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 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

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

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

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

Aims. To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. Methods. EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt Staphylococcus aureus biofilms on Matrigel-coated glass and two materials widely used in orthopaedic implants (Ti-6Al-4V and highly cross-linked polyethylene (HXLPE)). To assess the efficacy of biofilm dispersion, crystal violet staining biofilm assay and colony counting after sonification and culturing were performed. The results were further confirmed and visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). We then investigated the efficacies of EDTA-NS irrigation in vivo in rat and pig models of biofilm-associated infection. Results. When 10 mM or higher EDTA-NS concentrations were used for ten minutes, over 99% of S. aureus biofilm formed on all three types of materials was eradicated in terms of absorbance measured at 595 nm and colony-forming units (CFUs) after culturing. Consistently, SEM and CSLM scanning demonstrated that less adherence of S. aureus could be observed on all three types of materials after 10 mM EDTA-NS irrigation for ten minutes. In the rat model, compared with NS irrigation combined with rifampin (Ti-6Al-4V wire-implanted rats: 60% bacteria survived; HXLPE particle-implanted rats: 63.3% bacteria survived), EDTA-NS irrigation combined with rifampin produced the highest removal rate (Ti-6Al-4V wire-implanted rats: 3.33% bacteria survived; HXLPE particle-implanted rats: 6.67% bacteria survived). In the pig model, compared with NS irrigation combined with rifampin (Ti-6Al-4V plates: 75% bacteria survived; HXLPE bearings: 87.5% bacteria survived), we observed a similar level of biofilm disruption on Ti-6Al-4V plates (25% bacteria survived) and HXLPE bearings (37.5% bacteria survived) after EDTA-NS irrigation combined with rifampin. The in vivo study revealed that the biomass of S. aureus biofilm was significantly reduced when treated with rifampin following irrigation and debridement, as indicated by both the biofilm bacterial burden and crystal violet staining. EDTA-NS irrigation (10 mM/10 min) combined with rifampin effectively removes S. aureus biofilm-associated infections both in vitro and in vivo. Conclusion. EDTA-NS irrigation with or without antibiotics is effective in eradicating S. aureus biofilm-associated infection both ex and in vivo. Cite this article: Bone Joint Res 2024;13(1):40–51

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

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

Aims. Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella. Methods. For the haematogenous infection, G. mellonella larvae were implanted with a Kirschner wire (K-wire), infected with S. aureus, and subsequently phages and/or gentamicin were administered. For the early-stage biofilm implant infection, the K-wires were pre-incubated with S. aureus suspension before implantation. After 24 hours, the larvae received phages and/or gentamicin. In both models, the larvae also received daily doses of phages and/or gentamicin for up to five days. The effect was determined by survival analysis for five days and quantitative culture of bacteria after two days of repetitive doses. Results. In the haematogenous infection, a single combined dose of phages and gentamicin, and repetitive injections with gentamicin or in combination with phages, resulted in significantly improved survival rates. In the early-stage biofilm infection, only repetitive combined administration of phages and gentamicin led to a significantly increased survival. Additionally, a significant reduction in number of bacteria was observed in the larvae after receiving repetitive doses of phages and/or gentamicin in both infection models. Conclusion. Based on our results, a single dose of the combination of phages and gentamicin is sufficient to prevent a haematogenous S. aureus implant-related infection, whereas gentamicin needs to be administered daily for the same effect. To treat early-stage S. aureus implant-related infection, repetitive doses of the combination of phages and gentamicin are required. Cite this article: Bone Joint Res 2024;13(8):383–391

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

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

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

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

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

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

Aims. There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). Methods. The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites. Results. Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF. Conclusion. Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections. Cite this article: Bone Joint Res 2022;11(11):787–802

Aims. Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). Methods. A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR. Results. The group with LIPUS and 0.35% PI exhibited decreased levels of serum biochemical markers, improved weightbearing scores, reduced reactive bone changes, absence of viable bacteria, and decreased inflammation compared to the Control group. Despite the greater antibiofilm activity observed in the PI group compared to the LIPUS and saline group, none of the monotherapies were successful in preventing reactive bone changes or eliminating the infection. Conclusion. In the rat model of PJI treated with DAIR, LIPUS combined with 0.35% PI demonstrated stronger antibiofilm potential than monotherapy, without impairing any local soft-tissue. Cite this article: Bone Joint Res 2024;13(7):332–341

Aims. Arthroplasty surgery of the knee and hip is performed in two to three million patients annually. Periprosthetic joint infections occur in 4% of these patients. Debridement, antibiotics, and implant retention (DAIR) surgery aimed at cleaning the infected prosthesis often fails, subsequently requiring invasive revision of the complete prosthetic reconstruction. Infection-specific imaging may help to guide DAIR. In this study, we evaluated a bacteria-specific hybrid tracer (. 99m. Tc-UBI. 29-41. -Cy5) and its ability to visualize the bacterial load on femoral implants using clinical-grade image guidance methods. Methods. 99m. Tc-UBI. 29-41. -Cy5 specificity for Stapylococcus aureus was assessed in vitro using fluorescence confocal imaging. Topical administration was used to highlight the location of S. aureus cultured on femoral prostheses using fluorescence imaging and freehand single photon emission CT (fhSPECT) scans. Gamma counting and fhSPECT were used to quantify the bacterial load and monitor cleaning with chlorhexidine. Microbiological culturing helped to relate the imaging findings with the number of (remaining) bacteria. Results. Bacteria could be effectively stained in vitro and on prostheses, irrespective of the presence of biofilm. Infected prostheses revealed bacterial presence on the transition zone between the head and neck, and in the screw hole. Qualitative 2D fluorescence images could be complemented with quantitative 3D fhSPECT scans. Despite thorough chlorhexidine treatments, 28% to 44% of the signal remained present in the locations of the infection that were identified using imaging, which included 500 to 2,000 viable bacteria. Conclusion. The hybrid tracer . 99m. Tc-UBI. 29-41. -Cy5 allowed effective bacterial staining. Qualitative real-time fluorescence guidance could be effectively combined with nuclear imaging that enables quantitative monitoring of the effectiveness of cleaning strategies. Cite this article: Bone Joint Res 2023;12(1):72–79

Introduction: One of the most important mechanisms S. epidermidis uses to establish infection on biomaterials is biofilm formation, in which adhesion and the production of polysaccharide intercellular adhesin (PIA) are key factors. Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to inhibit S. epidermidis biofilm formation and may be useful in prevention or treatment of implant infections (. 1. ,. 2. ). The potential of these drugs was evaluated by determining the effects of the NSAIDs on bacterial growth, adhesion to bare and conditioned polymethyl-methacrylate (PMMA), on biofilm development, and on established biofilms. Methods: A PIA-deficient mutant and wild type strain (gift of Prof. D. Mack, Hamburg) and 3 clinical isolates of S. epidermidis were used. The NSAIDs were salicylic acid, acetylsalicylic acid, ibuprofen and phenylbutazone. Their effects on bacterial growth rate and viability were assessed. For adhesion assay, bacteria were exposed to a 1mM concentration of each drug and allowed to adhere for 1h to bare or human plasma – conditioned PMMA before being sonicated and quantified by chemiluminescence and culture. For biofilm assays, bacteria were grown on silicone discs in the presence of various drug concentrations for 4 days before being sonicated and quantified as above. Mature (4 day) biofilms were also exposed to the drugs for a further 4 days and quantified similarly, to assess the effect on established biofilms. Results: All NSAIDs tested significantly (P< 0.05) reduced the growth rate and viability of each strain, in a concentration – dependent manner. Reduction of adhesion was observed on bare PMMA suggesting interference with either vitronectin – binding protein or charge / hydrophobic interactions. This was independent of the effect on growth. However, adhesion to plasma – conditioned PMMA, presumably mediated by MSCRAMMs, was not significantly affected. Reduction of biofilm formation was observed for all strains and was concentration – dependent, suggesting that inhibition of PIA synthesis was not responsible. There was a significant effect on established biofilms, this was also concentration dependent. Conclusions:. All four NSAIDs reduced S. epidermidis growth rate and viability, but at concentrations above those achievable therapeutically. The effect on adherence was confined to unconditioned PMMA. The effect on biofilm formation and on established biofilms appeared to be related to that on growth and viability. On these grounds, NSAIDs appear to have a limited prospect for use in prevention or treatment of S. epidermidis biomaterial-related infection. However, catheter coating, NSAID-antibiotic combinations, and potential for other types of infection may have greater prospects

