Background. Gram stain microscopy is a routinely requested investigation in the evaluation of septic arthritis in both paediatric and adult patients. Recent evidence suggests that gram stain microscopy has poor diagnostic accuracy in adults with a sensitivity of only 45%, however the diagnostic accuracy remains unknown in children. We sought to establish the diagnostic utility of gram stain microscopy in the diagnosis of septic arthritis in children. Methods. We conducted a retrospective review of all patients of 16 years and under that underwent aspiration and washout of suspected septic joints in theatre from March 2005 to February 2011. Theatre data were cross referenced with microbiology results and analysed by simple descriptive methods in Excel. Results. We identified 23 paediatric patients undergoing washout or aspiration of a suspected septic joint during the time period studied. 17 (74%) of the patients were female and the average age was 2 years (Range 1 month–16 years). The most commonly affected joints were Knees (12/23, 52%) and Hips (7/23, 30%), with the remainder of infections occurring sporadically. There were 9 cases of culture confirmed septic arthritis (39% of all washouts), and these occurred in 5 knees (56%), 3 Hips (33%) and 1 ankle (11%). Organisms were Staphylococcus Aureus (2/8), Coagulase Negative Staphylococcus (2/9), Streptococcus pneumoniae (2/9), Group B Streptococcus (2/9) and Group A Streptococcus (1/9). Gram stain microscopy identified organisms in 3 aspirates both of which were confirmed on extended culture (Sensitivity 33%, Specificity 100%). Conclusion. Gram stain microscopy identified only 33% of culture positive cases of septic arthritis within this study. Our results suggest that gram stain microscopy lacks the diagnostic accuracy to be used in the exclusion of septic arthritis in the paediatric population

The detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. Twenty one fully grown 12-week-old male Sprague-Dawley rats were used in this study. The rats were assigned to one of three groups randomly. One group was selected to be the baseline control group with intact intervertebral discs (n=7). In the other two groups, the vertebrae were immobilized with an external fixation device that fixed four caudal vertebrae (C7-C10) for 4 and 8 weeks, respectively. Four K-wires were fixed in parallel using two aluminum alloy cuboids which do not compress or stretch the target discs. The immobilized discs were harvested and then stained with hematoxylin/eosin, scanned using atomic force microscopy to obtain the modulus at both nano and micro scales, and analyzed the gene expression with real-time quantitative polymerase chain reaction. Significance of differences between the study groups was obtained using a two-way analysis of variance (ANOVA) with Fisher's Partial Least-Squares Difference (PLSD) to analyze the combined influence of immobilization time and scanning region. Statistical significance was set at P≤0.05. Compared to the control group, the inner layer of annulus fibrosus presented significant disorder and hyperplasia after immobilization for 8 weeks, but not in the 4 week group. The fibrils in inner layer showed an alteration in elastic modulus from 91.38±20.19MPa in the intact annulus fibrosus to 110.64±15.58MPa (P<0.001) at the nano scale after immobilization for 8 weeks, while the corresponding modulus at the micro scale also underwent a change from 0.33±0.04MPa to 0.47±0.04MPa (P<0.001). The upregulation of collagen II from 1±0.03 in control to 1.22±0.03 in 8w group (P = 0.003) was induced after immobilization, while other genes expression showed no significant alteration after immobilization for both 4 and 8 weeks compared to the control group (P>0.05). The biomechanical properties at both nano and micro scales altered in different degrees between inner and outer layers in annulus fibrosus after immobilization for different times. Meanwhile, the fibril arrangement disorder and the upregulation of collagen II in annulus fibrosus were observed using hematoxylin/eosin staining and real-time RT-PCR, respectively. These results indicate that immobilization not only influenced the individual collagen fibril at the nano scale, but also suggested alterations of micro-biomechanics and cell response. This work provides a better understanding of IVD degeneration after immobilization and benefits to the clinical treatment related to disc immobilization

