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. 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

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. 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 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

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

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. 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

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

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

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

Objectives. Periprosthetic joint infection following joint arthroplasty surgery is one of the most feared complications. The key to successful revision surgery for periprosthetic joint infections, regardless of treatment strategy, is a thorough deep debridement. In an attempt to limit antimicrobial and disinfectant use, there has been increasing interest in the use of acetic acid as an adjunct to debridement in the management of periprosthetic joint infections. However, its effectiveness in the eradication of established biofilms following clinically relevant treatment times has not been established. Using an in vitro biofilm model, this study aimed to establish the minimum biofilm eradication concentration (MBEC) of acetic acid following a clinically relevant treatment time. Materials and Methods. Using a methicillin-sensitive Staphylococcus aureus (MSSA) reference strain and the dissolvable bead assay, biofilms were challenged by 0% to 20% acetic acid (pH 4.7) for ten minutes, 20 minutes, 180 minutes, and 24 hours. Results. The MBEC of acetic acid was found to be: 15%, 11%, 3.2%, and 0.8% following a ten-minute, 20-minute, 180-minute, and 24-hour treatment, respectively. Conclusion. This study found that the MBEC of acetic acid following a 10- or 20-minute treatment time exceeded its safety threshold, making these concentrations unsuitable as a topical debridement adjunct. However, a clinically acceptable concentration (5%) was still found to eliminate 96.1% of biofilm-associated MSSA following a 20-minute treatment time. Cite this article: S. T. J. Tsang, P. J. Gwynne, M. P. Gallagher, A. H. R. W. Simpson. The biofilm eradication activity of acetic acid in the management of periprosthetic joint infection. Bone Joint Res 2018;7:517–523. DOI: 10.1302/2046-3758.78.BJR-2018-0045.R1

Aim. Despite the expanding research focusing on bacterial biofilm formation, specific histochemical biofilm stains have not been developed for light microscopy. Therefore, pathologists are often not aware of the presence of biofilm formation when examining slides for diagnosing bacterial infections, including orthopaedic infections. The aim of the present study was to develop a combined histochemical and immunohistochemical biofilm stain for simultaneous visualization of Staphylococcus aureus bacteria and extracellular matrix in different colours using light microscopy. Methods. Infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with the biofilm forming S. aureus strain S54F9. The infection time was 5 and 15 days, respectively. First, 25 common histochemical protocols were used in order to find stains that could identify extracellular biofilm matrix. Hereafter, the histochemical protocols for Alcian Blue pH3, Luna and Methyl-pyronin green were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. Finally, the three new combined protocols were applied to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. For all combined protocols applied on all types of tissue (porcine and human) the number of double stained bacterial aggregates were counted. On the same sections the percentage of extracellular matrix of representative bacterial aggregates was calculated by image analysis. Results. Simultaneous visualization of bacterial cells and extracellular matrix in different colours was detected in both porcine and human tissue sections with all three combined protocols. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain i.e. if it was Alcian blue pH3 (colouring polysaccharides), Luna or Methyl green-pyronin (both colouring extracellular DNA), respectively. In the porcine models, 10 percent of the bacterial aggregates in a 10× magnification field revealed both the extracellular matrix and bacteria simultaneously in two different colours. For the human case, this was seen in 90 percent of the bacterial aggregates. The percentage of extracellular matrix of representative bacterial aggregates was 60 and 20 percent in the human and porcine tissues, respectively. Conclusions. The amount of S. aureus biofilm extracellular matrix increased with infection time. A combination of histochemical and immunohistochemical staining is a practical method for identification and evaluation of S. aureus biofilm in orthopaedic infections

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

Prosthetic joint infection (PJI) is a serious complication following joint replacement. Antiseptic solutions are often used for intraoperative wound irrigation particularly in cases of revision for PJI. Antiseptic irrigation is intended to eradicate residual bacteria which may be either free floating or in residual biofilm although there is no clear clinical efficacy for its use. Also, reviewing the scientific literature there is discordance in in vitro results where some studies questions antiseptic efficacy whilst others suggest that even at low concentration antiseptic agents are effective at eradicating bacterial biofilms. The aim of this in vitro study was to establish the efficacy of undiluted antiseptic agents at eradication of a typical PJI forming biofilm and determine the importance of an antiseptic neutralisation step in this assessment. Mature Staphylococcus epidermidis biofilms grown on TiAl6V4 discs were submerged in chlorohexidine (CHL) gluconate 4%, povidone-iodine (PI) 10% or phosphate-buffered saline (PBS) control solution. The discs were then rinsed, the biofilm bacteria suspended in solution using sonication and vortexing, and the viable count (CFU/ml) of the bacterial suspensions determined. The rinse/suspension solution was either (a) PBS or (b) Dey-Engley neutralization broth (NB). When PBS was used to rinse/suspend the biofilm a highly significant, 7.5 and 4.1, mean log reduction in biofilm vitality was observed from the control, for CHL 4% and PI 10%, respectively. However, when NB was the rinse/suspension solution the apparent antiseptic biofilm eradication efficacy was replaced with a statistically significant but clinically irrelevant less the one log-reduction in biofilm vitality. Clinical antiseptic agents are ineffective at eradicating S. epidermidis biofilm in an in vitro PJI model and absence of a neutralisation step gives the false impression of efficacy. Antiseptics alone are an ineffective treatment for biofilm related PJI and no substitute for meticulous debridement

Periprosthetic joint infection (PJI) remains one of the most devastating complications that can occur following total joint arthroplasty. Failure rate of standard treatment for PJI is estimated to be around 40% at two years post revision surgery. A major clinical challenge contributing to treatment failure and antibiotics tolerance is the biofilm formation on implant surfaces. Lytic bacteriophages (phages) can target biofilm associated bacteria at localized sites of infection by penetrating and disrupting biofilm matrices; furthermore, phage replication within the biofilm leads to high local concentrations resulting in a powerful therapeutic effect. The aim of this study is to test if phage cocktail has better antimicrobial effect than vancomycin or a single agent phage against biofilm forming MRSA clinical strain Staphylococcus aureus (S. aureus). S. aureus BP043 was utilized in this study. This strain is a PJI clinical isolate, methicillin resistant (MRSA) and biofilm-former. Three lytic phages, namely, 44AHJD, Team1 and P68, known to infect S. aureus, were tested for their efficiency against S. aureus BP043. The ability of the phages to eliminate S. aureus BP043 planktonic or biofilm cultures was tested either as singular phages or as a cocktail of the three phages. Planktonic cells were adjusted to ∼ 1×109 CFU/mL in tryptic soy broth (TSB) and each phage was added alone or as a cocktail at ∼ 1×109 PFU/mL with moi of 1 (a multiplicity of infection). Bacterial growth was assessed by measuring optical densities at 24hr and was compared to the control of S. aureus BP043 with no phage. BP043 biofilms was grown for 24hr on plasma sprayed titanium (Ti-6Al-4V) alloy disc surfaces. Mature biofilms were then treated with one of the three phages or a cocktail of the 3 phages for 24hr at ∼ 1×109 PFU/mL in TSB. Then, biofilms were dislodged, and bacterial survival was assessed by plating on tryptic soy agar plates. Survival in treated biofilms was compared to control biofilm that was exposed only to TSB. Planktonic cells growth in the presence of phage 44AHJD was reduced significantly (p <0.0001) after 24hr compared to the control. The other two phages did not show a similar pattern when used alone. The reduction in growth was more pronounced when the three phages were combined together (p <0.0001, compared to the control, p=0.011 3, 44AHJD alone versus 3 phages). Exposing BP043 biofilm to the phage cocktail resulted in more than three logs (CFU/mL) reduction in bacterial load residing in the biofilm while no effect was detected when either vancomycin or each phage was used solely. We have demonstrated that the usage of lytic phage cocktail contributes to better clearance of planktonic cultures of the S. aureus MRSA isolate. More importantly, viable bacteria in the biofilms that were grown on plasma sprayed titanium discs were reduced by more than 37% when a phage cocktail was used compared to using a single phage or vancomycin. This work is aimed at gathering preclinical evidence for using phage as a new therapeutic avenue to treat PJI

