Aims. Biofilm infections are among the most challenging complications in orthopaedics, as bacteria within the biofilms are protected from the host immune system and many antibiotics. Halicin exhibits broad-spectrum activity against many planktonic bacteria, and previous studies have demonstrated that halicin is also effective against Staphylococcus aureus biofilms grown on polystyrene or polypropylene substrates. However, the effectiveness of many antibiotics can be substantially altered depending on which orthopaedically relevant substrates the biofilms grow. This study, therefore, evaluated the activity of halicin against less mature and more mature S. aureus biofilms grown on titanium alloy, cobalt-chrome, ultra-high molecular weight polyethylene (UHMWPE), devitalized muscle, or devitalized bone. Methods. S. aureus-Xen36 biofilms were grown on the various substrates for 24 hours or seven days. Biofilms were incubated with various concentrations of halicin or vancomycin and then allowed to recover without antibiotics. Minimal biofilm eradication concentrations (MBECs) were defined by CFU counting and resazurin reduction assays, and were compared with the planktonic minimal inhibitory concentrations (MICs). Results. Halicin continued to exert significantly (p < 0.01) more antibacterial activity against biofilms grown on all tested orthopaedically relevant substrates than vancomycin, an antibiotic known to be affected by biofilm maturity. For example, halicin MBECs against both less mature and more mature biofilms were ten-fold to 40-fold higher than its MIC. In contrast, vancomycin MBECs against the less mature biofilms were 50-fold to 200-fold higher than its MIC, and 100-fold to 400-fold higher against the more mature biofilms. Conclusion. Halicin is a promising antibiotic that should be tested in animal models of orthopaedic infection. Cite this article: Bone Joint Res 2024;13(3):101–109

Aims. Electromagnetic induction heating has demonstrated in vitro antibacterial efficacy over biofilms on metallic biomaterials, although no in vivo studies have been published. Assessment of side effects, including thermal necrosis of adjacent tissue, would determine transferability into clinical practice. Our goal was to assess bone necrosis and antibacterial efficacy of induction heating on biofilm-infected implants in an in vivo setting. Methods. Titanium-aluminium-vanadium (Ti6Al4V) screws were implanted in medial condyle of New Zealand giant rabbit knee. Study intervention consisted of induction heating of the screw head up to 70°C for 3.5 minutes after implantation using a portable device. Both knees were implanted, and induction heating was applied unilaterally keeping contralateral knee as paired control. Sterile screws were implanted in six rabbits, while the other six received screws coated with Staphylococcus aureus biofilm. Sacrifice and sample collection were performed 24, 48, or 96 hours postoperatively. Retrieved screws were sonicated, and adhered bacteria were estimated via drop-plate. Width of bone necrosis in retrieved femora was assessed through microscopic examination. Analysis was performed using non-parametric tests with significance fixed at p ≤ 0.05. Results. The width of necrosis margin in induction heating-treated knees ranged from 0 to 650 μm in the sterile-screw group, and 0 to 517 μm in the biofilm-infected group. No significant differences were found between paired knees. In rabbits implanted with sterile screws, no bacteria were detected. In rabbits implanted with infected screws, a significant bacterial load reduction with median 0.75 Log10 colony-forming units/ml was observed (p = 0.016). Conclusion. Induction heating was not associated with any demonstrable thermal bone necrosis in our rabbit knee model, and might reduce bacterial load in S. aureus biofilms on Ti6Al4V implants. Cite this article: Bone Joint Res 2024;13(12):695–702