With its high wear and corrosion resistance, CoCrMo alloy has been widely used for metal-on-metal total hip replacements (THRs). However, the use of the metal-on-metal implants has dropped substantially as a result of several alerts issued by the Medicines and Healthcare products Regulatory Agency (MHRA) due to concern on metal ion release [1]. However, some of the first generation of metal-on-metal THRs have lasted for more than 20 years [2]. It is far from clear why some MoM joints have survived, while other failed. It is known that dynamic changes occur at the metal surface during articulation. For example, a nanocrystalline layer has been reported on the topmost surface of both in vivo and in vitro CoCrMo THRs [3, 4] but it is not known whether this layer is beneficial or detrimental. The current work focuses on the sub-surface damage evolution of explanted MoM hips, which is compared to in vitro tested CoCrMo hip prostheses. Site-specific TEM cross-section of both in vivo and in vitro CoCrMo samples were prepared by focused ion beam (FIB) in situ lift-out method (Quanta 200 3D with Omniprobe, FEI, the Netherlands). TEM of the FIB specimens was performed on various microscopes. Routine bright field imaging was performed on a Tecnai 20 (FEI, the Netherland) operating at 200 kV, while high resolution transmission electron microscopy (HRTEM) of the nanocrystalline layer and other surface species was undertaken on a Jeol 2010F (Jeol, Japan) operating at 200 kV. A nanocrystalline layer (which was not present on the starting surfaces) was observed on both explanted in vivo and in vitro tested materials. For the explanted joints, the nanocrystalline layer was thin (a few 100 nm) and the extent did not appear to correlate with the local wear rate. For in vitro samples, the nanocrystalline layer was thicker (up to micron). HRTEM from this layer are shown in Fig. 1 and Fig. 2. The nanocrystallite size was ∼5 nm and appeared to be a mixture of face centred cubic and hexagonal close packed phases. The formation of the nanocrystalline layer and its correlation with wear behaviour are discussed

Aim. Staphylococcus aureus (SA) can cause various infections and is associated with high morbidity and mortality rates of up to 40%. Antibiotic treatment often fails to eradicate SA infections even if the causative strain has been tested susceptible in vitro. The mechanisms leading to this persistence is still largely unknown. In our work, we to reveal SA interactions with host cells that allow SA to persist at the site of infection. Method. We established a sampling workflow to receive tissue samples from patients requiring surgical debridement due to SA bone-and joint or soft-tissue infections. We developed a multiplex immunofluorescent staining protocol which allowed us to stain for SA, leukocytes, neutrophils, macrophages, B-cells, T-cells, DAPI and cytoplasmatic marker on the same sample slide. Further, distance of SA to cell nuclei was measured. Interaction of immune cells and SA on a single cell level was investigated with high-resolution 3D microscopy. We then validated our findings applying fluorescence-activated cell sorting (FACS) on digested patient samples. Finally, we aimed to reproduce our ex vivo patient results in an in vitro co-culture model of primary macrophages and clinical SA strains, where we used live cell microscopy and high-resolution microscopy to visualize SA-immune cell interactions and a gentamicin protection assay to assess viability of SA. Results. Here, we revealed that CD68+ macrophages were the immune cells closest to SA with a mean distance of 56μm (SD=36.4μm). Counting the amount of SA, we found in total >7000 single SA in nine patients. Two-thirds of SA were located intracellularly. Two-thirds of the affected immune cells with intracellular SA were macrophages. The distribution of intra- to extracellular SA was independent of ongoing antibiotic therapy and underlying infection type. FACS confirmed these findings. In our co-culture model, intracellular SA remained alive for the whole observation period of eight hours and resided in RAB5+ early phagosomes. Conclusions. Our study suggests an essential role of intracellular survival in macrophages in SA infections. These findings may have major implication for future treatment strategies

Aim. Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria. Method. 19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples. Results. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infectious samples as a common finding in both cell types. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number could be found as a sign for increased mitochondrial fission. Conclusions. The results show that mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This suggests that manipulation of mitochondrial dynamics in a way of inhibiting mitochondrial fission may improve bone cell survival and exploit bone cells regenerative potential to aid in the treatment of osteomyelitis

Aim. Multispecies biofilms are associated with difficult periprosthetic joint infections (PJI), particularly if they have different antibiotic sensitivities. We aimed to determine if we could generate and kill a multispecies biofilm consisting of a Gram negative and Gram positive pathogen in-vitro with antibiotic loaded calcium sulfate beads containing single or combination antibiotics. Methods. To establish whether we could co-culture mixed species biofilms various combinations of Pseudomonas aeruginosa (PA), Enterococcus faecalis (EF), Staphylococcus aureus (SA) and Enterobacter faecalis (EF) were grown together on 316L stainless steel coupons and agar plates. Based on this screen we focused on PA + EF and challenged them with high purity calcium sulfate beads (Stimulan Rapid Cure) loaded with vancomycin (V), alone tobramycin (T) alone or vancomycin and tobramycin in combination (V+T). Bioluminescence, light imaging, plate count, confocal microscopy and scanning electron microscopy were used to quantify growth. Results. On 316LSS the V loaded bead reduced both EF and PA by approximately 2 logs compared to unloaded control beads. A T alone loaded bead eliminated PA from the dual species biofilm and caused a 2-log reduction in EF. The V+T-beads reduced PA by 9-logs and EF by 8.3 logs. In terms of total CFUs V+T beads reduced the bioburden by 8.4 logs compared to V or T alone. which resulted in 2.1 and 2.6 log reductions respectively. (* P<0.05, *** P<0.001). On agar PA dominated the culture for the unloaded and V loaded beads. However, when challenged with a T loaded bead both species were able to coexist and a zone of killing was generated in both species in the multispecies biofilms. However, this zone was smaller and included more tolerant variants than the zone generated by V+T-loaded beads. Conclusions. There were species proportion differences between biofilms grown on agar and 316LSS demonstrating the importance of growth conditions on species interactions. Antibiotics against strains with differing sensitivities can shift species interactions. High purity calcium sulfate beads containing tobramycin a broad-spectrum Gram positive and negative antibiotic vancomycin, a Gram-positive targeted antibiotic killed a larger percentage of a multispecies in an in-vitro biofilm than either single gram-specific antibiotic alone, demonstrating the advantage of using combination antibiotics for treating multispecies biofilms