Aim. Cutibacterium acnes (CA) is one of the crucial actors in spine instrumentation or shoulder prosthesis. Its population is subdivided into 6 major phylotypes: IA1, IA2, IB, IC, II and III. Recent methods for discriminating subpopulations within CA phylotypes highlight the predominance of SLST types H1 to 6 or K1 to 20 in bone and joint infection (BJI). The impact of their ability to produce a biofilm during the development of the infection (with resistance / tolerance to antibiotics used for treatment) remains little studied. Method. The purpose of this study was to determine whether the ability to establish a biofilm varied according to the different subtypes of clinical strains of CA previously characterized and involved in BJI (hip, knee and shoulder prosthesis). The BioFilm ring test (BioFilm Control®) method with index determination, called BFI (BioFilm Index) inversely proportional to the level of biofilm production was used (BFI = 0.00 indicates a high production of biofilm versus BFI = 20.00 indicates zero production). The BFI was determined after 3 h (T3) and 6 h (T6) incubation. The strains used came from patients, 5 belonging to the IA1 phylotype (SLST A1 and D1 types) and 4 to different phylotypes (IA2, IB, II and III). Results. The results show that the kinetics of establishment of an early CA biofilm turns out to be phylotype dependent. The most productive strains are those belonging to phylotype II (BFI T3 = 5.73, BFI T6 = 0.00) and to type SLST D1 belonging to phylotype IA1 (BFI T3 = 4.07, BFI T6 = 0.00). The other strains did not demonstrate saturated BFI, even after 6 h of incubation. Conclusions. The exact role of CA, as well as its ability to produce a biofilm in the pathophysiology of BJI, remains poorly understood and the prolonged use of antibiotics to treat these infections is necessary, especially if devices have not been removed, with potential risk of increasing antibiotic resistance and therapeutic failures. CA's different phylotypes demonstrate different biofilm production capabilities, which could have an impact on the antibiotic efficacy suggesting the interest of effective anti-biofilm molecules on metabolically less active strains

Infected wounds are a major problem for patients and health care systems. The inflammation triggers expression of high levels of extracellular protease activities which degrade newly formed granulation tissue. The expression of host-derived proteases had been studied in wound healing extensively. In contrast, the contribution of bacterial proteases in impaired healing acute and chronic wounds is poorly understood as is how bacterial proteases can be blocked. In this study the expression of P. aeruginosa proteases was studied. P. aeruginosa is associated with poor healing and sufficiently common in wound infections to merit closer study. We used in vitro biofilm and planktonic culture models to analyze the culture-dependent expression of different P. aeruginosa proteases and how protease modulating polymers can inhibit activities. P. aeruginosa (PAO1, DSM 22644) was grown in LB. o. medium (aerated planktonic cultures) or in a biofilm culture model (dialysis tubing on LB. o. plates). The supernatant of planktonic or wash fluids from biofilm cultures were analyzed for protease activity. Global extracellular protease activities increased in a time- and culture condition-dependent manner (for planktonic cultures 180 ng/ml trypsin equivalent 8h, 330 ng/ml 24h, 490 ng/ml 48h; biofilm cultures 190 ng/ml trypsin equivalent 8h, 420 ng/ml 24h, 170 ng/ml 48h). Enzyme zymography revealed in biofilm cultures predominant bands at 50 kD (8h, 24h, 48h), 90 kD (24h) and > 200 kD (8h, 24h, 48h). In planktonic cultures the pattern was different 50 kD (8h), 90 kD (8h, 24h, 48h), 130 kD (24h, 48h) and > 200 kD (8h, 24h). Two different polyacrylate superabsorbers could inhibit P. aeruginosa protease activities. Favor PAC 300 blocked protease activity by 60% and SXM 9170 by 35%. These data demonstrate complex, culture-dependent expression of extracellular proteases in P. aeruginosa, a microorganism associated with poor wound healing outcomes. From a therapeutic perspective polyacrylate superabsorbers strongly inhibited global protease activities. In the next steps the protease expression pattern needs to be analyzed in P. aeruginosa wounds and correlated with healing progression

Biofilm-associated infections in wounds or on implants are difficult to treat. Eradication of the bacteria is nearly always impossible, despite the use of specific antibiotics. The bactericidal effects of high-energy extracorporeal shock waves on Staphylococcus aureus have been reported, but the effect of low-energy shock waves on staphylococci and staphylococcal biofilms has not been investigated. In this study, biofilms grown on stainless steel washers were examined by electron microscopy. We tested ten experimental groups with Staph. aureus-coated washers and eight groups with Staph. epidermidis. The biofilm-cultured washers were exposed to low-energy shock waves at 0.16 mJ/mm. 2. for 500 impulses. The washers were then treated with cefuroxime, rifampicin and fosfomycin, both alone and in combination. All tests were carried out in triplicate. Viable cells were counted to determine the bactericidal effect. The control groups of Staph. aureus and Staph. epidermidis revealed a cell count of 6 × 10. 8. colony-forming units/ml. Complete eradication was achieved using the combination of antibiotic therapy (single antibiotic in Staph. aureus, a combination in Staph. epidermidis) and shock wave application (p < 0.01). We conclude that shock waves combined with antibiotics could be tested in an in vitro model of infection

Soft tissue biopsies may prove culture negative in biofilm prosthetic infections. Identification of the causative bacteria could be achieved by either scraping of the prosthetic surface or by sonication of the entire implant. These techniques have not been thoroughly studied in experimental models where the biofilm is developed in vivo. In a novel rat biofilm model we compared scraping and sonication as methods for dislodging biofilm bacteria. Twenty plates of steel alloy (5×7×1mm), with a surface roughness (Ra) of 0.35 (0.19–0.51) μm, were inserted into 20 standardised pieces of sheep costae, weight: 1.2 (1.0–1.5) gram. To each bone graft was added 50 μL of a Staphylococcus epidermidis suspension containing 1.4 (1.1–1.7)×104 CFU. Ten Sprague Dawley rats were operated with implantation of the bone graft subfascially on each side of the interscapular region. After two weeks the grafts were excised. The plates were removed from the grafts and rinsed twice in saline. Aliquots of 50 μL were cultured. 10 plates were scraped, followed by vortex mixing of the knife blade; and 10 plates were sonicated at 30 kHz for five minutes. 50 μL of the saline used for a) vortex mixing of the knife blade, and b) sonication, was seeded on agar. After overnight incubation the number of CFU was counted. The total number of CFU recovered after scraping and sonication were 2(0–13) × 102 and 298(8–878) × 102, respectively (p< 0, 01). Compared to the number of CFU in the rinsing fluid, no increase was observed after scraping. For each plate that was sonicated there was a 38 (3–300) fold increase in the number of CFU. First, sonication is a superior technique for dislodging biofilm bacteria in an in vivo model, compared to scraping. Secondly, the present experimental model is a promising method for developing biofilm in vivo

Maggot therapy as an ancient method is succesfully used for treatment of acute and chronic wound infections in traumatology and orthopaedics. In this study, for the first time, the influence of sterile maggot excretions of Lucilia sericata on Pseudomonas aeruginosa (PAO1) biofilm formation on three common used orthopaedic materials was investigated. Sterile maggot excretions were collected according to a standardized method and the protein concentration was measured. The influence of the excretions on PAO1-biofilm formation was tested on comb-like devices, especially designed for these experiments, made from polyethylene, titanium and stainless steel. These combs were made to fit into a flat-bottom 96-wells microtiter plate. In the wells a suspension of PAO1-bacteria, nutrient medium and maggot excretions were pipetted. In the control wells, no excretions were added. Combs were placed in the wells and incubated for 24 hours at 37°C. The formed biofilms were stained in crystal violet and eluted with ethanol. The Optical Density (OD 595 nm) was read to quantify biofilm formation. The experiments were conducted with excretions from young maggots (Instar-1 maggots) and full grown maggots (Instar-3 maggots). All experiments were done in quadruplicate. The following can be concluded: PAO1-biofilm formation is the strongest on polyethylene and the weakest on stainless steel. Sterile maggot excretions are effective at preventing initial biofilm formation (p≤0.013) as well as preventing additional accumulation after its initiation (p≤0.038). The excretions even cause a significant breakdown of an existing biofilm (p≤0.028). Excretions from full grown maggots are more effective than those from young maggots. This study shows for the first time that sterile maggot excretions of Lucilia sericata inhibit biofilm formation, prevent its further grow and break down existing biofilms. While biofilm formation on orthopaedic materials is a severe complication, this experimental study could indicate a new treatment for biofilm formation on infected biomaterials