Aims. This study investigated vancomycin-microbubbles (Vm-MBs) and meropenem (Mp)-MBs with ultrasound-targeted microbubble destruction (UTMD) to disrupt biofilms and improve bactericidal efficiency, providing a new and promising strategy for the treatment of device-related infections (DRIs). Methods. A film hydration method was used to prepare Vm-MBs and Mp-MBs and examine their characterization. Biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were treated with different groups. Biofilm biomass differences were determined by staining. Thickness and bacterial viability were observed with confocal laser scanning microscope (CLSM). Colony counts were determined by plate-counting. Scanning electron microscopy (SEM) observed bacterial morphology. Results. The Vm-MBs and Mp-MBs met the experimental requirements. The biofilm biomass in the Vm, Vm-MBs, UTMD, and Vm-MBs + UTMD groups was significantly lower than in the control group. MRSA and E. coli biofilms were most notably damaged in the Vm-MBs + UTMD group and Mp-MBs + UTMD group, respectively, with mean 21.55% (SD 0.08) and 19.73% (SD 1.25) remaining in the biofilm biomass. Vm-MBs + UTMD significantly reduced biofilm thickness and bacterial viability (p = 0.005 and p < 0.0001, respectively). Mp-MBs + UTMD could significantly decrease biofilm thickness and bacterial viability (allp < 0.001). Plate-counting method showed that the numbers of MRSA and E. coli bacterial colonies were significantly lower in the Vm-MBs + UTMD group and the Mp, Mp-MBs, UTMD, Mp-MBs + UTMD groups compared to the control group (p = 0.031). SEM showed that the morphology and structure of MRSA and E. coli were significantly damaged in the Vm-MBs + UTMD and Mp-MBs + UTMD groups. Conclusion. Vm-MBs or Mp-MBs combined with UTMD can effectively disrupt biofilms and protectively release antibiotics under ultrasound mediation, significantly reducing bacterial viability and improving the bactericidal effect of antibiotics. Cite this article: Bone Joint Res 2024;13(9):441–451

Aims. Periprosthetic joint infections (PJIs) are among the most devastating complications after joint arthroplasty. There is limited evidence on the efficacy of different antiseptic solutions on reducing biofilm burden. The purpose of the present study was to test the efficacy of different antiseptic solutions against clinically relevant microorganisms in biofilm. Methods. We conducted an in vitro study examining the efficacy of several antiseptic solutions against clinically relevant microorganisms. We tested antiseptic irrigants against nascent (four-hour) and mature (three-day) single-species biofilm created in vitro using a drip-flow reactor model. Results. With regard to irrigant efficacy against biofilms, Povidone-iodine treatment resulted in greater reductions in nascent MRSA biofilms (logarithmic reduction (LR) = 3.12; p < 0.001) compared to other solutions. Bactisure treatment had the greatest reduction of mature Pseudomonas aeruginosa biofilms (LR = 1.94; p = 0.032) and a larger reduction than Vashe or Irrisept for mature Staphylococcus epidermidis biofilms (LR = 2.12; p = 0.025). Pooled data for all biofilms tested resulted in Bactisure and Povidone-iodine with significantly greater reductions compared to Vashe, Prontosan, and Irrisept solutions (p < 0.001). Conclusion. Treatment failure in PJI is often due to failure to clear the biofilm; antiseptics are often used as an adjunct to biofilm clearance. We tested irrigants against clinically relevant microorganisms in biofilm in vitro and showed significant differences in efficacy among the different solutions. Further clinical outcome data is necessary to determine whether these solutions can impact PJI outcome in vivo. Cite this article: Bone Joint J 2021;103-B(5):908–915

Aims. Here we used a mature seven-day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or antibiotics or non-contact induction heating, and which combinations could eradicate S. aureus in mature biofilms. Methods. Mature biofilms of S. aureus (ATCC 29213) were grown on titanium alloy (Ti6Al4V) coupons for seven days and were subjected to the following treatments or their combinations: antibiotics, mechanical cleaning, or heat shock by induction heating of 60°C for one minute. Experiments were repeated at least five times. Results. In the untreated biofilm, growth up to 1.8×10. 11. colony-forming units (CFU)/cm. 2. was observed. Treatment with ciprofloxacin, flucloxacillin, vancomycin, cefuroxime, and amoxicillin all with rifampicin gave 6.0 log, 6.1 log, 1.4 log, 4.8 log, and 3.6 log reduction in CFU/cm. 2. , respectively. Mechanical cleaning alone resulted in 4.9 log reduction and induction heating in 7.3 log reduction. There was an additional effect of ciprofloxacin, flucloxacillin, and induction heating when used in combinations. There was no additional effect for mechanical cleaning. No bacterial growth could be detected after induction heating followed by seven days of ciprofloxacin with rifampicin. Conclusion. Mechanical cleaning, antibiotics, and non-contact induction heating reduced the bacterial load of mature S. aureus biofilms with approximately 5 log or more as a single treatment. The effect of mechanical cleaning on mature S. aureus biofilms was limited when used in combination with antibiotics and/or induction heating. Cite this article: Bone Joint Res 2022;11(9):629–638