Aim. To compare the performance of sonication and chemical methods (EDTA and DTT) for biofilm removal from artificial surface. Method. In vitro a mature biofilms of Staphylococcus epidermidis (ATCC 35984) and P. aeruginosa ATCC®53278) were grown on porous glass beads for 3 days in inoculated brain heart infusion broth (BHI). After biofilm formation, beads were exposed to 0.9% NaCl (control), sonication (40 kHz, 1 min, 0.2 W/cm. 2. ), EDTA (25 mM/15 min) and DTT (1 g/L/15 min). Quantitative and qualitative biofilm analysis were performed with viable counts (CFU/ml) and microcalorimetry using time to detection (TTD), defined as the time from insertion of the ampoule into the calorimeter until the exponentially rising of heat flow signal exceeded 100 μW, which is inversely proportional to the amount of remaining bacterial biofilm on the beads. All experiments were performed in triplicate. Results. Mean colony counts obtained after treatment S. epidermidis biofilms with EDTA and DTT was similar to those after 0.9% NaCl (control) – 6.3, 6.1 and 6.0 log CFU/mL, respectively. Sonication detected significantly higher CFU counts with 7.5 log (p<0.05). Concordant results were detected with microcalorimetry: significantly less (p<0.05) biofilm after treatment with sonication compared to EDTA and DTT (12 h vs 6h and 6h, respectively). The same results were observed when P. aeruginosa biofilms were treated. Mean colony counts dislodged after treatment with EDTA and DTT was similar to those after 0.9% NaCl (control) – 5.2, 5.3 and 5.0 log CFU/mL, respectively. Sonication detected significantly higher CFU counts with 6.5 log (p<0.05). Microcalorimetry reviled concordant results: significantly less (p<0.05) biofilm after treatment with sonication in comparison with EDTA and DTT (11 h vs 6h and 6h, respectively). Conclusions. Chemical methods showed no difference in biofilm dislodging compared to normal saline. Sonication is superior to chemical methods (DTT or EDTA) for biofilm detection. Sonication may be improved by combination of two or more chemical dislodgement methods

Background. Periprosthetic joint infection (PJI) is a devastating complication and interest exists in finding lower cost alternatives to current management strategies. Current strategies include a two-stage revision with placement of an antibiotic spacer and delayed placement of a new arthroplasty implant. This study aimed to show that biofilm residue can be reliably eradicated on infected implants, safely allowing re-implantation in a spacer. Methods. Strains of Staphylococcus aureus MRSA252 or Staphylococcus epidermidis RP62A were grown on cobalt-chrome discs. For each strain, discs were divided into 5 groups (5 discs each) and exposed to several sterilization and biofilm eradication treatments: (1) autoclave, (2) autoclave + sonication; (3) autoclave + saline scrub; (4) autoclave + 4% chlorhexidine (CHC) scrub; and (5) autoclave + sonication + CHC scrub. Sterilization and biofilm eradication were quantified with crystal violet assays and scanning electron microscopy (SEM). Results. Relative to non-treated controls, autoclaving alone reduced biofilm load by 33.9% and 54.7% for MRSA252 and RP62A strains, respectively. On average, the most effective sterilization and biofilm removal treatment was the combined treatment of autoclaving, sonication and CHC-scrub for MRSA252 (100%) and RP62A (99.8%). High resolution SEM revealed no cells or biofilm for this combined treatment. Conclusions. Using two commonly encountered bacterial strains in PJI, infected cobalt-chrome implants were sterilized and eradicated of residual biofilm with a combination of autoclaving, sonication and CHC scrubbing. This protocol is time efficient, can be done in the OR and provides a basis for reuse of infected implants as articulating spacers in PJI

Aim. Most orthopedic infections are due to the microbial colonization of abiotic surfaces, which evolves into biofilm formation. Within biofilms, persisters constitute a microbial subpopulation of cells characterized by a lower metabolic-activity, being phenotipically tolerant to high concentrations of antibiotics. Due to their extreme tolerance, persisters may cause relapses upon treatment discontinuation, leading to infection recalcitrance hindering the bony tissue regeneration. Using isothermal microcalorimetry (IMC), we aimed to evaluate in vitro the presence of persisters in a methicillin-resistant Staphylococcus aureus (MRSA) biofilm after treatment with high concentrations of vancomycin (VAN) and their ability to revert to a normal-growing phenotype during incubation in fresh medium without antibiotic. Moreover, the ability of daptomycin to eradicate the infection by killing persisters was also investigated. Method. A 24h-old MRSA ATCC 43300 biofilm was exposed to 1024 µg/ml VAN for 24h. Metabolism-related heat of biofilm-embedded cells, either during or after VAN-treatment, was monitored in real-time by IMC for 24 or 48h, respectively. To evaluate the presence of VAN-derived “persisters” after antibiotic treatment, beads were sonicated and detached free-floating bacteria were further challenged with 100xMIC VAN (100 µg/ml) in PBS+1% Cation Adjusted Mueller Hinton Broth (CAMHB).. Suspensions were plated for colony counting. The resumption of persister cells' normal growth was analysed by IMC on dislodged trated cells for 15h in CAMHB. Activity of 16 µg/ml daptomycin was assessed against persister cells by colony counting. Results. When incubated with 1024 µg/ml VAN, MRSA biofilm produced undetectable heat, suggesting a strong reduction of cell viability and/or cellular metabolism. However, the same samples re-inoculated in fresh medium produced a detectable and delayed metabolism-related heat signal, similarly to that generated by persister cells. The following exposure to 100xMIC VAN resulted in neither complete killing nor bacterial growth, strongly supporting the hypothesis of a persistent phenotype. IMC analysis indicated that VAN-treated biofilm cells resumed normal growth with a ∼3h-delay, as compared to the untreated growth control. Daptomycin treatment yielded a complete eradication of persister cells selected after VAN treatment. Conclusions. Hostile environmental conditions (e.g. high antibiotic bactericidal concentrations) select for persister cells in MRSA biofilm after 24h-treatment in vitro. A staggered treatment vancomycin/daptomycin allows complete biofilm eradication. These results support the use in clinical practice of a therapeutic regimen based on the combined use of antibiotics to kill persisters and eradicate MRSA biofilms. IMC represents a suitable technique to detect persisters and characterize in real-time their reversion to a metabolically-active phenotype