Aim. To evaluate the ability of different combinations of antibiotic loaded cement to inhibit bacteria growth and biofilm formation. Method. Cement beads were aseptically prepared using Palacos R (plain 40g PMMA cement) or Palacos R+G (40g PMMA cement containing industrially added 0.5g of gentamicin), with or without supplementary antibiotics as follows: Palacos R; Palacos R+G; Palacos R plus 1g / 2g daptomycin; Palacos R+G plus 1g / 2g of daptomycin; Palacos R plus 1g / 2g vancomcyin; and Palacos R+G plus 1g / 2g vancomycin. After production, each antibiotic loaded acrylic cement (ALAC) combination was allocated into two groups (group 1 and 2). The group 2 cement beads were initially eluted in broth at 37. o. C for 72hours then transferred to fresh broth containing a known concentration of bacteria. The group 1 samples were not eluted but directly immerse in culture broth containing bacteria. All samples were thereafter incubated at 37. o. C for 24 hours. After incubation, group 1 samples were visually assessed for bacterial growth, while for the group 2 samples, biofilm formation were quantified using ultrasonication and viable bacteria counting technique. Three proficient biofilm forming Staphylococcus epidermidis bacterial strains (1457, 1585-RA and 5179-R1) were used for all experiments and the bacteria counts were expressed as colony forming units / ml (CFU/ml). Results. In the group 1 samples, all the ALAC combinations were able to inhibit growth of all the three biofilm bacteria strains assessed except the gentamicin only samples in which biofilm growth were observed within 24hours. Meanwhile, in group 2, bacterial growth and biofilm formation by all three bacterial strains were observed on all the ALAC combinations, with the least biofilm formation being on the Palacos R+G plus 2g daptomycin combinations (mean CFU/ml: 1.04E +06) and the greatest on the gentamicin only cement (mean CFU/ml: 2.3E +07). Conclusions. Our study demonstrates that the highest antimicrobial activity of ALAC is seen in the first 24 hours. However, after 72 hours of antibiotic release, fresh bacterial exposure in fresh broth resulted in varying degrees of biofilm colonisation of all ALAC surfaces. Nonetheless, the overall biofilm formation was least on the gentamicin / daptomycin combinations and the results were statistically significant when compared to plain cement (p < 0.05, two tail t-test)

Aim. Staphylococcus aureus is the first causative agent of bone and joints infections (BJI). It causes difficult-to-treat infections because of its ability to form biofilms, and to be internalized and persist inside osteoblastic cells. Recently, phage therapy has emerged as a promising therapy to improve the management of chronic BJI. In the present study, we evaluated the efficacy of an assembly of three bacteriophages previously used in a clinical case report (Ferry, 2018) against S. aureus in in vitro models of biofilm and intracellular osteoblast infection. Methods. Using HG001 S. aureus, the bactericidal activities of the assembly of the three bacteriophages (Pherecydes Pharma) used alone or in association with vancomycin or rifampicin were compared by quantifying the number of viable bacteria in mature biofilms and infected osteoblasts after 24h of exposure. Results. The activity of bacteriophages against biofilm-embedded S. aureus was dose-dependent. Synergistic effects were observed when bacteriophages were combined to antibiotics at the lowest concentrations, with no significant bactericidal activity in monotherapy. In the human osteoblast infection model, we were able to show that phage penetration into osteoblasts was only possible when the cells were infected, suggesting a S. aureus dependent Trojan horse mechanism. The intracellular inoculum in osteoblasts treated with bacteriophages or vancomycin was significantly higher than in cells treated with lysostaphin, used as control condition of rapid killing of bacteria released in the extracellular media after death of infected cells and absence of intracellular activity. These results suggest that bacteriophages are probably both i) inactive in the intracellular compartment and ii) unable to kill all bacteria released after cell lysis into the extracellular medium fast enough to prevent them from reinfecting other osteoblasts. Conversely, the intracellular inoculum recovered from cells treated with vancomycin+bacteriophages was significantly lower than the one inside cells treated with vancomycin or bacteriophages alone, suggesting that this combination allowed a better control of released bacteria in the extracellular media. Finally, bacteriophages did not increase the activity of rifampicin in this model. Conclusion. In conclusion, we showed that the bacteriophages tested were highly active against S. aureus in mature biofilm but had no activity against bacteria internalized in osteoblasts. Additional studies using animal models of BJI and well-conducted clinical trials are needed to further evaluate phage therapy and its positioning in the management of these infections

Introduction: Propionibacterium acnes (P. acnes), a common anaerobic skin commensal, has been implicated in biomaterial-related infections (BRI). Bacteria can adhere to biomaterial surfaces and grow as a bio-film held together by exopolymer, exhibiting increased antimicrobial resistance. To our knowledge, images of P. acnes biofilms have not previously been published. We have demonstrated the ability of P. acnes to adhere to surgical steel and to develop a biofilm on this material. However its ability to adhere to and develop a biofilm on titanium, a commonly used surgical implant material, has not been fully investigated. Aims:. To determine the quantitative adherence and biofilm development of P. acnes on titanium compared to surgical steel. To assess the subsequent effect of penicillin, the therapeutic drug of choice, on mature P. acnes biofilms. Method: Six clinical isolates of P. acnes were assayed for adherence to materials with and without plasma glycoprotein conditioning film by chemiluminescence and culture. Biofilm development was assessed by chemiluminescence, fluorescence microscopy, environmental (ESEM) and scanning electron microscopy (SEM). Mature biofilms were exposed to plasma concentrations of penicillin and quantified by chemiluminescence and culture. Unpaired student’s t tests and univariate linear regression models were calculated using SPSS software (version 12). Results: Univariate linear regression showed that P. acnes adherence to titanium was 18% (p=0.001) greater than to steel. Adherence was reduced by the presence of the conditioning film on titanium by 28% (p=0.001), but this made no significant difference to P. acnes adherence to steel. P. acnes biofilms were clearly demonstrated, along with bacterial expolymer, showing an interesting similarity to biofilms of S. epidermidis. P. acnes grows as a thick biofilm on both materials held together by exopolymer and our preliminary results suggest that biofilms on titanium might be less susceptible to antimicrobials after 24 hours of penicillin treatment; a reduction of 94% on steel and 81% on titanium (p=0.057, p=0.39 resp). Conclusions: P. acnes adheres to steel and titanium, a crucial first step in BRI. Greater numbers of P. acnes adhere to titanium than to steel. The naked surface of titanium is microporous, assisting adhesion. A conditioning film reduces P. acnes adherence to titanium but not to steel. P. acnes develops as a biofilm on steel and titanium. Results indicate that pathogenesis of P. acnes infection on titanium is more successful than on steel. P. acnes biofilms on titanium may be harder to eradicate with antimicrobial agents

Direct observations have shown that the bacteria and fungi that cause device-related and other chronic infections grow in well developed biofilms on the surfaces of biomaterials and of compromised tissues. This mode-of-growth confers on the microorganisms an inherent resistance to host defenses, and antibiotic therapy, and makes these infections very difficult to detect because biofilm bacteria do not produce colonies when plated on the agar media used in routine cultures. We have initiated two comprehensive studies of total joint prostheses, and of non-unions secondary to trauma, in which we use DNA-based (Ibis and 454) methods for the detection of bacteria and an iterative process in which we locate and visualize biofilms using FISH probes and confocal microscopy. The DNA-based detection system confirms culture results, but detects more organisms and determines their sensitivity to antibiotics, and appears to be useful in the management of both types of infection. The use of confocal microscopy and FISH probes to visualize and map biofilms, in relation to orthopedic hardware and affected tissues, confirms the Ibis data and provides useful insights into the etiology of orthopedic infections

Introduction. According to the Australian registry 2014, periprosthetic joint infection (PJI) is the fourth important reason for revision of a primary total hip arthroplasty (THA). PJI is frequently caused by commensal strains of the skin such as Staphylococcus aureus or Staphylococcus epidermis. Deep infection is depending on many factors, such as implant surface chemical and physical behaviour, device design, host site, surgery and host response. Nevertheless, a lack of knowledge is seen concerning the specific effects of different surfaces on the biological response of different biomaterials. In addition, it is difficult to discriminate the material chemico-physical properties by the topological features, such as surface roughness. Indeed, it has been widely demonstrated that surface composition, electric charge, wettability and roughness of implant surfaces have a strong influence on their interactions with biological fluids and tissues. Therefore, also bearing surface properties can influence the incidence of PJI, just shown recently. Objectives. To verify the capability of ceramic bearings to reduce bacteria biofilm adhesion by means of their surface chemico-physical properties. Methods. The surface chemico-physical properties of the most common materials in THA as monolithic alumina, zirconia platelet toughened alumina (ZPTA), zirconia (TZP), titanium alloy (Ti6Al4V), stainless-steel and cobalt alloy (Co28Cr6Mo) were compared. All materials were characterized using x-ray photoelectron spectroscopy (XPS), fourier transform spectroscopy (FTIR), x-ray diffraction (XRD) and zeta-potential. Additionaly wettability by contact angle measurement with various media as simulated body fluid (SBF), bacterial broth, cell culture media and fetal bovine serum (FBS) was determined. Furthermore, the surface protein adsorption amount was evaluated by bicinchoninic acid (BCA) assay analysis using FBS as protein source. Selective protein adsorption was also evaluated by electroforetic technique. The specimens' surface anti-bacterial adhesion activity was evaluated by Staphylococcus aureus biofilm formation after 24h by colonies forming units count. Cytocompatibility was assessed using human primary osteoblasts cell culture and MTT assay. Results. The surface of all tested materials was found to be electronegative at physiological pH by means of zeta-potential measurement. Nevertheless, monolithic alumina and ZPTA have the isolectric point at lower pHs and adsorbed a larger amount of proteins (albumin and fibronectin) in comparison with metal surfaces. Such feature might be correlated with bacteria biofilm growth, since the ceramic surfaces were also less colonized by Staphylococcus aureus in comparison to metal surfaces (p<0.005) while they maintained the ability to promote osteoblasts adhesion and proliferation. The above results were confirmed by XPS technique where the ceramic surfaces had less hydroxyl groups and consequently were less prone to adhere with biological species as the bacteria. No correlation was observed using the FTIR and XRD surface characterization techniques. Conclusions. The ceramic bearing surfaces were found to reduce the bacteria biofilm adhesion, because of their surface chemico-physical properties