Aims. Biofilm formation is intrinsic to prosthetic joint infection (PJI). In the current study, we evaluated the effects of silver-containing hydroxyapatite (Ag-HA) coating and vancomycin (VCM) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation. Methods. Pure titanium discs (Ti discs), Ti discs coated with HA (HA discs), and 3% Ag-HA discs developed using a thermal spraying were inoculated with MRSA suspensions containing a mean in vitro 4.3 (SD 0.8) x 10. 6. or 43.0 (SD 8.4) x 10. 5. colony-forming units (CFUs). Immediately after MRSA inoculation, sterile phosphate-buffered saline or VCM (20 µg/ml) was added, and the discs were incubated for 24 hours at 37°C. Viable cell counting, 3D confocal laser scanning microscopy with Airyscan, and scanning electron microscopy were then performed. HA discs and Ag HA discs were implanted subcutaneously in vivo in the dorsum of rats, and MRSA suspensions containing a mean in vivo 7.2 (SD 0.4) x 10. 6.   or 72.0 (SD 4.2) x 10. 5.   CFUs were inoculated on the discs. VCM was injected subcutaneously daily every 12 hours followed by viable cell counting. Results. Biofilms that formed on HA discs were thicker and larger than those on Ti discs, whereas those on Ag-HA discs were thinner and smaller than those on Ti discs. Viable bacterial counts in vivo revealed that Ag-HA combined with VCM was the most effective treatment. Conclusion. Ag-HA with VCM has a potential synergistic effect in reducing MRSA biofilm formation and can thus be useful for preventing and treating PJI. Cite this article:Bone Joint Res. 2020;9(5):211–218

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

Aim. Prosthetic joint infections (PJI) remain a great challenge in orthopedic surgery with a high mortality rate. It is particularly complicated by biofilms and infections caused by Methicillin-resistant Staphylococcus aureus (MRSA). It concurrently shields bacteria from host immune responses and confers resistance to antibiotics. This study aims to investigate the efficacy of radioimmunotherapy as an innovative therapeutic modality to address the challenges posed by MRSA and its biofilm. Method. We induced specific monoclonal antibodies 4497-IgG1 as carriers, which target wall teichoic acids (WTA) existing on MRSA and its biofilm. Radionuclides actiniumr-225 (. 225. Ac, α-emitter) and lutetium-177 (. 177. Lu, β-emitter) were conjugated with mAbs using DOTA as chelator. Quality control was assessed using thin layer chromatography and immunoreactivity assays. . 225. Ac- and . 177. Lu-labelled 4497-IgG1 were employed to evaluate the susceptibility of MRSA and its biofilm to the radioimmunotherapy in vitro. Planktonic MRSA and biofilms, at concentrations of 10. 8. and 10. 7. CFU/mL, were incubated at 37°C for 60 minutes in PBS containing either . 225. Ac-mAb (0 - 14.8 kBq) or . 177. Lu-mAb (0 - 14.8 MBq). Radiolabelled dunituximab and free radionuclides serve as isotype-matched negative control. The bacterial viability and metabolic activity were subsequently quantified using CFU and XTT assays. Results. The radiochemical purity of the . 225. Ac-mAbs and . 177. Lu-mAbs complex were determined to be 95.4% and 96.16%. Immunoreactivity fractions of them were measured at 81.8% and 80.8%. . 225. Ac-mAbs and . 177. Lu-mAbs exhibited significant and dose-dependent antimicrobial effects on both planktonic MRSA and biofilm. . 225. Ac- and . 177. Lu-4497IgG1 at doses of 7.4 kBq and 7.4 MBq resulted in more than 4-log reduction in bacterial counts. In biofilms, 2-log reduction at the highest . 225. Ac radioactivity of 14,8kBq. The . 177. Lu complex showed a strong dose-dependent effect, with a reduction of up to 4-log. The XTT assay confirmed these findings, showing a decrease in metabolic activity corresponding to a decrease in bacterial counts, and a slight increase in metabolic activity at the lower dose. Conclusions. Our study demonstrates the efficacy of . 225. Ac and . 177. Lu-labelled 4497-IgG1 antibodies in mediating dose-dependent bactericidal effects against planktonic MRSA and biofilms in vitro. This indicates that radioimmunotherapy could be a potential targeted therapeutic strategy against MRSA and its biofilm. Further research in preclinical and clinical settings is warranted to validate and refine these findings on biofilm-associated implant infections