Background. Although described as a commensal bacterium with low pathogenicity, Cutibacterium acnes involvement has been reported in many clinical entities: infections associated with devices, such as shoulder prosthetic joint infections, osteosynthesis, breast implants or cerebrospinal fluid shunts. Various studies show that C. acnes grows as a biofilm, contributing to its persistence by allowing its escape from the action of the immune system and antibiotics. Purpose. Our aim was to assess the activity of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) on eight different well-characterized C. acnes strains after growth in biofilm mode. Methods. Eight susceptible strains of C. acnes were selected for this study, including two reference strains (ATCC6919 and ATCC11827) and six clinical strains. All C. acnes strains were studied using two different methods to study the biofilm production at different time points: the BioFilm Ring Test. ®. technique (early stages of adhesion) and the Crystal Violet (CV) method (mature biofilm). In a second step, the impact of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) was studied. For the CV technique, two types of tests were performed: preventive tests (addition of active substances and bacteria at the same time) and curative challenge tests (addition of active substances on a biofilm already formed after 48h). Transmission electron microscopy was performed to investigate the morphology modifications. Results. C. acnes isolates from phylotypes IA. 1. and IA. 2. , seem to produce more mature biofilm in the first stages of adhesion than other phylotypes. Curative assays were performed to evaluate the efficacy of antibiotics and Myrtacine. ®. on mature biofilm. Significant efficacy of Myrtacine. ®. at 0.03% was observed for C. acnes strains. Moreover, the combination of Myrtacine. ®. and doxycycline appears to decrease the total biofilm biomass. The effect of doxycycline as a preventive measure was minimal. On the contrary, a similar use of Myrtacine. ®. as early as 0.001% showed significant efficacy with a significant decrease in total biofilm biomass for all C. acnes strains. Transmission electron microscopy revealed a significantly decreased biofilm growth in treated bacteria with Myrtacine. ®. compared to untreated bacteria. Moreover, the total number of bacteria decreased as the concentration of Myrtacine. ®. increased suggesting also an antimicrobial effect. Conclusion. These results confirm the difference in biofilm producing ability depending on C. acnes phylotypes. These results suggest that Myrtacine. ®. may be a promising alternative antibacterial and anti-biofilm agent like peroxide de benzoyle to prevent shoulder prosthetic joint infection involving planktonic and biofilm C. acnes

Title. Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model. Aim. To assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model. Method. All animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 10. 5. CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours. Results. 1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the mouse femur. Proper placement of all implants were confirmed with in vivo X-rays, and ex vivo photos. We empirically derive the ROI during each imaging session by aggregating the imaged volume which ranges from (636.4um × 636.4um × 151um) = 0.625 +/- 0.014 mm. 3. of bone marrow in a global GFP-transgenic mouse. 2) IV-MLSM imaging acquisition of the “race for the surface”.In vitro MPLSM images of implants partially coated with USA300LAC (RFP-MRSA) were verified by SEM image. Results from IV-MLSM of RFP-MRSA and GFP. +. host cell colonization of the contaminated implants illustrated the mutually exclusive surface coating at 3hrs, which to our knowledge is the first demonstration of “the race for the surface” between bacteria and host cells via intravital microscopy. 3) Quantifying the “race for the surface” with CFU verification of S. aureus on the implant. 3D volumetric rendering of the GFP. +. voxels and RFP+ voxels within the ROI were generated in Imaris. The voxel numbers suggeste that the fight for the surface concludes ∼3hrs post-infection, and then transitions to an aggressive MRSA proliferation phase. The results of WT control demonstrate a significant increase in CFU by 12hrs post-op for both stainless steel (P<0.01) and titanium (P<0.01). Conclusions. We developed IV-MLSM to quantify the “Race for the Surface” between host cells and contaminating S. aureus in a murine femur implant model. This race is remarkably fast, as the implant surface is completely covered with 3hrs, peak bacterial growth on the implant occurs between 2 and 12 hours and is complete by 12hrs