Background. The main reasons for hip prosthesis failure are aseptic loosening and periprosthetic joint infection (PJI). The real frequency of PJI is probably largely underestimated because of non-standardized definition criteria, diagnostic procedure, treatment algorithm and other confounders. Therefore, data from joint registries are not reflecting the frequency of PJI and can be misleading; particularly low-grade PJI can be frequently misdiagnosed as aseptic failure. Therefore, prospective clinical studies with standardized protocol, comprehensive diagnostic procedure and sufficient follow-up should be performed. Sonication of explanted prosthesis is highly sensitive for detection of biofilms on prosthetic surface and allows quantitative analysis of biofilm formation. We hypothesize that by using sonication, ceramic components (BIOLOX®delta, BIOLOX®forte) will show higher resistance against biofilm adhesion compared to polyethylene (PE) and metal (CoCrMo). Methods. In this prospective multicentre study (level of evidence: Ia), we included all consecutive adults ≥18 years of age, who underwent explantation of the hip prosthesis for infection or aseptic reason. Excluded were patients in whom part of the prosthetic components were retained. A standardized and comprehensive diagnostic algorithm was applied, including sonication of all removed prosthetic components for qualitative and quantitative microbiological analysis (ultrasound bath 40 kHz, 1 W/cm2, 1 min). Individual components (metal, PE, ceramic) were separately placed in sterile boxes for investigation. All patients were simultaneously included in the European Prosthetic joint infection cohort (EPJIC, . www.epjic.org. ) to ensure long-term follow-up. Results. Up to date, 79 patients were included, of whom 47 (60%) were diagnosed with aseptic failure and 32 (40%) with PJI. Mean age was 73 years (27–87 years), 32 (41%) were males. Table 1 summarizes the demographic characteristics. In 32 patients with PJI, most frequently isolated organisms were coagulase-negative staphylococci (n=12, 38%), Staphylococcus aureus (n=7, 22%) and Propionibacterium acnes (n=4, 13%), followed by enterococci (n=2; 6%) and gram-negative bacilli (n=2; 6%); 2 infections (6%) were polymicrobial and 3 were culture-negative (9%). Table 2 summarizes the microbiological results from sonication of removed components. Causative microorganism could be detected in sonication fluid from polyethylene in 100%, from metal in 92% and from ceramic in 69%. Significantly lower bacterial counts expressed as colony-forming units (CFU) were detected in sonication fluid from ceramic components (230 CFU/ml) than from PE (6’250 CFU/ml) and metal components (5’870 CFU / ml) (p < 0.01). Conclusions. These first results support the hypothesis that significantly less biofilm biomass is formed on ceramic surface, compared to PE and metal surfaces, potentially indicating higher ceramic “resistance” against bacterial adhesion. These findings should be confirmed with non-microbiological investigation such as imaging (fluorescent in situ hybridization, confocal laser scanning or electron microscopy). Furthermore, in 6 of 32 patients (19%) with PJI, an aseptic loosening was preoperatively suspected. Infection was found only by systematic application of an optimized diagnostic method, particularly sonication of the removed implant. Final study results are expected to be available by the end of 2016

Introduction:. Periprosthetic infections that accompany the use of total joint replacement devices cause unwanted and catastrophic outcomes for patients and clinicians. These infections become particularly problematic in the event that bacterial biofilms form on an implant surface. Previous reports have suggested that the addition of Vitamin E to ultra-high-molecular-weight polyethylene (UHMWPE) may prevent the adhesion of bacteria to its surface and thus reduce the risk of biofilm formation and subsequent infection. 1–3. In this study, Vitamin E was blended with two types of UHMWPE material. It was hypothesized that the Vitamin E blended UHMWPE would resist the adhesion and formation of clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) biofilms. Methods and Materials:. Five sample types were manufactured, machined and sterilized (Table 1). To determine if MRSA biofilms would be reduced or prevented on the surface of the Vitamin E (VE) loaded samples (HXL VE 150 kGy and HXL VE 75 kGy) in comparison to the other three clinically relevant material types, each was tested for biofilm formation using a flow cell system. 4. Direct Bacterial Quantification – An n = 7 samples of each material type were placed individually into a chamber of the flow cell. A solution of 10% modified brain heart infusion (BHI) broth containing 10. 5. MRSA cells/mL was flowed through each chamber. Using previously established protocols,. 4–7. after 48 hours of growth, each sample was removed, and the number of colony forming units (CFU) determined using a 10-fold dilution series. SEM Imaging – Using the same protocol as above, after the 48-hour incubation period, an n = 7 of each material type were fixed in 2.5% glutaraldehyde, dehydrated in ascending concentrations of ethanol, coated with carbon and imaged using scanning electron microscopy (SEM). Results:. Results indicated that the Vitamin E blended materials did not resist the attachment/formation of MRSA biofilms to any greater degree than the other three material types. All materials had greater than 10. 7. CFU/cm. 2. (Figure 1). SEM images corroborated with the quantification data (Figure 2). Discussion:. In contrast to previously published results,. 1–3. these data indicated that Vitamin E blended UHMWPE may not have the ability to prevent biofilm formation of a clinical MRSA isolate from occurring

Aim. Aim of this study was to evaluate the ability of Sb-1 to enhance the antibiotic activity (tested in combination) degrading the biofilm matrix (impairing the freely diffusion of antimicrobials) and specifically targeting “persister” cells (biofilm sub-population tolerant to most antibiotics and responsible for the infection recalcitrance) of methicillin-resistant Staphylococcus aureus. Method. MRSA ATCC 43300 24h-old biofilm was treated for 18h with Sb-1 titers (from 10. 4. to 10. 6. pfu/ml). Biofilm matrix was evaluated by confocal laser scanning microscopy after staining with wheat germ agglutinin conjugate with Alexafluor488 (WGA488) to label exopolysaccharide matrix and Syto 85 to label bacterial cells. Persister status was induced using two different protocols: i) by exposing stationary phase S. aureus to 400 µg/ml carbonyl cyanide m-chlorophenylhydrazone (CCCP) in PBS for 3h at 37°C and ii) by treatment of 24h old biofilm with 512 µg/ml ciprofloxacin for further 24h at 37°C. Then, induced persister cells and non-induced controls (10. 6. CFU/ml) were treated with 10. 4. PFU/ml and 10. 7. PFU/ml Sb-1 for 3h, followed by CFU counting. Alternatively, bacteria were washed and incubated in fresh BHI medium for the resumption of normal growth and the bacterial growth assessed after further 24 hours. Results. Sb-1 showed a dose-dependent reduction of exopolysaccharide components of MRSA biofilm matrix at sub-inhibiting phage titers. With 10. 6. PFU/ml Sb-1, no fluorescent signal related to WGA488 was detected, although bacterial viability was not impaired. Higher Sb-1 titer (10. 7. PFU/ml) determined a strong reduction (ranging between 2.5 – 5 log CFU/ml) of persister cells. By contrast, in presence of 10. 4. PFU/ml Sb-1, no reduction was observed in persister cells. However, persister cells pre-treated with 10. 4. pfu/ml Sb-1 were completely killed when bacteria were inoculated after phage treatment in fresh medium, reverting to a normal-growing phenotype. Conclusions. Due to its ability to degrade the MRSA exopolysaccharide matrix at sub-inhibitory concentrations and kill persister cells, directly at higher titers or indirectly with lower titers, Sb-1 phage is a valid therapeutic option to be used alone or in combination with current antibiotics for the successful eradication of methicillin resistant S. aureus biofilm associated with prosthetic joint infections