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)

Aims. Induction heating is a noninvasive, nonantibiotic treatment modality that can potentially be used to cause thermal damage to the bacterial biofilm on the metal implant surface. The purpose of this study was to determine the effectiveness of induction heating on killing Staphylococcus epidermidis from biofilm and to determine the possible synergistic effect of induction heating and antibiotics. Methods. S. epidermidis biofilms were grown on titanium alloy (Ti6Al4V) coupons for 24 hours (young biofilm) and seven days (mature biofilm). These coupons with biofilm were heated to temperatures of 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, and 90°C for 3.5 minutes and subsequently exposed to vancomycin and rifampicin at clinically relevant concentrations. Results. For the young biofilm, total eradication was observed at 65°C or higher for 3.5 minutes followed by 24 hours of vancomycin 10 mg/l and rifampicin 1 mg/l. For the mature biofilm, total eradication was observed at 60°C for 3.5 minutes followed by 24 hours of vancomycin 10 mg/l and rifampicin 1 mg/l. Total eradication was also observed at 60°C for 3.5 minutes followed by 24 hours of vancomycin 1 mg/l and rifampicin 1 mg/l followed by another thermal shock of 60°C for 3.5 minutes (two thermal shocks). Conclusion. Induction heating of Ti6Al4V coupons is effective in reducing bacterial load in vitro for S. epidermidis biofilms. Induction heating and antibiotics have a synergistic effect resulting in total eradication of the biofilm at 60°C or higher for clinically relevant concentrations of vancomycin and rifampicin. Cite this article:Bone Joint Res. 2020;9(4):192–199

Aim. In trauma surgery, the development of biomaterial-associated infections (BAI) is one of the most common complications affecting trauma patients, requiring prolonged hospitalization and the intensive use of antibiotics. Following the attachment of bacteria on the surface of the biomaterial, the biofilm-forming bacteria could initiate a chronic implant-related infection. Despite the use of conventional local and systemic antibiotic therapies, persistent biofilms involve various resistance mechanisms that contribute to therapeutic failures. The development of in vivo chronic BAI models to optimize antibiofilm treatments is a major challenge. Indeed, the biofilm pathogenicity and the host response need to be finely regulated, and compatible with the animal lifestyle. Previously, a Galleria mellonella larvae model for the formation of an early-stage biofilm on the surface of a Kirschner (K)-wire was established. In the present study, two models of mature biofilm using clinical Staphylococcus aureus strains were assessed: one related to contaminated K-wires (in vitro biofilm maturation) and the second to hematogenous infections (in vivo biofilm maturation). Rifampicin was used as a standard drug for antibiofilm treatment. Method. In the first model, biofilms were formed following an incubation period (up to 7 days) in the CDC Biofilm Reactor (CBR, BioSurface Technologies). Then, after implantation of the pre-incubated K-wire in the larvae, rifampicin (80 mg/kg) was injected and the survival of the larvae was monitored. In the second model, biofilm formation was achieved after an incubation period (up to 7 days) inside the larvae and then, after removing the K-wires from the host, in vitro rifampicin susceptibility assays were performed (according to EUCAST). Results. The first model indicate that in vitro biofilm maturation affects the bacterial pathogenicity in the host, depending on the S. aureus strain used. Furthermore, the more the biofilm is matured, the more the rifampicin treatment efficiency is compromised. The second model shows that, despite the fast in vivo biofilm formation in the host, the number of bacteria, either attached to the surface of the K-wire surface or in surrounding tissue of the larvae, was not increased over time. Conclusions. Altogether, these results allow the establishment of biofilm models using G. mellonella larvae in order to understand the impact of biofilm maturation on both the bacterial pathogenicity and the efficiency of antibiofilm treatments