Aim. Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin resistant Enterococci (VRE) have emerged as multi-drug resistant Gram-negative pathogens associated with Periprosthetic Joint Infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ABLCB) to inhibit bacterial growth, biofilm formation and eradicate preformed biofilms of K. pneumoniae and E. faecalis. Method. Three strains of K. pneumoniae (carbapenem resistant BAA1705, New Delhi metallo-beta-lactamase producing BAA2146 [NDM-1], a carbapenemase producing BAA2524) and a vancomycin resistant strain of E. faecalis (ATCC51299) were used. 4.8mm diameter ABLCBs (Stimulan Rapid Cure, Biocomposites) were loaded with vancomycin (VAN) & gentamicin (GEN) at 500 and 240 mg/10cc pack or VAN & rifampicin (RIF) at 1000 and 600 mg/10cc pack respectively and placed onto tryptic soy agar (TSA) plates spread with each of the four strains independently and incubated for 24 hours at 37°C. The beads were transferred daily onto fresh TSA medium spread with the test cultures. The zone of inhibition was recorded until no inhibition was observed. Biofilm prevention efficacy was investigated in 6 well plates. Bacterial cells (5×10. 5. CFU/mL in tryptic soy broth) were treated with ABLCBs. Media was removed and challenged with bacteria daily for 7 days. CFU counts were taken after 1, 2, 3 and 7 days. For biofilm killing, ABLCB were added to 3 day formed biofilms in 6 well plates. CFU counts were estimated at 1, 3 and 7 days with daily media exchange. Results. ABLCB demonstrated effective initial eluting concentrations depending on the strains. The NDM-1 strain of K. pneumoniae had lower sensitivity than other strains towards VAN & RIF and resistant towards VAN & GEN. E. faecalis was sensitive to both combinations. For repeat challenges, ABLCBs prevented colonisation and reduced biofilm formation, except for the NDM-1 strain which grew in the presence of VAN & GEN. Preformed biofilms were more difficult to reduce with antibiotics than in the prevention assay. Biofilm growth was observed at 1 week of contact with ABLCBs, despite negative cultures at earlier time points for K. pneumoniae and E. faecalis. However, there was a significant killing (2–3 logs, P<0.05) of biofilm bacteria with all antibiotic combinations compared to unloaded beads. Conclusions. This study provides evidence that local release of antibiotics from ABLCBs may be useful in the treatment of multidrug resistant strains of K. pneumoniae and E. faecalis (CRE and VRE) associated with PJIs. In-vitro results do not necessarily correlate to clinical results

Aim. Staphylococcus aureus and Pseudomonas aeruginosa are ubiquitous pathogens often found together in polymicrobial, biofilm-associated infections. The mixed-species biofilm are significantly more resistant to antimicrobial treatment and are associated with failures. Bacteriophages present a promising alternative to treat biofilm-related infections due to their rapid bactericidal activity on multi-drug resistant bacteria. In this study, we assess the simultaneous or sequential application of phages and ciprofloxacin on the mixed-species biofilm in vitro. Method. Ciprofloxacin was tested alone and in combination with Pyo-bacteriophage cocktail against P.aeurginosa ATCC 27853 and MRSA ATCC 43300 mixed-species biofilm. In order to evaluate the effect of combined treatment on biofilm-embedded cells, mature biofilms were grown on porous glass beads with MRSA (10. 6. CFU/ml) and P.aeruginosa (10. 3. CFU/ml) and incubated for 24h at 37° C in LB broth. The beads were then washed and placed in fresh LB in the presence of sub-eradicating titers/concentrations of phages and ciprofloxacin (corresponding to 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 × MBEC. biofilm. ), respectively, simultaneous or in order (pretreated with phages for 3-6-12-24 hours) at 37°C. In all cases, heat flow produced by the viable cells still embedded in the biofilm was measured for 48 hours by isothermal microcalorimetry. Results. Simultaneous or sequential treatment with pyo-bacteriophage (10. 5. and 10. 6. PFU/ml) and ciprofloxacin, producing a synergistic effect resulting in the complete eradication of the biofilm was evaluated. When sub-eradicating concentrations of ciprofloxacin together with sub-eradicating titers of phages simultaneously used to treat mixed-species biofilm, a delay and/or reduction of heat flow produced by bacteria was observed. The same effect was seen when mix-biofilm was pre-treated with phages for 3 hours and 24 hours, respectively. However, antibiotic introduction after 6 and 12 hours resulted in a high synergistic eradicating effect with pyo-bacteriophage. The concentration of ciprofloxacin decreased dramatically from >512 μg/ml to < 16 μg/ml. Conclusions. While MBEC of ciprofloxacin against mixed-species biofilm of Pseudomonas aeruginosa and Staphylococcus aureus was above drug concentrations reachable in clinical practice, the co-administration with bacteriophage strongly reduced the antibiotic doses needed to eradicate biofilm. There is a specific time delay in antibiotic introduction to reach the eradication of mix-species biofilm. These results have implications for optimal combined treatment approaches

Aim. The primary aim of this in vitro study was to test the efficacy of daptomycin to eradicate staphylococcal biofilms on various orthopedic implant surfaces and materials. The secondary aim was to quantitatively estimate the formation of staphylococcal biofilm on various implant materials with different surface properties. Method. We tested six clinically important biomaterials: cobalt chrome alloy, pure titanium, grid-blasted titanium, porous plasma-coated titanium with/without hydroxyapatite, and polyethylene. Two laboratory strains of bacteria commonly causing PJI were used, namely Staphylococcus aureus* and Staphylococcus epidermidis*. After overnight incubation with biofilm formation, the test samples were washed and individually exposed to increasing daptomycin concentrations (4–256 mg/l) during 24-hours. Samples were subsequently sonicated in order to detect dislodged biofilm bacteria on blood agar plates by viable growth and transferred to a microcalorimeter*** for real-time measurement of growth related heat flow during 24-h incubation. Minimal biofilm eradication concentration (MBEC) was determined as the lowest concentration of antibiotic required to eradicate the biofilm bacteria on the sample. The time to detection expressed as the heat flow >50 µW (TTD-50) indirectly quantifies the initial amount of biofilm bacteria, with a shorter TTD-50 representing a larger amount of bacteria. Results. MBEC of S. aureus biofilm on smooth metallic surfaces (median 6 mg/l, range 4–8 mg/l) was significantly lower than the rough/porous metallic surfaces (median 128 mg/l, range 32–256 mg/l; p<0.001). Variations of MBEC in experiments with S. epidermidis biofilms on test samples with smooth or rough/porous surface was found non-significant (p=0.25). Mean TTD-50 (±SD) of S. aureus biofilms on rough/porous metallic samples (2.3 ±1.1 hours) was significantly lower than smooth metallic samples (6.7 ±0.4 hours, p<0.001) and polyethylene (5.3 ±0.5 hours, p<0.001). Mean TTD-50 with S. epidermidis biofilm on smooth metals (3.9 ± 1.0 hours) was also significantly higher than their rough/porous counterparts (2.0 ± 1.0 hours, p=0.010). Conclusions. Growth of biofilm bacteria on orthopedic materials are variably influenced by exposure to the potent antimicrobial effect of high-dose daptomycin. In this study, the main factor decisively influencing biofilm quantity and daptomycin susceptibility of staphylococcal biofilms was the irregular surface topography. * ATCC® 29213™. ** ATCC® 35984™. *** TAM III