Background and Purpose: The remarkably low wear of metal-on-metal (MOM) bearings involving cobalt-chromium (Co-Cr) alloys has led to a resurgence in its use. However, consequences of these wear particles and the corrosion products are for the most part unclear. Recent research efforts towards the bacteriological influences of the MOM-degradation products suggested that particulate MOM debris promotes planktonic bacterial growth. On the other hand, extremely high concentrations of metal ions, derived from salts, have shown to possess bacteriostatic effects (growth reduction) on planktonic growth and on biofilm formation. The effects of salt-derived metal ions were found to be inhibitory and not bactericidal (lethal to bacteria). However, these two findings were both found under static growth conditions and no studies have investigated these findings under more clinically resembling dynamic growth conditions. In addition, influences of Co-Cr particles on biofilm formation have not yet been studied. Therefore, the aim of this study was to evaluate how Co-Cr particles and Co-Cr ions affect biofilm formation under static and dynamic growth conditions. Methods: A collection of clinically isolated bacterial strains were exposed to Co-Cr particles and Co-Cr ions in concentrations as found in serum and above as found in adjacent tissue. The experiments were conducted as well under static, as under dynamic growth conditions. Biofilm formation in wells, stained with live/dead viability staining and visualized by confocal laser scanning microscopy, was analyzed with COMSTAT, yielding biovolume, biofilm thickness, and live/dead ratio of the bacteria within the biofilm. Results: Co-Cr particle concentrations of 20 g/L reduced biofilm formation significantly. Moreover, these particle concentrations were found to be bactericidal (killed the bacteria). The live/dead ratio decreased when culturing was done under dynamic growth conditions when compared to the static growth condition. Under both growth conditions, biofilm formation was inhibited at concentrations of 10/5 mg/L Co-Cr ions, as reported to occur in synovial fluids. Co-Cr ion concentrations up to 1/0,5 mg/L revealed no consistent influence on biofilm formation. Interpretation: Long-term clinical data on infection rates for Co-Cr MOM-bearings are not yet available, but the current results suggest that Co-Cr ions may yield these prostheses less prone to biofilm formation and subsequent infection

A phenomenon of methicillin resistance in methicillin sensitive Staphylococcus aureus has been noted in organisms living in biofilm induced by the state of cell wall deficiency. The rate and the amount of biofilm formed by the cell wall deficient organisms far exceeds that of cell wall patent organisms. Once removed from the biofilm the S. aureus had the same sensitivities of the original organism. Cell wall deficient organisms outside the biofilm did not demonstrate the methicillin resistance. A known laboratory strain (ATCC 9144) was induced into a cell wall deficient state and allowed to form biofilm. The rate of formation and amount formed was compared with that formed by cell wall patent organisms. Before inducing cell wall deficiency sensitivity to methicillin was demonstrated using standard microbiological technique. Using an oxacillin containing plate as a culture medium: the biofilm, cell wall deficient organisms and the cell wall competent organisms were inoculated onto separate media. Organisms from the biofilm were isolated and grown free of the biofilm on blood agar. Any growth on the oxacillin containing plate would demonstrate methicillin resistance. There was no growth on the plates containing the cell wall competent or cell wall deficient organisms. There was however growth on the plate inoculated with bio-film, however when organisms were isolated from the biofilm, there was no growth on the media. Antibiotic sensitivities of the original inoculant and the organisms isolated from the biofilm were the same. The biofilm, induced as a result of cell wall deficiency, offers a form of structural protection to the Staphylococcus aureus without altering the resistance pattern of organism. Standard microbiological techniques would therefore report the organism as methicillin sensitive, however clinically the organism may behave as a methicillin resistant organism. The state of cell wall deficiency encourages the formation of biofilm in S. aureus. In-vitro the state of cell wall deficiency is induced using high osmolality media or sub-lethal doses of cell wall active antibiotics. Both these states are found in clinical practice

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. Finally we demonstrate that pbp4, a key gene in cell wall synthesis, is down-regulated under sodium and potassium stress. 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. Gene transcription was assessed using RT-PCR. Results: There was a positive correlation between biofilm 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. A key gene involved in cell wall production (pbp4) was down-regulated under sodium (p = 0.03) and potassium (p = 0.03) stress. 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 prostheses may be more likely in areas of major tissue trauma such as large resections and revisions. Furthermore, cellular stress leads to increased resistance to penicillin, a cell wall active antibiotic, in naïve cells which may nullify prophylaxis and complicate bacterial eradication in vivo. Finally we postulate a link between the experimental rise in penicillin resistance and the down-regulation of pbp4 demonstrated by RT-PCR under the same halide stresses

The use of sub-lethal doses of cell wall active antibiotics to induce cell wall deficiency in S aureus has been described. Cell Wall Deficient S aureus show an increased in-vitro ability to form biofilm. Cephalosporins(cell wall active antibiotics.) are commonly used at time of arthroplasty surgery as antimicrobial prophylaxis. Adherence is fundamental step in biofilm formation. The adherence of cell wall deficient S aureus versus ‘wild type’ S aureus to glass was investigated. Slides comparing the two types of organisms were analysed using fluoroscopy and J-image software. The ability to adhere to plastic was investigated using a micro-titre based absorption test. In a third investigation a centrifugal force was used to quantify the adherence ability of the cell wall deficient organisms to the glass slides. The cell wall deficient organisms demonstrated an increased ability to adhere to glass compared to the ‘wild type’. After exposure, there was on average twenty times more cell wall deficient organisms per unit area compared to the ‘wild-type’. The micro-titre plates were similar. After incubation, the absorption of each well was measured. Compared to the ‘wild type’ there was a significantly increased absorption in wells containing the cell wall deficient organisms. Showing an increased ability to adhere to plastic. The third technique quantified the ability to adhere using a centrifugal force. The slides were exposed to ‘wild type’ and cell wall deficient organisms, however before staining they were placed in a centrifuge. On analysis there were five cell wall deficient S aureus per field of view, compared to 0.5 ‘wild-type’. An increased ability of cell wall deficient S aureus to adhere to surfaces has been shown. Adherence is fundamental to biofilm formation. The significance to orthopaedics is that the inadequate use of Cephalosporins at time of operation may be facilitating chronic infections

Background: Bacteria form a biofilm on the surface of orthopaedic devices, causing persistent and infection. Little is known about biofilms formation on bone grafts and bone substitutes. We analyzed various representative materials regarding their propensity for biofilm formation caused by Staphylococcus aureus. Methods: As bone graft beta-tricalciumphosphate (b-TCP, CyclOsTM) and as bone substitute a tantalum metal mesh (trabecular metalTM) and PMMA (Pala-cosTM) were investigated. As test organism S. aureus (strain ATCC 29213) was used. Test materials were incubated with bacterial solution of 105 colony-forming units (cfu)/ml at 37°C for 24 h without shaking. After 24 h, the test materials were removed and washed 3 times in normal saline, followed by sonication in 50 ml Ringer solution at 40 kHz for 5 minutes. The resulting sonication fluid was plated in aliquots of 0.1 ml onto aerobe blood agar with 5% sheep blood and incubated at 37°C with 5% CO2 for 24 h. Then, bacterial counts were enumerated and expressed as cfu/ml. All experiments were performed in triplicate to calculate the mean ± standard deviation. The Wilcoxon test was used for statistical calculations. Results: The three investigated materials show a differing specific surface with b-TCB> trabecular metal> PMMA per mm2. S. aureus formed biofilm on all test materials as confirmed by quantitative culture after washing and sonication. The bacterial counts in sonication fluid (in cfu/ml) were higher in b-TCP (5.1 x 106 ± 0.6 x 106) and trabecular metal (3.7 x 106 ± 0.6 x 106) than in PMMA (3.9 x 104 ± 1.8 x 104), p< 0.05. Conclusion: Our results demonstrate that about 100-times more bacteria adhere on b-TCP and trabecular metal than on PMMA, reflecting the larger surface of b-TCP and trabecuar metal compared to the one of PMMA. This in-vitro data indicates that bone grafts are susceptible to infection. Further studies are needed to evaluate efficient approaches to prevent and treat infections associated with bone grafts and substitutes, including modification of the surface or antibacterial coating