Aim. To evaluate the efficiency of pulse lavage combined with electrical fields to remove biofilm from a metallic surface. Method. Using a 12-well culture plate designed for the application of electrical fields, strains of S. epidermidis were incubated at each well for 24 hours at 37ºC. After incubation, supernatant culture medium was removed, and each well was filled with 3ml of normal saline. Six different models were compared: a) control, b) low-pressure pulse lavage, c) high-pressure pulse lavage, d) pulsed electrical fields, e) low-pressure pulse lavage in combination with pulsed electrical fields, and f) high-pressure pulse lavage in combination with pulsed electrical fields. In all cases, exposure time was set to 25 seconds. In the electrical field models, 50 pulses were applied. After exposure, each bottom electrode was scraped carefully to release adhered bacteria. Subsequently, different dilutions of biofilm removed were spread onto Müller Hinton agar plates and incubated for 24h at 37 ºC, and colony-forming units (CFU) per milliliters were counted. Bacterial counts were then compared to the control model. Results. High-pressure pulse lavage combined with pulsed electrical fields showed the greatest biofilm removal with reductions of up to 11.9 logarithms when compared to the control group. The lowest reduction was achieved by low-pressure pulsed lavage (4.7 logs). All reductions showed statistically significant differences. Conclusion. The results of our comparative study between different models demonstrates high reduction rates for biofilm removal. Further in vivo studies are needed to evaluate the capacity of the combination of high-pressure pulse lavage with pulsed electrical fields in removing bacterial biofilm in real conditions

Prosthetic joint infection (PJI) is an important cause of arthroplasty failure. There is no method to disclose the presence or map the distribution of the in vivo biofilm on infected arthroplasty despite the recognition that such a tool would aid intraoperative decision making and improve novel implant design. The aim of this study was to test the efficacy of four dyes to disclose bacterial biofilm in an in vitro setting. Four dyes with known affinity to bacterial biofilm were assessed to determine their efficacy to disclose biofilms in an in vitro model of PJI. Three dyes (Methylene Blue, Indocyanine Green and Rose Bengal) have established clinical utility and the other, Thioflavin T, is known to fluoresce in the presence of amyloid a known biofilm constituent. The efficacy of the dyes to discriminate between biofilms of different mass and vitality (high, low or the non-inoculated control) was determined after three minutes exposure of the biofilm to the dyes by calculating the amount of dye bound to the biofilm via sonication and spectrophotometry, quantification of the dye through standardised photographic imaging of the stained biofilm and the calculation of inter-observer agreement. Each experiment was performed in triplicate for each dye and repeated three times. For each of the disclosure dyes assessed there was significant difference demonstrated between the amount of dye bound to the high and low mass biofilms (p<0.05) as well as in the amount of dye quantified in photographic and fluorescent image assessment between biofilms of differing mass (p<0.01). There was excellent agreement between three observers, for each disclosure dye, in determining the biofilm mass of each stained disc (Kappa>0.91). This study demonstrates the efficacy of biofilm disclosure dyes in an in vitro PJI model which could one day be used to disclose and map the clinical biofilm in vivo

Aim. Periprosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty. While research has focused on developing better tests for disease diagnosis, treatment options have stayed relatively constant over the years with high failure rates ranging from 30%–50% and are due in part to the protective biofilm produced by some bacterial species. Current treatment options are compromised by the presence of biofilm, emphasizing the need for novel treatment strategies to be developed. Our group has developed a novel treatment (PhotothermAA) which has demonstrated in vitro its ability to target bacterial biofilm. The purpose of this study was to test this PhotothermAA technology in vivo in a rabbit model of PJI for its efficacy in eradicating biofilm. Method. Rabbits were fitted with a titanium implant into the tibial plateau and inoculated with 5×10. 6. CFU Xen36 (luminescent Staphylococcus aureus). At two weeks, rabbits underwent irrigation and debridement and treatment with PhotothermAA gel for two hours and subsequently laser heated using an 808 nm laser for 10 minutes. Gel was washed out and implant was removed for quantitative biofilm coverage analysis via scanning electron microscopy (SEM, n=3 for control and n=2 for PhotothermAA treated). Periprosthetic tissue was collected before and after treatment for toxicity studies via hemotoxylin and eosin (H&E) staining and scored for necrosis by three blinded reviewers (n=5 per group). Student's t-test was used for statistical analysis. Results. Implants isolated after PhotothermAA gel treatment had less biofilm coverage on the surface of the implant compared to non-treated control via SEM analysis (36.9% vs. 55.2%, p<0.14). PhotothermAA gel treatment and subsequent laser treatment was not harmful to surrounding tissue as no increase in necrotic tissue was observed. Conclusions. PhotothermAA gel and laser treatment safely decreases biofilm coverage on infected knee implants in a rabbit PJI model