Aims. This study aims to enhance understanding of clinical and radiological consequences and involved mechanisms that led to corrosion of the Precice Stryde (Stryde) intramedullary lengthening nail in the post market surveillance era of the device. Between 2018 and 2021 more than 2,000 Stryde nails have been implanted worldwide. However, the outcome of treatment with the Stryde system is insufficiently reported. Methods. This is a retrospective single-centre study analyzing outcome of 57 consecutive lengthening procedures performed with the Stryde nail at the authors’ institution from February 2019 until November 2020. Macro- and microscopic metallographic analysis of four retrieved nails was conducted. To investigate observed corrosion at telescoping junction, scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDX) were performed. Results. Adjacent to the nail’s telescoping junction, osteolytic changes were observed in bi-planar radiographs of 20/57 segments (35%) after a mean of 9.5 months (95% confidence interval 7.2 to 11.9) after surgery. A total of 8/20 patients with osseous alterations (40%) reported rest and ambulation pain of the lengthened segment during consolidation. So far, 24 Stryde nails were retrieved and in 20 (83%) macroscopic corrosion was observed at the nail’s telescoping junction. Before implant removal 11/20 radiographs (55%) of lengthened segments with these 20 nails revealed osteolysis. Implant retrieval analysis by means of SEM showed pitting and crevice corrosion. EDX detected chromium as the main metallic element of corrosion. Conclusion. Patients are exposed to the risk of implant-related osteolysis of unclear short- and long-term clinical consequences. The authors advocate in favour of an early implant removal after osseous consolidation. Cite this article: Bone Joint Res 2021;10(7):425–436

Aim. Quadrupled hamstring anterior cruciate ligament plasties (4xHp) have been described as having a higher risk of infection than bone patellar tendon bone plasties (BPTBp). There are 2 theories that might explain this phenomenon. One is the presence of sutures in a 4xHp that could act as a foreign body, The other is the more complex preparation of a 4xHp that might lead to higher contamination rates during the process. The objective of the present study was to evaluate the formation of biofilm in these plasties and to compare it between a 4xHp and a BPTBp. The hypothesis was that the presence of sutures in 4xHp would increase the amount of biofilm present in them in comparison to BPTBp. Method. A descriptive in vitro study was conducted. One 4xHp and one BPTBp were prepared. They were subsequently divided into 8 fragments. Three of them were reserved for negative control, and the rest were contaminated with a strain of S. Epidermidis (ATCC 35984) 10–5. Finally, a quantitative analysis was carried out by means of microcalorimetry and sonication with plating. Additionally, a qualitative analysis was carried out by means of electron microscopy. Results. In isothermal microcalorimetry, both contaminated plasties showed the same growth dynamics with a population peak (200uW) at 8h. No significant differences were found between the bacterial growth profiles of 4xHp and BPTBp. The product of sonication was plated and the number of colony forming units per milliliter (CFU/ml) was counted at 24 hours. No significant differences were detected between the 4×Hp (mean +/− sem = 3,5×107 +/− 3450000) and the BPTBp (4,6 ×107 +/− 1,455e+7). With a p value of 0.6667, there were no differences of significance (Mann-Whitney test). In the samples analyzed with electron microscopy, no specific biofilm growth pattern was identified upon comparing BPTBp with 4xHp. Conclusions. There were no significant differences at either the quantitative or qualitative level when comparing bacterial growth in BPTBp and 4xHp. Therefore, the presence of sutures in 4xHp cannot be established as a predisposing factor to higher infection rates. These findings may be justified in the sense that the plasties themselves already behave like foreign bodies. Therefore, the presence of sutures does not increase the possibility of biofilm forming on their surface

Aim. Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella. Method. The bacteriophages (191219) were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages, and S. aureus EDCC 5055(1×10. 7. CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37. o. C and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analysing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well plate. The eradication of biofilm was analysed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts at 2h, 4h and 24h time points of post treatment. In addition, we have analysed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Finally, experiments are performed to prove the effect of bacteriophages to clear in vivo biofilm using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis. Results. Our results demonstrate the in vitro efficacy of bacteriophages against planktonic S. aureus. Transmission electron microscopy (TEM) experiments revealed severe infection of bacteria by bacteriophages. Bacteriophages also eradicated in a dose-dependent manner in vitro S. aureus biofilm formation and were active against intracellular S. aureus in an osteoblastic cell line. TEM analysis visualized the effect of the bacteriophages on S. aureus inside the osteoblasts with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria infection model, single administration of phages failed to show improvement in survival rates, but exhibited some synergistic effects with gentamicin or rifampicin, which was not statistically significant. Conclusions. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections. Further preclinical and clinical trials are required to establish adequate treatment protocols