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

Copal bone cement loaded with gentamicin and clindamicin was developed recently as a response to the emerging occurrence of gentamicin-resistant strains in periprothetic infections. The objective of this study was to compare the in vitro antibiotic release and antimicrobial efficacy of gentamicin/clindamicin-loaded Copal bone cement and gentamicin-loaded Palacos R-G bone cement, as well as biofilm formation on these cements. In order to determine antibiotic release, cement blocks were placed in phosphate buffer and aliquots were taken at designated times for measurement of antibiotic release. In addition, the bone cement discs were pressed on agar to study the effects of antibiotic release on bacterial growth. Biofilm formation on the different bone cements was also investigated after 1 and 7 days using plate counting and confocal laser scanning microscopy (CLSM). Experiments were done with a gentamicin-sensitive S. aureus and a gentamicin-resistant CNS. Antibiotic release after 672 h from Copal bone cement was more extensive (65% of the clindamycin and 41% of the gentamicin incorporated) than from Palacos R-G (4% of the gentamicin incorporated). The higher antibiotic release from Copal resulted in a stronger and more prolonged inhibition of bacterial growth on agar. Plate counting and CLSM of biofilms grown on the bone cements showed that antibiotic release reduced bacterial viability, most notably close to the cement surface. Moreover, the gentamicin-sensitive S. aureus formed gentamicin-resistant small colony variants on Palacos R-G, and therefore, Copal was much more effective in decreasing biofilm formation than Palacos R-G. Biofilm formation on bone cement could be more effectively reduced by incorporation of a second antibiotic, next to gentamicin. Antibiotic release from the cements had a stronger effect on bacteria close to the cement than on bacteria at the outer surface of the bio-film. Clinically, bone cement with two antibiotics may be more effective than cement loaded with only gentamicin. The clinical efficacy of antibiotic loaded bone cements in combination with systemic antibiotics can be explained because antibiotics released from cements kill predominantly the bacteria in the bottom of the biofilm, whereas systemic antibiotics can only deal with bacteria at the outer surface of the biofilm

Introduction The aim of this study was firstly to investigate the prevalence of icaABCD-operon which codes the production of the polysaccharide intracellular adhesin(PIA), responsible for biofilm production, in a collection of clinically significant staphylococci isolated from orthopaedic infections and secondly to assess the relationship between biofilm production and the presence or not of ica-operon. First Step – Material & Methods Between 1/2003 and 12/2005 200 CoNS were isolated from orthopaedic patients associated with soft tissue and bone infections(group I) and 200 CoNS from blood cultures of hospitalized patients from different wards of the same Hospital(group II). Identification was carried out by Gram-stain, catalase and coagulase tests and the API Staph System. Detection of icaADBC genes was performed by PCR. Production of biofilm was tested by the method of Christensen. Results In group I, 62(31.37%) carried the entire ica-operon; from these isolates biofilm formation was detected in 35(17.5%). 5 isolates, despite biofilm production, did not carry any gene of ica-operon. In group II, 70(35.5%) carried entire the ica-operon; biofilm formation was detected in 37(18.5%) of these isolates. 3 S. capitis, 1 S. epidermidis and 1 S. hominis carried only the icaADB, icaA and icaB genes respectively. Second Step – Material & Methods Based on the observation of PIA-production only in (50%) of ica(+) CoNS, 20 S. epidermidis isolates recovered from clinical specimens (pus) of orthopaedic patients and belonging to distinct PFGE clones, were selected on the basis of the presence of the entire ica operon. Nevertheless, only 10 of them produced biofilm. Nucleotide sequence analysis of ica-operon was carried out in all isolates; expression of icaADBC genes was also tested by RT-PCR. Results Sequencing analysis revealed that all isolates carried an intact ica-operon, without point mutations. Concerning icaADBC mRNA production, all genes of ica-operon were expressed in biofilm-producing isolates, whereas in the no-biofilm producing strains the icaA and icaC genes were not expressed, while a faint expression was observed for the icaB and icaD genes. Discussion Biofilm-forming capacities of CoNS from orthopaedic infections was not significantly greater than those from other infections (p> 0,05). The capacity of ica-operon(+) staphylococcal isolates to form biofilm seems to be dependent on the expression of ica-genes, specifically of icaA and icaC. The inability of ica(+) isolates to produce biofilm emphasizes that some unknown mechanisms influence icaADBC expression. Finally, the recognition of biofilm-producing CoNS without carrying any gene of ica operon underlined the existence of unidentified also mechanisms controlling biofilm production, apart from icaADBC expression

Background: Bacteria form biofilms on the surface of orthopaedic devices, causing persistent infections. Monitoring biofilm formation on bone grafts and bone substitutes is challenging due to heterogeneous surface characteristics. We analyzed various bone grafts and bone substitutes regarding their propensity for in-vitro biofilm formation caused by S. aureus and S. epidermidis. Methods: Beta-tricalciumphosphate (β-TCP, Chro-nOsTM), processed human spongiosa (TutoplastTM) and PMMA (EndobonTM) were investigated. PE was added as a growth control. As test strains S. aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) were used. Test materials were incubated with defined bacterial solution (105 colony-forming units (cfu)/ml) at 37°C for 24 h without shaking. After 24 h, the test materials were removed and washed 3 times in PBS, followed by a standardised sonication protocol (Trampuz et al. 2007, NEJM). The resulting sonication fluid was plated in aliquots of 100μl onto aerobe blood agar with 5% sheep blood and incubated at 37°C with 5% CO2 for 24 h. Bacterial counts were enumerated and expressed as cfu/ml. Sonicated samples were transferred to a microcalorimeter (TA Instrument) and heat flow at 37°C was continuously monitored over a 24h period with a precision of 0.0001°C and a sensitiviy of 200μW. All experiments were performed in triplicates to calculate the mean ± standard deviation. ANOVA analysis was used for statistical calculations. Results: For S. aureus bacterial counts (log10 cfu/ sample) were significantly higher (p< 0.001) for the porous (β-TCP 7.67 ± 0.17, Tutoplast 7.65 ± 0.15) than for the solid samples (PMMA 6.12 ± 0.18, PE 5.17 ± 0.22). Bacterial density (log10 cfu/surface) was 10^1–10^2 times higher for the S. epidermidis than for the S. aureus. In calorimetry the shape of the heat flow curves was characteristic for the individual strain and was not influenced by the test materials. The time to detection (TTD) was shortest for β-TCP for both strains and TTD was always shorter for S. aureus than S. epidermidis with corresponding material. Cfu/sample calculated from the calorimetric data was concordant with the standard culturing method. Conclusion: Our results demonstrate biofilm formation with both strains on all tested materials. The calorimetry in all cases was able to detect quantitatively the amount of biofilm. Further studies are needed to see whether calorimetry is a suitable tool also to monitor approaches to prevent and treat infections associated with bone grafts and bone substitutes

S epidermidis and P aeruginosa are recognised major biofilm pathogens in medical device contamination and chronic wounds. Within biofilms, bacteria are enclosed in a polymeric matrix that cements them to each other and to the surface and protects them by increasing resistance to host immunity, antibiotics and biocides. Staph and pseudomonas spp biofilm were grown on glass coupons for 48 hours and the coupons randomly inserted into the wound model for 24 hours and subjected to TNP and the following:. No instillation. 0.1% w/v formulated hypocholorous acid (FHA) instillation. Saline instillation. Betadine instillation. Betadine and saline instillations were for 30 minutes, while FHA was for three minutes, every eight, four and two hours per day. The biocides were at sub-lethal concentrations. The coupons were then extracted to avoid damaging the biofilms and effect of TNP was assessed by colony forming units and electron microscopy. The results show that lower frequency of instillation did not have significant effect on bacterial load for both types of bacteria. Increase in frequency of instillations resulted in no growth of pseudomonas while increase in frequency of instillations resulted in a significant decrease in growth of staph spp. Frequent flushing of the wound model resulted in a loss of biofilm bacteria for both Pseudomonas and Staph epi. The biocides combined with TNP were more effective in killing Pseudomonas compared with Staph epi

Aim: The aim of the project was to discover if bacteria were implicated in non-union of fractures of the tibia and femur, which had been treated with intramedullary nailing. Method: 40 intramedullary nails removed from tibial and femoral fractures were retrieved for the purpose of the study. 20 of these nails were from fractures, which had successfully united and were removed for mainly anterior knee pain or discomfort at screw sites. These nails formed the control group for the project. 20 nails were removed from fractures which had failed to unite prior to further operative intervention such as exchange nailing or the application of an Ilizarov frame. These fractures had no clinical evidence of infection and formed the study group for the project. The nails were subjected to ultrasound in the research laboratory to dislodge adherent bacterial formed as biofilm from the surface of the nail. Using both standard culture techniques and non-culture techniques, any dislodged bacteria were isolated and identified. Non-culture techniques involved the use of specific monoclonal antibody labelled immunofluorescence microscopy. Isolated bacteria were tested for the sensitivities of commonly used antibiotics in orthopaedic practice according to NCCLS guidelines. Results: We discovered that bacteria were detected in up to 70% of the nails removed from fractures, which had failed to unite, using both standard culture and non-culture techniques. Also, we discovered that bacteria were detected in up to 30% of the nails removed from fractures that had united. The organisms identified were mainly the coagulase negative staphylococcus epidermidis and the gram-positive anaerobe proprionibacterium acnes. The antibiotics gentamicin, erythromycin, cefotaxime and cefomandole performed poorly against the isolated bacteria. Vancomycin, ciproxin and fucidin however performed better. Conclusion: Bacteria formed as biofilm on intramedullary nails may have a significant role in the development of non-union fractures treated by this method. The bacteria isolated showed worrying resistance to commonly used antibiotics in orthopaedic practice, in particular, those used as prophylaxis