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

Introduction. The use of antibiotic-loaded polymethylmethacrylate bone-cement spacers during two-stage exchange procedures is the standard in the treatment of patients with delayed prosthetic joint infection. The real antimicrobial activity of these spacers is unclear because the adherence of bacteria to cement might result in clinical recurrence of infection. The purpose of the study is to evaluate the in vitro formation of Pseudomonas Aeruginosa (PA) and Staphylococcus spp. biofilm on antibiotic-loaded bone cement. Materials and methods. Cement disks (diameter = 6 mm) impregnated with gentamicin and colistin were submerged in bacterial suspensions of Methicillin-resistant Staphylococcus Aureus(MRSA), Staphylococcus epidermidis (SE), and PA. Negative controls (specimen disks without antibiotic) were similarly prepared. Biofilm formation was visualized by confocal scanning laser microscopy (CSLM), after staining the discs with the live/dead BacLight viability stain containing SYTO 9 dye and propidium iodide. Images from five randomly selected areas were acquired for each disc. Sequential optical sections of 2 µm were collected in sequence along the z-axis over the complete thickness of the sample. The resulting stacks of images were analyzed, quantified and rendered into three-dimensional (3D). The biofilm thickness on antibiotic bone cement compared with the controls was automatically evaluated. Results. CSLM showed living bacteria and bacterial biofilm on the surface of all cement disks, either antibiotic-loaded or controls. Mean biofilm thickness on the controls was 29.6 µm for MRSA, 32.3 µm for SE, and 59.7 µm for PA. The 3D rendering showed decrease in the biofilm thickness for all bacterial strains on gentamicin- and colistin-impragnated cement disks as compared with the controls. The incorporation of gentamicin into cement resulted in a 54%, 74%, and 45% reduction in the bacterial biofilm thickness for MRSA, PA and SE, respectively. The use of colistin leaded to a 51 % reduction in the PA biofilm thickness. Conclusion. The bacterial viability and biofilm formation are reduced by adding antibiotics to bone cement but antibiotic-loaded bone cement does not completely inhibit the formation of an infectious biofilm in vitro

Summary Statement. A study to evaluate biofilm development on different coatings of UHMWPE was performed. We observed a species-specific effect, with S. aureus affected mainly by DLC-F and S. epidermidis by DLC. These data correlates with previous adherence studies. Introduction. Prosthetic joint infection is intimately related to bacterial biofilms on implant biomaterials. Recently, diamond-like carbon (DLC) coating has been suggested to improve the antibacterial performance of medical grade GUR1050 ultra high molecular weight polyethylene (UHMWPE) supplied by Orthoplastics bacup, UK versus collection and clinical staphylococcal strains. The aim of this study was to make an approximation towards the actual impact of such coatings in biofilm formation. Material and Methods. Biofilm formation by two collection laboratory strains (S. aureus 15981[4] and S. epidermidis ATCC 35984) was evaluated with raw UHMWPE and two UHMWPEs coated with DLC, and fluorine doped DLC (F-DLC). The coated surfaces were obtained by plasma enhanced chemical vapour deposition, as previously described. All the sterilised surfaces were exposed to ≈10. 8. colony forming units/mL during 48 hours at 35° C, with total medium exchange at 24 hours without shaking. Surfaces were carefully washed with PBS (X 3) and then stained with Backlight. ©. live/dead stain for 15 minutes. Confocal Laser Scanning Microscopy was used for sampling the surfaces and studying biofilm, for which eight random series of photographs (Named SERIES) and four predefined biofilm series (Named BIOFILM) were taken. Biofilm thickness (microns) and covered surface by live/dead bacteria (%) were determined for both SERIES and BIOFILM. Assays were made in triplicates. Photographs were analyzed by ImageJ software, and data, by a Mann-Withney test. Results. Biofilm thickness and bacterial coverage per surface type in SERIES as well as BIOFILM for S. aureus and S.epidermidis, respectively is shown. A diminution of these two variables was observed in the coated surfaces versus raw UHWMPE with statistically significant reductions (p≤0.0001). F-DLC was the most effective coated surface versus S. aureus, with the least biofilm thickness and the highest proportional percentage of dead bacteria, and so DLC was versus S. epidermidis. Of interest, the proportion of dead S. epidermidis was higher in raw UHMWPE. Discussion & Conclusions. Staphylococcal biofilm formation on UHMWPE surfaces is irregular. Both biofilm thicknesses as bacterial coverage were lower in DLC and F-DLC. These preliminary data correlate to our previous bacterial adherence findings and support the better anti-adherence performance of DLC coated UHMWPEs. Fluorine is suggested to exhibit a bacteria-dependant behavior, but at present its role is quite far to be known. Further studies using clinical strains of both species are needed to evaluate the accuracy of these results

Aim. The increase of antimicrobial resistance reduces treatment options for implant-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Bacteriophages present a promising alternative to treat biofilm-related infections due to their rapid bactericidal activity and activity on multi-drug resistant bacteria. In this study, we investigated the synergistic activity of lytic bacteriophage Sb-1 with different antibiotics against MRSA biofilm, using a real-time highly sensitive assay measuring growth-related heat production (microcalorimetry). Methods. Rifampin, fosfomycin, vancomycin and daptomycin were tested alone and in combination with S. aureus specific phage, Sb-1, against MRSA (Staphylococcus aureus*). MRSA biofilm was formed on porous glass beads (Φ 4 mm, pore size 60 µm) and incubated for 24 h at 37° C in BHI. After 3 times washing biofilms were exposed first to different titers of bacteriophages, ranging from 102 to104 plaque-forming unite (pfu)/ml and after 24h treated again with subinhibitory concentration of antibiotics (corresponding to 1/4, 1/8, 1/16, 1/32 × MHICbiofilm). After 24h antibiotic treatment, the presence of biofilm on glass beads was evaluated by isothermal microcalorimetry for 48h. Heat flow (µW) and total heat (J) were measured. Results. MHICs of rifampin, fosfomycin, daptomycin and vancomycin when tested alone were 256 μg/ml, >4096 μg/ml, 128μg/ml and 2048μg/ml, respectively. Synergistic activity against biofilm MRSA was observed when vancomycin was tested at subinhibitory concentrations 512 μg/ml, 256 μg/ml, 128 μg/ml and 64 μg/ml in combination with subinhibitory titers of Sb-1 at 102, 103, 104 pfu/ml. Complete inhibition of heat production was observed only in combination with a higher titer of Sb-1 (104 pfu/ml). High synergistic activities were also observed in the presence of rifampin, fosfomycin and daptomycin. Conclusions. While MHICs of antibiotics against MRSA biofilm were above drug concentrations reachable in clinical practice, the co-administration with bacteriophage Sb-1 strongly reduced the antibiotic doses needed to eradicate MRSA biofilm. The use of bacteriophage and antibiotics in combination represent an effective strategy to treat implant-associated infections

Recent evidence suggests that the microbial community, its spatial distribution and activity play an important role in the prolongation of treatment and healing of chronic infections. Standard bacterial cultures often underestimate the microbial diversity present in chronic infections. This lack of growth is often due to a combination of inadequate growth conditions, prior usage of antibiotics and presence of slow-growing, fastidious, anaerobic or unculturable bacteria living in biofilms. Thus, diagnosis of chronic infections is challenged by lack of appropriate sampling strategies and by limitations in microbiological testing methods. The purpose of this study was to improve sampling and diagnosis of prosthetic joint infections (PJI) and chronic wounds, especially considering the biofilm issue. Systematic sampling, sonication of prosthesis and extended culture were applied on patients with chronic wounds and patients with suspected PJIs. Optimized DNA extraction, quantitative PCR, cloning, next generation sequencing and PNA FISH were applied on the different types of specimens for optimized diagnosis. For further investigation of the microbial pathogenesis, in situ transcriptomics and metabolomics were applied. In both chronic wounds and PJIs, molecular techniques detected a larger diversity of microorganisms than culture methods in several patients. Especially in wounds, molecular methods identified more anaerobic pathogens than culture methods. A heterogeneous distribution of bacteria in various specimens from the same patient was evident for both patient groups. In chronic wounds, multiple biopsies from the same ulcer showed large differences in the abundance of S. aureus and P. aeruginosa at different locations. Transcriptomic and metabolomic analyses indicated the important virulence genes and nutrient acquisition mechanisms of Staphylococcus aureus in situ. As an example, diagnosis and treatment of a patient with a chronic biofilm prosthesis infection persisting for 7 years will be presented. Our studies show that diagnosis of chronic biofilm related infections required multiple specimen types, standardized sampling, extended culture and molecular analysis. Our results are useful for improvement of sampling, analysis and treatment in the clinic. It is our ambition to translate studies on bacterial activity into clinical practice in the future