Aim. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules involve in biofilm. Due to ethical restrictions, the use mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections. This model organism is easy to handle, cheap and ethical restriction free and could be used for the high through put screening of antimicrobial compounds to treat biofilm. To promote the use of this model in basic research we aimed to validate this based on the typical biofilm features such as less susceptible to the antibiotics, complexity of the biofilm structure and gene expression profile of biofilms. Method. G. mellonella larvae are maintained at 30oC on artificial diet in an incubator. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. After sterilization with 100% alcohol, these K-wires were pre-incubated in S. aureus bacterial suspension (5×10. 6. CFU/ml) for 30 min, washed in PBS and implanted inside the larva after with help of scalpel. The larvae were incubated at 37. o. C for two day for the survival analysis. To analyze the less susceptibility of the biofilms towards antibiotics, the larvae were treated with gentamicin and compared survival with planktonic infection in G. mellonella. To reveal the complex structure of biofilm, the implants were removed and processed for the MALDI analysis. Whole genome-based transcriptome of biofilm was performed to explore the changes in transcriptional landscapes. Results. The results are very promising to validate the use of G. mellonella as in vivo model to study the biofilm formation on implanted materials. The gentamicin treatment could rescue the larvae from the planktonic infection, but not from the biofilm infection on the implants. Further, the MALDI analysis could reveal the complex structure and components of S. aureus biofilm formed on the implant inside the larvae. Finally, the transcriptomic analysis revealed the gene expression changes that can be compared to normal biofilm expression profile. Conclusions. Further, comparison of these results with other in vivo models such as rat and mouse as well as acute and chronic clinical samples from patients with implant-associated bone infections could validate and relevant use of this model to study S. aureus biofilm infections

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

Aim. The efficacy of various irrigation solutions in removing microbial contamination of a surgical wound and reducing the rate of subsequent surgical site infection (SSI), has been demonstrated extensively. However, it is not known if irrigation solutions have any activity against established biofilm. This issue is pertinent as successful management of patients with periprosthetic joint infection (PJI) includes the ability to remove biofilm established on the surface of implants and necrotic tissues. The purpose of this study was to evaluate the efficacy of various irrigation solutions in eradicating established biofilm, as opposed to planktonic bacteria, in a validated in vitro model. Method. Established biofilms of Staphylococcus aureus and Escherichia coli were exposed to different irrigation solutions that included Polymyxin 500,000U/L plus bacitracin 50,000U/L, Vancomycin 1g/L, Gentamicin 80mg/L, Normal saline 0.9%, off-the-shelf Betadine 0.3%, Chlorhexidine 0.05%, Benzalkonium 1.3g/L, Sodium hypochlorite 0.125%, and Povidone-iodine 0.5%. Each experiment was conducted in a 96-well microtiter plate with a peg lid and standardized per the MBEC assay manufacturer's protocol. Following 2 minutes of solution exposure to the irrigation solution, residual biofilms were recovered by sonication. Outcome measures for antibiofilm efficacy were residual colony forming units (CFU) and optical density (690nm). Experiments were conducted in 24 replicates and the observations recorded by two blinded observers. Statistical analysis involved t-tests with Bonferonni adjustment. Results. Povidone-iodine 0.5%, Betadine 0.3%, Benzalkonium 1.3g/L, and Sodium hypochlorite 0.125% were significantly more efficacious against S.aureus biofilm versus all other solutions (p<0.001). Against E.coli biofilm, Povidone-iodine-0.5%, Benzalkonium-1.3g/L and Sodium hypochlorite-0.125% were also most effective compared to other irrigation solutions (p<0.001). Polymyxin-bacitracin, Gentamicin, Vancomycin, and Saline solutions had minimal activity against both E.coli and S.aureus biofilms (p<0.001). Similar trends were observed using both experimental endpoints (CFU and Turbidity) and both investigators (interrater reliability; r=0.99). Conclusion. This in vitro study observed that topical antibiotic solutions do not have any activity against established biofilms. Irrigations solutions containing adequate amount of povidone-iodine, betadine, sodium hypochlorite, and benzalkonium appear to have activity against established biofilm by gram positive and gram negative organisms. The use of these irrigation solutions may need to be considered in patients with established PJI