The determination of the cause of prosthetic failures in total hip arthroplasties can be difficult. Pre-operative imaging, including plain x-rays, tri-phse bone scan and MRI imaging have not been able to discern septic from aspectic causation. White blood cell scans, once thought specific for infection when positive, has demonstrated positivity in”wear and debris” reactions. Labs including WBC, Sed Rate, CRP can be elevated in septic, as well as, aseptic failures. Although frozen section is reliable showing acute inflammation for infection, chronic active inflammation which often is seen with chronic infections, can also be seen in aspectic failures. Cultures are often falsely positive, but in chronic infection it may be associated with less than 80% retrieval. Five cases of acetabular loosening associated with radiolucencies around a prosthesis were studied. These cases had rapid failures and were thought to be secondary to an oil residual left after processing of an in growth prosthesis. All patients had a radiolucent zone around the bone prosthetic intersurface. The patients had increasing pain on weight bearing and a positive bone scan. Frozen section at the time of surgery demonstrated chronic inflammation and was culture negative. The acetabular prosthesis and associated parts were placed immediately in 80% Etoh and Tris buffer. Combinations of confocal laser microscopy with live-dead stains, FISH Probes for Staph., or scanning electron microscopy was performed looking for biofilm. All five of the prosthesis or related parts showed the presence of bacterial biofilm. One of these had cement covering the porous portion. These results demonstrate our inability to discern aseptic from septic loosening in total hip arthroplasty by the current clinical means

Prosthetic joint infections are difficult to treat due to bacterial biofilm. Our group has developed a linezolid elution system by human cancellous bone delivering high concentrations the first 48 hours (Giannitsioti et al. 53rd ICAAC, 2013: A-1050). We tested the activity of this system to inhibit growth of one ica expressing isolate of Staphylococcus epidermidis (MRSE). At a first step, sterile mesh cylinders containing bone particles of the femoral head of healthy volunteers (MCB) were impregnated into 3mg/ml linezolid for 1, 24 and 48 hours. Then log-phase inocula of 103, 105 and 107cfu/ml were exposed to MCB at 370C for 8 days with regular readings of bacterial growth. MCB were transferred into fresh Muller-Hinton Broth (MHB) every 24h to avoid material corrosion. At a second step, to simulate the ability of the system against biofilm-coated MCB, MCB without linezolid were incubated with 103 and 105 cfu/ml for 1 and 24h. MCB were daily transferred into fresh MHB containing 100μg/ml on day 1, 15 μg/ml on day 2, 3 μg/ml on day 3 and 0.5 μg/ml on day 4. 24h linezolid impregnated MCB achieved rapid bacterial killing of the 105 cfu/ml bacterial suspension followed by re-growth (Figure, n= 5). Similar results were observed for 1h and 48h impregnation and for both tested inocula. When biofilm-coated MCB generated by 24h exposure to 105 cfu/ml were exposed to linezolid, rapid bacterial killing was achieved followed by re-growth. Linezolid local elution may inhibit biofilm-producing MRSE only when locally eluted concentrations exceed 10μg/ml

Periprosthetic joint infection (PJI) is a difficult complication requiring a comprehensive eradication protocol. Cure rates have essentially stalled in the last two decades, using methods of antimicrobial cement joint spacers and parenteral antimicrobial agents. Functional spacers with higher-dose antimicrobial-loaded cement and antimicrobial-loaded calcium sulphate beads have emphasized local antimicrobial delivery on the premise that high-dose local antimicrobial delivery will enhance eradication. However, with increasing antimicrobial pressures, microbiota have responded with adaptive mechanisms beyond traditional antimicrobial resistance genes. In this review we describe adaptive resistance mechanisms that are relevant to the treatment of PJI. Some mechanisms are well known, but others are new. The objective of this review is to inform clinicians of the known adaptive resistance mechanisms of microbes relevant to PJI. We also discuss the implications of these adaptive mechanisms in the future treatment of PJI.

Cite this article: Bone Joint J 2022;104-B(5):575–580.

Periprosthetic joint infection (PJI) remains an extremely challenging complication. We have focused on this issue more over the last decade than previously, but there are still many unanswered questions. We now have a workable definition that everyone should align to, but we need to continue to focus on identifying the organisms involved. Surgical strategies are evolving and care is becoming more patient-centred. There are some good studies under way. There are, however, still numerous problems to resolve, and the challenge of PJI remains a major one for the orthopaedic community. This annotation provides some up-to-date thoughts about where we are, and the way forward. There is still scope for plenty of research in this area.

Cite this article: Bone Joint J 2022;104-B(11):1193–1195.

The first aim of the study was to investigate if bacteria were implicated in non-union of fractures of the tibia and femur, which had been treated with intramedullary nailing. The second aim was to evaluate the antimicrobial susceptibility of bacteria isolated from the retrieved intramedullary nails. Forty intramedullary nails removed from tibial and femoral fractures were retrieved for the purpose of the study. Twenty of these nails were from fractures, which had successfully united and 20 were removed from fractures which had failed to unite prior to further operative intervention. There was no evidence of clinical infection in either of the two groups. The nails were subjected to ultrasound in the research laboratory to dislodge adherent bacteria formed as biofilm from the surface of the nail. Using both standard culture techniques and non-culture techniques (Immunofluorescence microscopy and PCR analysis) any dislodged bacteria were isolated and identified. Isolated bacteria were tested for antimicrobial susceptibility to commonly used antibiotics in orthopaedic practice according to NCCLS guidelines. Bacteria were detected in 15 out of 20 [75%] of the nails removed from fractures, which had developed a non-union, and in 5 out of 20 [25%] of fractures that had united, using both standard culture techniques and non-culture techniques. The bacterial isolates identified were mainly Staphylococcus epidermidis and the Gram-positive anaerobe Proprionibacterium acnes. Vancomycin was the most effective antibiotic, with 2 out of 34 [6%] isolates being resistant. Erythromycin was the least effective, with 21 out of 34 [62%] isolates being resistant. Based on overall Minimum Bactericidal Concentrations at which 90% of all strains were killed, Vancomycin was the most active bactericidal agent tested followed in decreasing order by fucidic acid, ciprofloxacin, gentamicin, cefamandole and erythromycin. Bacteria were detected more commonly in the fracture non-union group than in the union group [p< 0.01]. Of the antibiotic agents tested Vancomycin was the most effective and Erythromycin was the least effective