Aims. Current treatments of prosthetic joint infection (PJI) are minimally effective against Staphylococcus aureus biofilm. A murine PJI model of debridement, antibiotics, and implant retention (DAIR) was used to test the hypothesis that PlySs2, a bacteriophage-derived lysin, can target S. aureus biofilm and address the unique challenges presented in this periprosthetic environment. Methods. The ability of PlySs2 and vancomycin to kill biofilm and colony-forming units (CFUs) on orthopaedic implants were compared using in vitro models. An in vivo murine PJI model of DAIR was used to assess the efficacy of a combination of PlySs2 and vancomycin on periprosthetic bacterial load. Results. PlySs2 treatment reduced 99% more CFUs and 75% more biofilm compared with vancomycin in vitro. A combination of PlySs2 and vancomycin in vivo reduced the number of CFUs on the surface of implants by 92% and in the periprosthetic tissue by 88%. Conclusion. PlySs2 lysin was able to reduce biofilm, target planktonic bacteria, and work synergistically with vancomycin in our in vitro models. A combination of PlySs2 and vancomycin also reduced bacterial load in periprosthetic tissue and on the surface of implants in a murine model of DAIR treatment for established PJI. Cite this article: Bone Joint J 2020;102-B(7 Supple B):3–10

Aim. We outline a treatment protocol for subjects with chronic periprosthetic joint infections (PJI) who elected not to have surgery. We developed a method of serial “fluid-depleting” aspirations with intra-articular gentamycin injections to affect the population of the biofilm community. We have experienced many treatment failures, as expected, but have also had a group of subjects who responded exceptionally well, requiring no surgical intervention. Our longest follow-up is 10 years. Method. From June 2009 to December 2018, 372 clinical cases of chronic PJI involving primary and revision TKA and THA were treated. Of these, 25 subjects were treated with an active suppression protocol, in lieu of surgery. The protocol entailed frequent aspirations and intra-articular antibiotic injections to quell the PJI inflammatory response. All aspirations were performed by the treating surgeon in the orthopaedic clinic without fluoroscopic guidance. Based on a subject's response to the protocol, he/she was identified as 1 of 3 classifications: 1) Ongoing Treatment – Biofilm Trained (OTBT), 2) Ongoing Treatment – Biofilm Untrained (OTBU), and 3) Treatment Failure (TF). OTBT subjects showed no clinical signs of infection. Serum biomarkers (CRP, ESR) remained consistently normal and subjects were not on oral suppressive antibiotics. Aspiration analysis and cultures remained negative. Maintenance treatment consisted of a fluid-depleting aspiration with an intra-articular gentamycin injection every 12–16 weeks. OTBU subjects showed improved clinical symptoms, lowered serum biomarkers, and lowered WBC counts, but still demonstrated objective signs of infection. TF subjects did not respond to the protocol and showed unchanged/worsening clinical symptoms. Results. Of the 25 subjects, 8 were THA's and 17 were TKA's. Of these cases, 21 (84%) were endoprosthetic replacements. 8 subjects (32%) were classified as OTBT, 6 (24%) as OTBU, and 11 (44%) as TF. All TF subjects were treated with a two-stage exchange protocol. Conclusions. This study is the first describing the potential of modifying bacterial biofilm in a chronic PJI. While our success rate was modest (32%), “Biofilm Trained” subjects demonstrated dramatic changes. Subjects led normal lives, only minimally disrupted by an aspiration and injection every 3–4 months. In the future, we are looking at different agents to modify the enveloping biofilm, including a pre-aspiration injection of EDTA to disrupt the biofilm surface, followed by injecting benevolent bacteria to transform the biofilm to a benevolent state. If able to achieve such a state in a consistent fashion, the impact on the patient and healthcare communities would be enormous

Aims. Biofilm formation is one of the primary reasons for the difficulty in treating implant-related infections (IRIs). Focused high-energy extracorporeal shockwave therapy (fhESWT), which is a treatment modality for fracture nonunions, has been shown to have a direct antibacterial effect on planktonic bacteria. The goal of the present study was to investigate the effect of fhESWT on Staphylococcus aureus biofilms in vitro in the presence and absence of antibiotic agents. Methods. S. aureus biofilms were grown on titanium discs (13 mm × 4 mm) in a bioreactor for 48 hours. Shockwaves were applied with either 250, 500, or 1,000 impulses onto the discs surrounded by either phosphate-buffered saline or antibiotic (rifampin alone or in combination with nafcillin). The number of viable bacteria was determined by quantitative culture after sonication. Representative samples were taken for scanning electron microscopy. Results. The application of fhESWT led to a ten-fold reduction in bacterial counts on the metal discs for all impulse numbers compared to the control (p < 0.001). Increasing the number of impulses did not further reduce bacterial counts in the absence of antibiotics (all p > 0.289). Antibiotics alone reduced the number of bacteria on the discs; however, the combined application of the fhESWT and antibiotic administration further reduced the bacterial count compared to the antibiotic treatment only (p = 0.032). Conclusion. The use of fhESWT significantly reduced the colony-forming unit (CFU) count of a S. aureus biofilm in our model independently, and in combination with antibiotics. Therefore, the supplementary application of fhESWT could be a helpful tool in the treatment of IFIs in certain cases, including infected nonunions. Cite this article: Bone Joint Res 2021;10(1):77–84

Infections are among the main complications connected to implantation of biomedical devices, having high incidence rate and severe outcome. Since their treatment is challenging, prevention must be preferred. For this reason, solutions capable of exerting suitable efficacy while not causing toxicity and/or development of resistant bacterial strains are needed. To address infection, inorganic antibacterial coatings, and in particular silver coatings, have been extensively studied and used in the clinical practice, but some drawbacks have been evidenced, such as scarce adhesion to the substrate, delamination, or scarce control over silver release. Here, antibacterial nanostructured silver-based thin films are proposed, obtained by a novel plasma-assisted technique, Ionized Jet Deposition (IJD). Coatings are obtained by deposition of metallic silver targets. Films thickness is selected based on previous results aimed at measuring extent and duration of silver release and at evaluating toxicity to host cells (fibroblasts). Here, composition (grazing incidence XRD) and morphology (SEM) of the obtained coatings are characterized for deposition onto different substrates, both metallic and polymeric. For heat sensitive substrates, possible alterations caused by coatings deposition in terms of morphology (SEM) and composition (FT-IR) is assessed. Then, a proof-of-concept study of the capability of these films to inhibit microbial biofilm formation is performed by using two different supports i.e., the Calgary Biofilm Device and the microplates. To the best of the Authors knowledge, this is the first study describing the application of specific anti-biofilm analyses to nanostructured coatings. In particular, anti-biofilm activities are tested against the following pathogenic strains: Escherichia (E.) coli NCTC12923, Staphylococcus (S.) aureus ATCC29213 and S. aureus 86. Among these, the strain 86 is not only pathogen but it also possesses several antibiotic resistance genes, allowing the evaluation of the utilization of nanostructured coatings as an alternative anti-microbial system to face the global threat of antibiotic resistance. Results indicate that films deposited from silver targets are composed of nanosized aggregates of metallic silver, indicating a perfect transfer of composition from the deposition target to the coatings. Results obtained here indicate that the films have significant antibacterial and antibiofilm activity. In addition, they prove that the system can be successfully applied for evaluation of coatings antibacterial efficacy for biomedical applications