Introduction. Introduction: Pin site infection is a common complication during treatment with a circular frame external fixator and increases time and support patients require from the limb reconstruction team. Wound swabs were not routinely sent by the clinical nurse specialists prior to this study, with most pin site infections treated as Staphylococcus aureus with flucloxacillin (clindamycin in penicillin allergy). The aim of this study was to ascertain whether routine sending of wound swabs in pin site infection would change antibiotic treatment. Materials & Methods. Materials and Method: Patients presenting at clinic or physiotherapy with clinical signs of pin site infection were assessed using the Maz Oxford Nuffield (MON) Pin Site Infection Grading System© (OUH, 2021). Antibiotics were commenced as per unit guidelines and swabs sent for microscopy, culture and sensitivity. Results. Results: There were forty patients treated with pin site infections. Swab results showed S. aureus in 60% (N=24), other organisms in 15% (N=6) and no growth or mixed skin organisms in 10% (N=10). Flucloxacillin was prescribed to 77.5% (n=31) of patients with the remaining 22.5% (n=9) of patients receiving clindamycin (due to allergy) or rifampicin and ciprofloxacin (due to a previous MRSA diagnosis). Antibiotic management was subsequently changed in only 5% (n=2) of patients due to one patient with a new MRSA diagnosis and one patient growing Enterobacter in three pin sites tested. Conclusions. Conclusion: This study has shown that routine swab testing of pin sites does not usually change antibiotic management but should still be used in multiple, severe or unresolving pin site infections

Aim. A novel anti-infective biopolymer implant coating was developed to prevent bacterial biofilm formation and allow on-demand burst release of anti-infective silver (Ag) into the surrounding of the implant at any time after surgery via focused high-energy extracorporeal shock waves (fhESW). Method. A semi-crystalline Poly-L-lactic acid (PLLA) was loaded with homogeneously dissolved silver (Ag) applied onto Ti6Al4V discs. A fibroblast WST-1 assay was performed to ensure adequate biocompatibility of the Ag concentration at 6%. The prevention of early biofilm formation was investigated in a biofilm model with Staphylococcus epidermidis RP62A after incubation for 24 hours via quantitative bacteriology. In addition, the effect of released Ag after fhESW (Storz DUOLITH SD1: 4000 impulses, 1,24 mJ/mm. 2. , 3Hz, 162J) was assessed via optical density of bacterial cultures (Escherichia coli TG1, Staphylococcus epidermidis RP62A, Staphylococcus aureus 6850) and compared to an established electroplated silver coating. The amount of released Ag after the application of different intensities of fhESW was measured and compared to a control group without fhESW via graphite furnace atomic absorption spectrometry (GF-AAS), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Results. The coating with 6% Ag reduced Staphylococcus epidermidis biofilm formation by 99.7% (mean±SD: 2.1×10^5 ± 3,9×10^5 CFU/µL) compared to uncoated controls (6.8×10^7 ± 4.9×10^7 CFU/µL); (p=0.0001). After applying fhESW the commercially available electroplated silver coating did not prevent the growth of all tested bacterial strains. Bacterial growth is delayed with 4% Ag and completely inhibited with 6% Ag in the novel coating, except for a small increase of S. aureus after 17 hours. SEM and EDS confirmed a local disruption of the coating after fhESW. Conclusions. This novel anti-infective implant coating has the potential to prevent bacterial biofilm formation. The on-demand burst release of silver via fhESW could be an adjunctive in the treatment of implant related infection and is of particular interest in the concept of single stage revision surgery

Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. To date, there are no animal models that can fully recapitulate how a biofilm is challenged in vivo in the setting of GN-PJI. The purpose of this study is to establish a clinically representative GN-PJI in vivo model that can reliably depict biofilm formation on titanium implant surface. We hypothesized that the biofilm formation on the implant surface would affect the ability of the implant to be osseointegrated. The model was developed using a 3D-printed, medical-grade titanium (Ti-6Al-4V), monoblock, cementless hemiarthroplasty hip implant. This implant was used to replace the femoral head of a Sprague-Dawley rat using a posterior surgical approach. To induce PJI, two bioluminescent Pseudomonas aeruginosa (PA) strains were utilized: a reference strain (PA14-lux) and a mutant strain that is defective in biofilm formation (DflgK-lux). PJI development and biofilm formation was quantitatively assessed in vivo using the in vivo imaging system (IVIS), and in vitro using the viable colony count of the bacterial load on implant surface. Magnetic Resonance Imaging (MRI) was acquired to assess the involvement of periprosthetic tissue in vivo, and the field emission scanning electron microscopy (FE-SEM) of the explanted implants was used to visualize the biofilm formation at the bone-implant interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration using microCT scans of the extracted femurs with retained implants in vitro, and indirectly assessed by identifying the gait pattern changes using DigiGaitTM system in vivo. A localized prosthetic infection was reliably established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14 and DflgK. This difference in the ability to persist in the model between the two strains was reflected on the gait pattern and implant osseointegration. We developed a novel uncemented hip hemiarthroplasty GN-PJI rat model. This model is clinically representative since animals can bear weight on the implant. PJI was detected by various modalities. In addition, biofilm formation correlated with implant function and stability. In conclusion, the proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapetics

Aim. The use of medical devices has grown significantly over the last decades, and has become a major part of modern medicine and our daily life. Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. For still not well understood reasons, the presence of a foreign body strongly increases susceptibility to infection. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. Formation of biofilms on the biomaterial surface is generally considered the main reason for these persistent infections, although bacteria may also enter the surrounding tissue and become internalized within host cells. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs). Method. Medical grade titanium implants (10×4×1 mm) were dip-coated in a solution of 10% (w/v) hyperbranched polymer, subsequently in a solution of 30% (w/v) polyethyleneimine and 10 mM sodium iodide, using a dip-coater, followed by a washing step for 10 min in ethanol. The QAC-coating was characterized using water contact angle measurements, scanning electron microscopy, FTIR, AFM and XPS. The antimicrobial activity of the coating was evaluated against S. aureus strain JAR060131 and S. epidermidis strain ATCC 12228 using the JIS Z 2801:2000 surface microbicidal assay. Lastly, we assessed the in vivo antimicrobial activity in a mouse subcutaneous implant infection model with S. aureus administered locally on the QAC-coated implants prior to implantation to mimic contamination during surgery. Results. Detailed material characterization of the titanium samples showed the presence of a homogenous and stable coating layer at the titanium surface. Moreover, the coating successfully killed S. aureus and S. epidermidis in vitro. The QAC-coating strongly reduced S. aureus colonization of the implant surface as well as of the surrounding tissue, with no apparent macroscopic signs of toxicity or inflammation in the peri-implant tissue at 1 and 4 days after implantation. Conclusions. An antimicrobial coating with stable quaternary ammonium compounds on titanium has been developed which holds promise to prevent BAI. Non-antibiotic-based antimicrobial coatings have great significance in guiding the design of novel antimicrobial coatings in the present, post-antibiotic era

Aim. Prosthetic joint infections pose a major clinical challenge. Developing novel material surface technologies for orthopedic implants that prevent bacterial adhesion and biofilm formation is essential. Antimicrobial coatings applicable to articulating implant surfaces are limited, due to the articulation mechanics inducing wear, coating degradation, and toxic particle release. Noble metals are known for their antimicrobial activity and high mechanical strength and could be a viable coating alternative for orthopaedic implants [1]. In this study, the potential of thin platinum-based metal alloy coatings was developed, characterized, and tested on cytotoxicity and antibacterial properties. Method. Three platinum-based metal alloy coatings were sputter-coated on medical-grade polished titanium discs. The coatings were characterized using optical topography and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Ion release was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). Cytotoxicity was tested according to ISO10993-5 using mouse fibroblasts (cell lines L929 and 3T3). Antibacterial surface activity, bacterial adhesion, bacterial proliferation, and biofilm formation were tested with gram-positive Staphylococcus aureus ATCC 25923 and gram-negative Escherichia coli ATCC 25922. Colony forming unit (CFU) counts, live-dead fluorescence staining, and SEM-EDS images were used to assess antibacterial activity. Results. Three different platinum-based metal alloys consisting of platinum-iridium, platinum-copper, and platinum-zirconium. The coatings were found 80 nm thick, smooth (roughness average < 60 nm), and non-toxic. The platinum-copper coating showed a CFU reduction larger than one logarithm in adherent bacteria compared to uncoated titanium. The other coatings showed a smaller reduction. This data was confirmed by SEM and live-dead fluorescence images, and accordingly, ICP-OES measurements showed low levels of metal ion release from the coatings. Conclusions. The platinum-copper coating showed low anti-adhesion properties, even with extremely low metal ions released. These platinum-based metal alloy coatings cannot be classified as antimicrobial yet. Further optimization of the coating composition to induce a higher ion release based on the galvanic principle is required and copper looks most promising as the antimicrobial compound of choice. Acknowledgments. This publication is supported by the DARTBAC project (with project number NWA.1292.19.354) of the research program NWA-ORC which is (partly) financed by the Dutch Research Council (NWO); and the AMBITION project (with project number NSP20–1-302), co-funded by the PPP Allowance made available by Health-Holland, Top Sector Life Sciences & Health to ReumaNederland