Aim. Diagnosis of prosthetic joint infection are often complicated by the presence of biofilm, which hampers bacteria dislodging from the implants, thus affecting sensitivity of cultures. In the last 20 years several studies have evidenced the usefulness of implant sonication to improve microbial recovery from biofilm formed on inert substrates. More recently, treatment of prosthetic joints and tissues with Dithiothreitol, a sulphur compound already used in routine diagnostic workflow for fluidification of respiratory samples, has proved to be not inferior to sonication in microbiological diagnosis of prosthetic joint infections. This study aimed to evaluate if the combination of the two treatments could further improve microbial retrieval from biofilm in an in vitro model. Method. Three isolates of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Eschericha coli and Pseudomonas aeruginosa responsible of prosthetic joint infections were used. They were grown onto 3 titanium discs (20 mm diameter) and incubated in 3 sterile plastic containers with 15 mL of Triptyc Soy Broth. After overnight incubation, not adhered cells were removed and fresh broth was added to each sample. After 48 hours incubation, the exausted broth was removed and one sample was used for sonication, one for treatment with 0,1% (v:v) Dithiothreitol and one treated with Dithiothreitol followed by sonication. Treated fluids were plated on Muller Hinton Agar plates for colony count. One-way ANOVA analysis was performed to evidence statistical differences between treatments. Results. Similar colony counts were observed for the 3 treatments: 10.1± 0.77 log CFU/mL for Dithiothreitol, 10.0 ± 0.75 for sonication and 10.1 ±0.73 for dithiothreitol + sonication. No statistical differences between the 3 treatments were evidenced by ANOVA analysis. Conclusions. Results seems to confirm that treatment with dithiothreitol is equivalent to sonication in recovering bacteria from biofilm grown on inert surface. Combining dithiotreitol treatment with sonication does not significantly improve bacterial recovery in respect to each treatment alone

Aims. Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models. Methods. Literature research was performed on PubMed and Embase databases. Keywords used for search criteria were “bone AND biofilm”. Information on the species of the animal model, bacterial strain, evaluation of biofilm and bone infection, complications, key findings on observations, prevention, and treatment of biofilm were extracted. Results. A total of 43 studies were included. Animal models used included fracture-related infections (ten studies), periprosthetic joint infections (five studies), spinal infections (three studies), other implant-associated infections, and osteomyelitis. The most common bacteria were Staphylococcus species. Biofilm was most often observed with scanning electron microscopy. The natural history of biofilm revealed that the process of bacteria attachment, proliferation, maturation, and dispersal would take 14 days. For systemic mono-antibiotic therapy, only two of six studies using vancomycin reported significant biofilm reduction, and none reported eradication. Ten studies showed that combined systemic and topical antibiotics are needed to achieve higher biofilm reduction or eradication, and the effect is decreased with delayed treatment. Overall, 13 studies showed promising therapeutic potential with surface coating and antibiotic loading techniques. Conclusion. Combined topical and systemic application of antimicrobial agents effectively reduces biofilm at early stages. Future studies with sustained release of antimicrobial and biofilm-dispersing agents tailored to specific pathogens are warranted to achieve biofilm eradication. Cite this article: Bone Joint Res 2022;11(10):700–714

Periprosthetic joint infections (PJI) are challenging complications following arthroplasty. Staphylococci are a frequent cause of PJI and known biofilm producers. Reoperations for PJI of the hip or knee between 2012 and 2015 performed at Sahlgrenska University Hospital were identified. Medical records were reviewed, and clinical parameters recorded for patients whose intraoperative bacterial isolates had been stored at the clinical laboratory. Staphylococcal strains isolated from reoperations due to first-time PJI were characterised by their ability to form biofilms using the microtiter plate test. The study group included 49 patients (70 bacterial strains) from first-time PJI, whereof 24 (49%) patients had recurrent infection. Strong biofilm production was significantly associated with recurrent infection. Patients infected with strong biofilm producers had a five-fold increased risk for recurrent infection. Strong biofilm production was significantly associated with increased antimicrobial resistance and PJI recurrence. This underscores the importance of determining biofilm production and susceptibility as part of routine diagnostics in PJI. Strong staphylococcal biofilm production may have implications on therapeutic choices and suggest more extensive surgery. Furthermore, despite the increased biofilm resistance to rifampicin, results from this study support its use in staphylococcal PJI