Aims. The aims of this study were to develop an in vivo model of periprosthetic joint infection (PJI) in cemented hip hemiarthroplasty, and to monitor infection and biofilm formation in real-time. Methods. Sprague-Dawley rats underwent cemented hip hemiarthroplasty via the posterior approach with pre- and postoperative gait assessments. Infection with Staphylococcus aureus Xen36 was monitored with in vivo photoluminescent imaging in real-time. Pre- and postoperative gait analyses were performed and compared. Postmortem micro (m) CT was used to assess implant integration; field emission scanning electron microscopy (FE-SEM) was used to assess biofilm formation on prosthetic surfaces. Results. All animals tolerated surgery well, with preservation of gait mechanics and weightbearing in control individuals. Postoperative in vivo imaging demonstrated predictable evolution of infection with logarithmic signal decay coinciding with abscess formation. Postmortem mCT qualitative volumetric analysis showed high contact area and both cement-bone and cement-implant interdigitation. FE-SEM revealed biofilm formation on the prosthetic head. Conclusion. This study demonstrates the utility of a new, high-fidelity model of in vivo PJI using cemented hip hemiarthroplasty in rats. Inoculation with bioluminescent bacteria allows for non-invasive, real-time monitoring of infection. Cite this article: Bone Joint J 2021;103-B(7 Supple B):9–16

Aim. To investigate the antimicrobial activity of a gentamicin-loaded bone graft substitute (GLBGS) in the prevention and eradication of bacterial biofilms associated with prosthetic joint infections (PJI). Method. The GLBGS (17,5 mg gentamicin/ml paste) with 40% hydroxyapatite/60% calcium sulfate. 1. was tested against biofilms of methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, methicillin-susceptible S. aureus (MSSA) ATCC 29213, Escherichia coli Bj HDE-1, S. epidermidis ATCC 12228 and Enterococcus faecalis ATCC 19433. For prevention studies, glass beads and different combinations of GLBGS were co-incubated for 24h at 37°C in CAMH broth with 1–5 × 10. 6. CFU/mL of bacteria. For eradication, biofilms were formed on glass beads for 24h at 37°C in CAMH broth. Then, beads were incubated with different combinations of GLBGS in medium at 37°C for 24h. For microcalorimetric measurements, beads were placed in ampoules and heat flow (µW) and total heat (J) were measured at 37°C for 24h. The minimal heat inhibitory concentration (MHIC) was defined as the lowest gentamicin concentration reducing the heat flow peak by ≥90% at 24h. Results. The GLBGS showed a good activity against all tested strains in both biofilm prevention and eradication. All MHIC values are reported in Table 1. Lower MHICs were observed when GLBGS was tested against E. coli (9.6 µg/mL prevention and 19.2 µg/mL eradication) and S. epidermidis (86 µg/mL and 38.8 µg/mL, respectively). For both prevention and eradication of MSSA, GLBGS MHIC was 631 µg/mL. E. faecalis biofilm formation was prevented with 631 µg/mL and eradicated with double concentration. MRSA showed a higher resistance to GLBGS up to 2516 µg/mL, both in biofilm prevention and eradication. Conclusions. This GLBGS is a valid composite for the prophylaxis and treatment of PJI. Further studies will be performed to evaluate the activity of higher concentrations of GLBGS against MRSA. 1. CERAMENT™|G, BONESUPPORT AB, Sweden

Aim. To evaluate bacterial adhesion and biofilm formation to metallic cerclage wire versus polymer cerclage system (SuperCable®). Methods. Experimental in vitro study to evaluate quantitative bacterial adherence to different cerclage wire materials. Two types of cerclage wires were compared: a metallic versus a polymer based wire (SuperCable®). A two-centimeter cerclage wire piece of each material was included in 2 mL of tryptic soy broth (TSB) culture media, inoculated with 10 microliters of a 0.5 McFarland of a Staphylococcus epidermidis strain and cultivated at 37°C during 2h for adhesion and 48h for biofilm formation. After this time, the cerclages were washed using a 1% phosphate buffered saline (PBS) and sonicated in new culture medium. After sonication, dilutions of each culture were spread in TSB agar and incubated 37°C during 24h. The number of colonies were counted and the cfu/cm2 was calculated. Results. There were no differences in the number of colonies counted at 2 hours. At 48 hours, the polymer cerclage system showed a clinically and statistically reduction of 95.2% in the biofilm formation of S. epidermidis. The highest bacterial counts were observed in metallic cerclages after 48h. Conclusion. In in vitro conditions, the polymer cerclage system may offer decreased biofilm formation compared with metallic cerclage wires. However, there are many other factors in in vivo conditions that could play a role in bacterial adhesion to cerclage wires. Further research is needed in order to recommend the use of polymer cerclage systems for septic revision surgery

Aim. To evaluate antimicrobial activity of Sb-1 and Pyo-bacteriophage in preventing and eradicating MRSA biofilm in vitro using isothermal micro calorimetry. Method. Two S. aureus specific bacteriophages, Sb-1 and Pyo-bacteriophage cocktail, were tested against S. aureus MRSA (ATCC 43300). MRSA biofilm was formed on porous glass beads and incubated for 24 h at 37° C in BHI, washed 3 times and exposed to different concentrations of bacteriophages. For biofilm prevention, MRSA (5×10. 6. CFUs/ml) was incubated with different phage titers. Glass beads were placed in the calorimeter and heat flow (µW) and total heat (J) were measured in real-time for 48h (eradication) or 24h (prevention). Results. Both tested bacteriophages rapidly inhibited the heat production of MRSA biofilm in a concentration-dependent manner during the first 24h, as shown for Sb-1 in Figure 1 A. After 48 h-expositions all the titers of bacteriophages show a strong reduction of biofilm viability (Figure 1B). MRSA biofilm was eradicated only by co-incubation with the highest Sb-1 phage titer (10. 7. PFUs/ml) (Figure 1A-B). In prevention experiments, significant reduction of MRSA heat production was already achieved at a lower titer (10. 2. PFUs/ml) of both Sb-1 and Pyo-bacteriophage and in the presence of 10. 4. PFUs/ml the heat production was completely abolished. Conclusions. Sb-1 and Pyo-bacteriophage are promising phages for treatment MRSA biofilms, as well as for preventing device colonization and controlling biofilms on surface. Their potential activity combined with antibiotics should be further investigated