Aim. There is growing evidence that bacteria encountered in periprosthetic joint infections (PJI) form surface-attached biofilms on prostheses, as well as biofilm aggregates embedded in synovial fluid and tissues. However, models allowing the investigation of these biofilms and the assessment of their antimicrobial susceptibility in physiologically relevant conditions are currently lacking. To address this, we developed a synthetic synovial fluid (SSF) model and we validated this model in terms of growth, aggregate formation and antimicrobial susceptibility testing, using multiple PJI isolates. Methods. 17 PJI isolates were included, belonging to Staphylococcus aureus, coagulase negative staphylococci, Cutibacterium acnes, Pseudomonas aeruginosa, enterococci, streptococci, Candida species and Enterobacterales. Growth and aggregate formation in SSF, under microaerophilic or anaerobic conditions, were evaluated using light microscopy. The biofilm preventing concentration (BPC) and minimum biofilm inhibitory concentration (MBIC) of relevant antibiotics (doxycyclin, rifampicin and oxacillin) were determined for the staphylococcal strains (n=8). To this end, a high throughput approach was developed, using a fluorescent viability resazurin staining. BPC and MBIC values were compared to the minimum inhibitory concentration (MIC) obtained with conventional methods. Results. The SSF model allowed all isolates to grow well under microaerophilic or anaerobic conditions. When cultured in SSF, all isolates formed biofilm aggregates, varying in size and shape along different species. A susceptibility testing method based on measuring resazurin-derived fluorescence was successfully developed, allowing high throughput determination of the BPC and the MBIC in SSF. For all staphylococci cultured in SSF a reduced susceptibility to the tested antibiotics was observed when compared to susceptibility data obtained in general medium. For rifampicin and doxycyclin the BPC was consistently higher than the MIC (two- to fourfold dilution difference for rifampicin and four- to sixfold dilution difference for doxycyclin). For oxacillin the MIC equaled the BPC for two isolates, while for the other isolates the BPC was higher than the MIC (two- to fourfold dilution difference). Expectedly, the MBIC was higher than the BPC and differences with the MIC were even more pronounced for all antibiotics tested (differences of six- to fourteenfold dilutions were observed). Conclusion. Our data indicate that the in vitro SSF model could provide more insight in how PJI-related pathogens form biofilms in physiologically relevant conditions. The BPC and MBIC were consistently and substantially higher than MIC. This model could be a valuable addition to evaluate the antimicrobial susceptibility in biofilms in a PJI context. Sources of funding: FWO-Vlaanderen (grant G066523N)

Aim. Polypropylene (PPE) synthetic mesh is increasingly used in knee arthroplasty surgery to salvage a disrupted extensor mechanism. Despite its clinical success, it is associated with a high rate of periprosthetic joint infection (PJI), which is hypothesized to be caused by bacterial biofilm. The purpose of the current study is to describe the progression of PPE-based biofilm formation over time and to determine if intraoperative antiseptic solutions could be used to effectively remove biofilm when treating PJI. Method. Commercially available knotted monofilament PPE mesh. 1. was cut into 10mm circular shape, immersed in tryptic soy broth (TSB) with methicillin-sensitive staphylococcus aureus and cultured individually in 48-well plates for 10 days to elucidate the biofilm grown on mesh over time. At every 24 hours, a triplicate of samples was retrieved and biofilm on the mesh was dislodged by sonicating at 52 kHz for 15 minutes and quantified by counting colony-forming units (CFUs) after overnight growth. The biofilm growth was also verified using scanning electron microscopy. The effect of saline and antiseptic solutions was verified by exposing 1) 0.05% chlorohexidine gluconate. 2. , 2) acetic acid-based mixture. 3. , 3) diluted povidone-iodine (0.35%), 4) undiluted povidone-iodine (10%). 4. , and 5) 1:1 combination of 10% povidone-iodine & 3% hydrogen peroxide on immature and mature biofilms for 3 minutes, created by culturing with bacteria for 24 hours and 72 hours respectively. All experiments were performed in quintuples and repeated. Antiseptic treatments that produced a three-log reduction in CFU counts compared to controls were considered clinically significant. Results. PPE-mesh produced reliable CFU counts at 24 hours and reached peak growth at 72 hours. For immature biofilm, all formulations of povidone-iodine produced significant reductions in CFU counts compared to controls. Although not meeting the established threshold, saline irrigation removed 86.5% of CFUs, while formulation based on chlorohexidine and acetic acid removed 99.2% and 99.7% respectively. For mature biofilm, formulations based on povidone-iodine and acetic acid produced significant reductions in CFU counts. Conclusions. Our findings suggest biofilm may form on mesh as early as 24 hours after bacterial exposure. Povidone-iodine formulations were consistently the most effective in removing biofilm on mesh surfaces. We recommend that surgeons consider using an antiseptic solution, preferably povidone-iodine-based, in addition to regular saline lavage when attempting to salvage a PPE mesh in the setting of PJI. 1. Marlex mesh (CR Bard, Davol Inc, Warwick, RI), . 2. Irrisept (Irrimax Corp, Gainesville, FL), . 3. Bactisure (Zimmer-Biomet, Warsaw, IN), . 4. Aplicare (Inc, Meriden, CT)

