their ability to form a biofilm. They were typed by agr (accessory gene regulator) group and genes coding for the 13 most relevant MSCRAMMs, Panton-Valentine leukocidin (PVL), PIA (polysaccharide intercellular adhesin), γ-haemolysin, the five most relevant Staphylococcal enterotoxins (SEA-SEE), exfoliative toxins A and B (ETA and ETB) and toxic shock protein (TST) were screened for by PCR. Results. The majority of the S. aureus isolates were methicillin susceptible (MSSA) with 83.4% for the OIRI and 93.2% for the NIRI. All isolates were able to produce a biofilm. A strong biofilm was produced in 13.8% of the OIRI isolates compared to 10.2% of the NIRI isolates. The difference between the isolates of the PJI versus the FFI was statistically significant (20% vs 8%; p=0.011). All four agr types were present in all groups. agrI predominated in the OIRI (42.4%) as well as in the NIRI (44.4%). Comparing OIRI with NIRI, agrII was present in a higher prevalence in OIRI (30.9% vs 14.8%) and agrIII in a lower incidence (21.2% vs 30.7%). Genes cna, clfA and bbp were exhibited predominantly by isolates from the NIRI, while the fnbB and the sdrE gene were more frequently observed among OIRI. Conclusions. Methicillin susceptible S. aureus (MSSA) was more prevalent than methicillin resistant S. aureus (MRSA) in this collection. Possible trends for the orthopaedic device associated infection groups FFI and PJI could be observed whereby isolates from PJI produced stronger biofilm than isolates from the FFI group. The agr type agrII, the fnbB gene and sdrE gene were more prevalent present in the OIRI compared to the NIRI. In contrast, agrIII, and the bbp gene were more prevalent in the NIRI than in the OIRI