Enterococcus faecalis is a rare but recognized cause of prosthetic joint infection. It is notorious for formation of biofilm on prosthetic surfaces. We hypothesized that a ‘serum factor’ was responsible for transformation of E. faecalis from its planktonic form to a biofilm existence upon making contact with prostheses. Using a novel ‘proteomic approach’, we studied the protein expression profiles of this bacterium when grown on an artificial surface in a serum environment against a control. E.faecalis 628 transconjugant formed by conjugation clinical strain (E55) and laboratory strain (JH2-2) was used to inoculate each of rabbit serum (RS) and Brain Heart Infusion (BHI) agar as a control and grown for 24 hours. Proteins were harvested for analysis in fractions including cell surface, membrane and cytosolic proteins. Recovered proteins were separated using 2-dimentional polyacrylamide gel electrophoresis (2D PAGE). Gels were stained and spots of interest harvested. These were analyzed using MALDI mass spectrometry followed by peptide mass fingerprinting using online database searches. Two surface exclusion proteins Sea1 and PrgA were only expressed from the serum culture. These proteins are both encoded by genes very close to the gene for enterococcal aggregation substance PrgB, which plays an integral role in biofilm formation. PrgA and PrgB are both encoded by the prgQ operon and hence expressed simultaneously upon activation of the operon. This tendency for serum only protein expression suggests the possibility of a pheromone-like activator in serum that could be a potential therapeutic target for management of biofilm associated E. faecalis prosthetic infections

Bacterial infection related to prosthetic replacement is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. In order to reduce the incidence of replacement-associated infections, we developed a novel coating technology of Hydroxyapatite (HA) containing silver (Ag). We reported the Ag-HA coating showed high antibacterial activity against E. coli, S. aureus and methicillin-resistant S. aureus (MRSA) under static condition. However, human bodies have a circulating body fluid, which is not a static condition. And the growth and the maturation of biofilm, which is said that a common course of persistent infections at a surgical site, are enhanced by the flow of broth in culture environment. Therefore, we evaluated whether the Ag-HA coating inhibits the biofilm formation on its surface or not by a biofilm-forming test under flow condition in this study. Ag-HA or HA powder was sprayed onto the commercial pure titanium disks using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin sensitive S. aureus (BF-MSSA; Seattle 1945) strain and the BF-MRSA (UOEH6) strain were used. The pre-culture bacterial suspension (about 10. 5. colony forming units; CFU) was inoculated onto the Ag-HA and HA coating disks. After cultivation at 37 °C for 1 h, the disks were rinsed twice with 500 μL sterile PBS (-) to eliminate the non-adherent bacteria. The number of the adherent bacteria on these disks was counted using culture method. After rinsing, the disks were transferred into petri-dish containing Trypto–Soy Broth (TSB) + 0.25% glucose with a stirring bar on the magnetic stirrer and they were cultured at 37°C for 7 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate on the surfaces of them was calculated using the NIH image software. The number of the bacteria on these disks was not so different between Ag-HA and HA coating after rinsing. After biofilm-forming test, the coverage of the biofilm of BF-MSSA was 2.1% and 81.0% on the Ag-HA and HA coatings, respectively. Similarly, in the case of BF-MRSA, it was 7.7% and 72.0% on the Ag-HA and HA coatings, respectively. Though bacteria slightly adhered, biofilm was hardly observed on the Ag-HA coating. The biofilm on the HA coating was extensive and mature. The inhibition effect of biofilm formation on the Ag-HA coating might be ascribed to the antibacterial effect by Ag ions released from the coating. Because Ag ions have a broad spectrum of antibacterial activity against pathogens, including biofilm forming bacteria, they inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria. Even in a flow condition, it was suggested that the AgHA shows the antibacterial activity, though the conditions in this work are different from those in living body

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

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

Aim. To investigate the local intra-operative concentration of gentamicin needed to prevent biofilm formation in a porcine model of implant-associated osteomyelitis. Method. In total 24 pigs were allocated to six groups. Group A (n=6) was inoculated with saline. Groups B (n=6), C (n=3), D (n=3), E (n=3) and F (n=4) were inoculated with 10 μL saline containing 10. 4. CFU of Staphylococcus aureus, however, different minimal inhibitory concentrations (MIC) of gentamicin were added to the inoculum of Groups C(160xMIC), D(1600xMIC), E(16000xMIC) and F(160000xMIC). The inoculums were injected into a pre-drilled implant cavity proximally in the right tibial bone. Following inoculation, a steel implant (2 × 15 mm) was placed in the cavity. The pigs were euthanized after five days. The implants were sonicated and swabs were taken from the implant cavity for microbiological evaluation. The peri-implant tissue was analyzed by histopathology including estimation of neutrophil infiltration. Results. The microbiological samples from Group A pigs were sterile. All implants and implant cavities of pigs inoculated with bacteria and bacteria + 160 or 1.600xMIC were positive for S. aureus. In each of the Groups E (16000xMIC) and F (160000xMIC) only one animal was found positive and 1/3 and 3/4 of the implants were sterile after sonication, respectively. All positive swabs were confirmed to be same spa-type as used for inoculation. By adding Groups C + D (<10000xMIC) and Groups E + F (>10000xMIC) a strong significant decrease (one-way ANOVA, P value = 0.001) of implant attached bacteria was only seen between the high MIC values and Group B (bacteria only). The histological examination demonstrated that 1600, 16000 and 160000 × MIC resulted in a peri-implant tissue reaction, including neutrophil estimation, comparable to saline inoculated animals. Patho-morphologically, it was not possible to distinguish between pigs inoculated with bacteria and bacteria + 160xMIC as both groups had a strong inflammatory response and an equal estimation of neutrophils. Discussion. The antibiotic susceptibility for prevention of an in vivo biofilm infection is influenced by body fluids, host immune response, extracellular host proteins like fibrin, tissue necrosis and development of an anaerobic environment. With the present in-vivo setup, we have demonstrated that local intra-operative gentamicin might be given in concentrations of more than 10000 times the MIC value in order to prevent biofilm formation by planktonic bacteria. Our study supports that biofilm susceptibility testing performed in-vitro is yet still unreliable for prediction of prophylactic and therapeutic success