Aim. To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5–15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum. Method. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5–15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry. In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 10. 4. CFU S. aureus (S54F9), Group B: seven days old 5–15 µm aggregate-inoculum; 10. 4. CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed. Results. The separation of aggregates into different size fractions was done successfully by the filtering method. Isothermal microcalorimetry showed, a delayed Time-to-peak metabolic activity of the aggregate-inoculum compared to the planktonic inoculum. S. aureus was isolated from subcutis, bone and implants from all animals in groups A and B. Both group A animals showed osteomyelitis at gross inspection with suppuration and sequestration, while groups B and C animals had no macroscopic lesions. From CT scans, both group A animals also showed positive signs of osteomyelitis, i.e., osteolysis, while only one animal in group B did, and none in group C. Histopathological examination of the bones showed more extensive inflammation in group A animals compared to those in group B, which showed more osteoid formation. Conclusions. Formation and separation of low metabolism bacterial aggregates into different size fractions was possible. The aggregates can be used as inoculum in the porcine IAO model, with microbiological re-isolation from both implants and tissue. Furthermore, the aggregates caused a less aggressive IAO, than the planktonic counterparts. Using aggregated bacteria as inoculum appears to be more relevant to the clinical situation of infecting bacteria

Aim. Antibacterial activity of coatings based on metal and metal oxide nanoparticles (NPs) often depends on materials and biotic targets resulting in a material-specific killing activity of selected Gram-positive and Gram-negative bacteria, including drug-resistant strains. In this perspective, the NPs loading amount, the relative elemental concentration inside the nanogranular building blocks and the deposition method are of paramount importance when the goal is to widen the antimicrobial spectrum, but at the same time to avoid high levels of metal content to limit undesired toxic effects. Aim of the present study was evaluation of the antimicrobial properties of two multielement nanogranular coatings composed of Titanium-Silver and Copper and of Magnesium-Silver and Copper. Method. Ti-Ag-Cu and Mg-Ag-Cu NPs were deposited on circular cover glasses (VWR) by Supersonic Cluster Beam Deposition. Biofilm-producer strains of Staphylococcus aureus (methicillin susceptible and resistant), Staphylococcus epidermidis (methicillin susceptible and resistant), Escherichia coli (fully susceptible and producer of extended spectrum beta lactamases), and Pseudomonas aeruginosa (susceptible and multidrug-resistant) were selected. The abilities of the selected strains to adhere, colonize and produce biofilm on the discs coated with Ti-Ag-Cu or Mg-Ag-Cu NPs were compared to uncoated circular cover glasses which were used as growth control. Cytotoxicity was also evaluated in order to assess the biocompatibility of the newly synthesized NPs. Results. In comparison to uncoated controls, both coatings showed significant anti-adhesive properties against S. aureus, S. epidermidis, and E. coli. Reduction in adhesion to Mg-Ag-Cu coated discs was observed also for P. aeruginosa isolates, although differences vs uncoated controls did not reach statistical significance. Biofilm formation was reduced on discs coated with Mg-Ag-Cu compared to Ti-Ag-Cu and, again, coatings had a milder effect on P. aeruginosa, probably due to its exceptional capability of attachment and matrix production. These results were confirmed by the evaluation of bacterial colonization on nanoparticles-coated discs by means of confocal laser scanning microscopy. A viability of 95.8% and 89.4% of cells cultured in the presence of Ti-Ag-Cu and Mg-Ag-Cu discs, respectively, when compared to negative controls was observed, thus excluding cytotoxic effects on eukaryotic cells. Conclusions. The newly synthesized Ti-Ag-Cu and Mg-Ag-Cu coatings are able to limit bacterial adhesion colonization and biofilm production, thus highlighting the safe use of multi-element families of NPs as new strategies against bacterial attachment to the surface of biomedical implants. However, further studies addressing activity against P. aeruginosa and including a wide number of isolates are warranted