Introduction. Support of appositional bone ingrowth and resistance to bacterial adhesion and biofilm formation are preferred properties for biomaterials used in spinal fusion surgery. Although polyetheretherketone (PEEK) is a widely used interbody spacer material, it exhibits poor osteoconductive and bacteriostatic properties. In contrast, monolithic silicon nitride (Si. 3. N. 4. ) has shown enhanced osteogenic and antimicrobial behavior. Therefore, it was hypothesized that incorporation of Si. 3. N. 4. into a PEEK matrix might improve upon PEEK's inherently poor ability to bond with bone and also impart resistance to biofilm formation. Methods. A PEEK polymer was melted and compounded with three different silicon nitride powders at 15% (by volume, vol.%), including: (i) α-Si. 3. N. 4. ; (ii) a liquid phase sintered (LPS) ß-Si. 3. N. 4. ; and (iii) a melt-derived SiYAlON mixture. These three ceramic powders exhibited different solubilities, polymorphic structures, and/or chemical compositions. Osteoconductivity was assessed by seeding specimens with 5 × 10. 5. /ml of SaOS-2 osteosarcoma cells within an osteogenic media for 7 days. Antibacterial behavior was determined by inoculating samples with 1 × 10. 7. CFU/ml of Staphylococcus epidermidis (S. epi.) in a 1 × 10. 8. /ml brain heart infusion (BHI) agar culture for 24 h. After staining with PureBlu™ Hoechst 33342 or with DAPI and CFDA for SaOS-2 cell adhesion or bacterial presence, respectively, samples were examined with a confocal fluorescence microscope using a 488 nm Krypton/Argon laser source. Images were also acquired using a FEG-SEM in secondary and backscattered modes on gold sputter-coated specimens (∼20–30Å). Hydroxyapatite (HAp) deposition was measured using a laser microscope. Raman spectra were collected for samples in backscattering mode using a triple monochromator using a 532 nm excitation source (Nd:YVO. 4. diode-pumped solid-state laser). Results. PEEK composites with 15 vol.% α-Si. 3. N. 4. , LPS ß-Si. 3. N. 4. , or the SiYAlON mixture showed significantly greater SaOS-2 cell proliferation (>600%, p<0.003, cf., Fig. 1(a)) and HAp deposition (>100%, p<0.003, cf., Fig. 1(b)) relative to monolithic PEEK. The largest increase in cell proliferation was observed with the SiYAlON composite, while the greatest amount of HAp was found on the LPS ß-Si. 3. N. 4. composite. Following exposure to S. epidermidis, the composite containing the LPS β-Si. 3. N. 4. powder showed one order of magnitude reduction in adherent live bacteria (p<0.003, cf., Fig. 1(c)) as compared to the PEEK monolith. It is interesting to note that the composite containing α-Si. 3. N. 4. exhibited the worst bacterial resistance (i.e., ∼100% higher than monolithic PEEK), suggesting that the bacteriostatic effectiveness of Si. 3. N. 4. bioceramics is apparently dependent upon the presence of selective sintering additives, viz. yttria and alumina. Conclusions. The addition of 15 wt.% of specific Si. 3. N. 4. powders to PEEK showed enhanced SaOS-2 cell adhesion, proliferation, and HAp deposition when compared to monolithic PEEK. These same composites also showed resistance to S. epi. adhesion and biofilm formation.. Although improvements in osteoconductivity have been previously observed by compounding or coating PEEK with HAp, titanium, or tantalum, these approaches did not provide anti-microbial properties. Compounding PEEK with Si. 3. N. 4. represents a significant advancement due to its ability to provide both improved bone apposition and resistance to biofilm formation. For any figures or tables, please contact the authors directly

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

Introduction: Staphylococcus aureus is a major cause of chronic infections and causes particular problems in relation to implanted prostheses. Biofilm formation on abiotic surfaces affords bacteria innate protection from opsonophagocytosis and antibiotic agents and complicates the eradication of infection from bone and implanted prostheses. Increased concentrations of sodium, the major extracellular cation, have previously been implicated in increased biofilm formation in Staphylococcus aureus. In this study we demonstrate that increased concentrations of potassium, the major intracellular cation, also causes a significant increase in biofilm formation. Furthermore we also show that halide stress also leads to a primary increase in penicillin resistance in Staphylococcus aureus. Methods: Staphylococcus aureus ATCC 9144 was cultured in broth supplemented with variable amounts of potassium chloride and sodium chloride. Biofilm formation was investigated in 96-well microtiter plates using a standard technique. Antibiotic resistance was investigated using graduated E-test strips. Results: There was a positive correlation between bio-film formation and increased concentrations of sodium and potassium. Biofilm formation was noted to be even greater under potassium stress than under sodium stress. Sodium stress also lead to a five-fold increase in penicillin resistance in naïve Staphylococcus aureus cells. Discussion: Cellular injury or insult can lead to cell necrosis and lysis. The intracellular concentration of potassium is 30 times higher than that of the surrounding extracellular fluid. Hence, cell necrosis leads to markedly increased local concentrations of potassium. These experiments show that an increase in potassium concentration leads to an increase in biofilm formation. This suggests that biofilm formation and hence infection of implanted pros-theses may be more likely in areas of major tissue trauma such as large resections and revisions. Furthermore, cellular stress leads to increased antibiotic resistance in naïve cells which may nullify prophylaxis and complicate bacterial eradication in vivo

Problems. Biofilm infections are increasingly associated with orthopedic implants. Bacteria form biofilms on the surfaces of orthopedic devices. The biofilm is considered to be a common cause of persistent infections at a surgical site. The growth and the maturation of biofilm are enhanced by the flow of broth in culture environment. In order to reduce the incidence of implant-associated infections, we developed a novel coating technology of hydroxyapatite (HA) containing silver (Ag). We previously reported that the Ag-HA coating inhibits biofilm formation under flow condition of Trypto Soy Broth + 0.25% glucose for 7 days. In this study, we evaluated whether the Ag-HA coating continuously inhibits the biofilm formation on its surface under flow condition of fetal bovine serum, which contains many in vivo substrates such as proteins. Materials and Method. The commercial pure titanium disks were used as substrates. Ag-HA or HA powder was sprayed onto the substrates using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin resistant Staphylococcus aureus (BF-MRSA; UOEH6) strain was used. The bacterial suspension (about 10. 5. colony forming units) was inoculated into 24-well sterile polystyrene tissue culture plates. The Ag-HA and HA coating disks were aseptically placed in the wells. After cultivation at 37°C for 1 hour, the disks were rinsed twice with 500 μL sterile PBS (−) to eliminate the non-adherent bacteria. After rinsing, the disks were transferred into petri-dish containing heat-inactivated FBS with a stirring bar on the magnetic stirrer and they were cultured at 37 °C for 24 hours, 7 and 14 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate (BCR) on the surfaces of them was calculated using the NIH image software. Results. Biofilm was hardly observed on the Ag-HA coating. However, the biofilm on the HA coating was extensive and mature (Fig. 1). At 24h after cultivation, BCRs of BF-MRSA were 2.1% and 19.8% on the Ag-HA and HA coatings, respectively. Similarly, they were 6.3% and 12.4% on the Ag-HA and HA coatings at 7 days. At 14 days they were 20.6% and 39.4% on the Ag-HA and HA coatings, respectively. These results demonstrate that BCRs on the Ag-HA coating were significantly lower than those on the HA coating (Fig. 2). Discussion. The Ag-HA coating continuously showed the inhibiting ability for biofilm formation under flow condition for 14 days. Ag ions inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria in the vicinity of the surface, although the release rate of Ag ions was high until 24h after immersion and decreased thereafter. The Ag-HA coating would be expected to contribute to reduction of implant-related biofilm infection

Biofilm development is a major factor in the pathogenesis of implant-related infections. However, there are only a low number of studies that analyses the ability of clinical isolates of bacteria to develop biofilm in vitro. Here we study biofilm development in several strains of Staphylococcus aureus and Coagulase-negative Staphylococcus (CNS) consecutively isolated from retrieved orthopaedic implants from patients diagnosed of implant-related infections. We have evaluated in vitro biofilm development using the crystal violet technique in microtiter plates. Biofilm development was confirmed by visual microscopy and Confocal Laser Scanning Microscopy. Staphylococcal strains were isolated from implant-related infections by sonication of retrieved prosthesis as previously published by our group, and identified using conventional methods. Twenty-seven strains (15 S. aureus, nine S. epidermidis, and one each of S. hominis, S. lugdunensis and S. warneri) were included in the study. Four strains of S. aureus (26.7 %) and one strain of S. epidermidis (8.3 %) did not develop biofilm in the test, showing OD lectures almost identical to the negative control. No statistical differences were detected between the two groups. The microscopic examination confirms this finding. Among the biofilm-producing strains, an important difference of the amount of biofilm produced was detected. One strain (S. aureus) produced biofilm in greater amount than all other strains, detectable even by visual examination of the plate. In conclusion, not all staphylococcal strains isolated from implant-related infections are able to develop biofilm in vitro. There must be other pathogenic factors that are important in the pathogenesis of implant-related infections and need to be studied in order to develop a better strategy for treat these infections