Use of allograft bone has become standard for bridging defects unlikely to heal by simple fixation and routinely used in revision arthroplasties for implant stabilization. Unfortunately, this decellularized allograft provides an ideal surface for bacterial colonization, necessitating repeated surgeries, extensive debridement and lengthy antibiotic treatments. With up to 18% infection rate following allograft surgeries, a need for more effective means to prevent this process is evident. We describe a novel modification of native bone allografts that renders their surface bactericidal while increasing the effectiveness of systemic antibiotic treatments. Allograft modification: Morselized human bone was washed extensively and sequentially coupled: 2X with Fmoc-aminoethoxyethoxyacetate (Fmoc-AEEA); deprotected with 20% piperidine in Dimethylformamide (DMF); and then coupled with vancomycin (VAN) for 12–16 hours. The VAN-bone was washed extensively with DMF and PBS for at least 1 day. VAN immuno-fluorescence: Control or VAN-bone was washed 5X with PBS, blocked with 10% FBS (1hr), incubated with rabbit anti-VAN IgG (4oC, 12h) followed by an Alexa-Fluor 488-coupled goat anti-rabbit IgG (1hr), and visualized by confocal laser microscopy. Antibiotic Activity. Equal dry weights of control and VANbone were sterilized with 70% ethanol, rinsed with PBS, and incubated with either Staphylococcus aureus (S. aureus) or Escherichia Coli (Ci=104 cfu) in TSB, 37oC, for 2, 5, 8 and 12 hrs. Antibiotic treatment: Clinical grade vancomycin was added to the solution with bacteria or following infection at a final concentration of 10μg/ml. Bacterial counts: Non-adherent bacteria were removed by washing and adherent bacteria suspended by sonication in 0.3% Tween-80 for 10mins followed by plating on 3M. ®. Petrifilms. Bacterial visualization: Non-adherent bacteria were removed by washing extensively with PBS and adherent bacteria stained with the Live/Dead BacLight Kit (20mins, RT) to cause viable bacteria to fluoresce green. Samples were visualized by confocal microscopy. In comparison to controls, VAN-bone consistently reduced the graft bacterial load by ~90% at all time points. After staining and visualization of adherent bacteria, biofilm formation was apparent on controls by 12 hrs and absent from VAN-bone. E.coli, a gramnegative organism that is not sensitive to VAN, readily colonized both control and VANbone, confirming retention of VAN specificity. We then evaluated VAN-bone activity in a system that modeled systemic antibiotic therapy and antibiotic prophylaxis. In the absence of solution antibiotics, VAN-bone exhibited a significant decrease in bacterial colonization as compared to controls. When 10 μg/ml VAN was added to the medium for the last 4 h (modeling systemic antibiotic therapy), colonization of control surfaces was reduced, while colonization of VAN-allograft was almost eliminated. When 10 μg/ml VAN was added concomitantly with S. aureus, VAN-bone colonization was undetectable, while colonization of control surfaces still occurred. We have previously described an antibiotic-tethered allograft that resists bacterial colonization. In this abstract, we test this technology with an vitro model of bone implantation in the presence of solution antibiotics. In these models, solution antibiotics failed to prevent infection of control bone while completely clearing the bacteria on VAN-bone. Furthermore, VAN bone exhibited high activity against S. aureus, a gram positive organism, whereas it was ineffective against E. coli, a gram negative organism. The specificity of the tethered antibiotic supported the view that the antibacterial properties of the allograft were related to the tethered antibiotic and not to undefined aspects of the attachment chemistry. In terms of antibacterial activity, when challenged with 104 CFU S. aureus (with concentrations reaching > 107 CFU by 24 h), the antibiotic -modified allograft consistently decreased bacterial colonization by > 90%; S. aureus inocula < 102 CFU resulted in no detectable colonization of the VAN-allograft. Thus, development of these allografts may not only combat allograft colonization but increase the effectiveness of prophylactic antibiotics to ultimately result in a new therapy for allograft-associated infection

Prosthetic joint infections represent complications connected to the implantation of biomedical devices. Bacterial biofilm is one of the main issues causing infections from contaminated orthopaedic prostheses. Biofilm is a structured community of microbial cells that are firmly attached to a surface and have unique metabolic and physiological attributes that induce improved resistance to environmental stresses including toxic compounds like antimicrobial molecules (e.g. antibiotics). Therefore, there is increasing need to develop methods/treatments exerting antibacterial activities not only against planktonic (suspended) cells but also against adherent cells of pathogenic microorganisms forming biofilms. In this context, metal-based coatings with antibacterial activities have been widely investigated and used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing the biofilm formation prevention efficacy. Additionally, standardized and systematic approaches to test antibacterial activity of newly developed coatings are still missing, while standard microbiological tests (e.g. soft-agar assays) are typically used that are limited in terms of simultaneous conditions that can be tested, potentially leading to scarce reproducibility and reliability of the results. In this work, we combined the Calgary Biofilm Device (CBD) as a device for high-throughput screening, together with a novel plasma-assisted technique named Ionized Jet Deposition (IJD), to generate and test new generation of nanostructured silver- and zinc-based films as coatings for biomedical devices with antibacterial and antibiofilm properties. During the experiments we tested both planktonic and biofilm growth of four bacterial strains, two gram-positive and two gram-negative bacterial strains, i.e. Staphylococcus aureus ATCC 6538P, Enterococcus faecalis DP1122 and Escherichia coli ATCC 8739 and Pseudomonas aeruginosa PAO1, respectively. The use of CBD that had the only wells covered with the metal coatings while the biofilm supports (pegs) were not sheltered allowed to selectively define the toxic effect of the metal release (from the coating) against biofilm development in addition to the toxic activity exerted by contact killing mechanism (on biofilms formed on the coating). The results indicated that the antibacterial and antibiofilm effects of the metal coatings was at least partly gram staining dependent. Indeed, Gram negative bacterial strains showed high sensitivity toward silver in both planktonic growth and biofilm formation, whereas zinc coatings provided a significant inhibitory activity against Gram positive bacterial strains. Furthermore, the coatings showed the maximal activity against biofilms directly forming on them, although, Zn coating showed a strong effect against biofilms of gram-positive bacteria also formed on uncoated pegs. We conclude that the metal-based coatings newly developed and screened in this work are efficient against bacterial growth and adherence opening possible future applications for orthopedic protheses manufacturing

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

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

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

Aim. Fungal periprosthetic joint infections are difficult to treat and often associated with a limited outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules for bacteria. However, in vivo modeling of biofilm-associated fungi models are very rare. Furthermore, due to ethical restrictions, 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 with bacteria. This model organism was not used for fungi biofilm infection yet. Thus, we aimed to establish G. mellonella as in vivo model to study fungal implant infections using Candida albicans as model organism and to test anti-fungal medication. Method. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. For the infection process, implants were pre-incubated in specified fungal growth culture Candida albicans at 1×10. 7. CFU/ml for 30 min at 150 rpm shaking conditions. Later, these implants were washed with 10ml PBS and implanted in the larvae as mentioned. To analyze the susceptibility of the implant-associated fungal infections towards anti fungal compounds, the larvae were treated with amphotericin B, fluconazole and voriconazole after 24h of implantation. The effect of anti-fungal compounds was measured in terms of survival observation for 5 days and fungal load in larvae on 2. nd. day. To reveal the fungal biofilm formation on implant, the implants were removed on day 3 and processed for SEM analysis. Results. Pre-incubated K-wire caused the Candida infection and observed the death of the larvae. The treatment with antifungal compounds recovered the larvae from the implant-infection, except in case of Voriconazole. However, the recovery with treatment of anti fungal compounds was not effective as the larvae with planktonic infection, which highlights typical biofilm phenotype. Further, the treatment with anti-fungal compounds with Amphotericin B and Fluconazole reduced the fungal load in larvae tissue. The SEM analysis revealed the formation fungal biofilm with hyphae and spores associated with larvae tissue on implant surface. Conclusions. The results from survival analysis, antifungal treatment and SEM analysis are very promising to use of G. mellonella as in vivo model to study fungal infections on implanted materials. Our study highlights the use of G. mellonella larvae as alternative in vivo model to study implant-associated fungal infections that reduces the use of the higher mammals

Introduction. Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. The purpose of this study is to establish a clinically representative GN-PJI model that can reliably recapitulate biofilm formation on titanium implant surface in vivo. We hypothesized that biofilm formation on an implant surface will affect its ability to osseointegrate. Methods. The model was developed using 3D-printed titanium hip implants, to replace the femoral head of male Sprague-Dawley rats. GN-PJI was induced using two bioluminescent Pseudomonas aeruginosa strains: a reference strain (PA14-lux) and a mutant biofilm-defective strain (ΔflgK-lux). Infection was monitored in real-time using the in vivo imaging system (IVIS) and Magnetic Resonance Imaging (MRI). Bacterial loads on implant surface and in periprosthetic tissues were quantified utilizing viable-colony-count. Field-emission scanning-electron-microscopy of the explanted implants was used to visualize the biofilm formation at the bone-implant-interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration in vitro using microCT scan, and indirectly assessed by identifying the gait pattern changes using DigiGait. TM. system in vivo. Results. Localized infection was established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14-lux and ΔflgK-lux. This difference in the ability to persist in the model between the two strains was reflected in the gait pattern and implant osseointegration. Conclusions. We developed a novel uncemented hip hemiarthroplasty, GN-PJI rat model. To date, the proposed in vivo biofilm-based model is the most clinically representative for GN-PJI since animals can bear weight on the implant and poor osseointegration correlates with biofilm formation. In addition, localized PJI was detected by various modalities. Clinical Relevance. The proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapeutics

Aim. The rise of multidrug-resistant bacteria and the decreasing efficacy of antibiotic therapy in successfully treating biofilm-associated infections are prompting the exploration of alternative treatment options. This study investigates the efficacy of different bioactive glass (BAG) formulations - alone or combined with vancomycin - to eradicate biofilm. Further, we study the influence of BAG on pH and osmotic pressure as important factors limiting bacterial growth. Method. Different BAG-S53P4 formulations were used for this study, including (a) BAG-powder (<45 μm), (b) BAG-granules (500–800 μm), (c) a cone-shaped BAG-scaffold and (d) two kinds of BAG-putty containing granules, with no powder (putty-A) or with additional powder (putty-B), and a synthetic binder. Inert glass beads were included as control. All formulations were tested in a concentration of 1750 g/ml in Müller-Hinton-Broth. Targeted bacteria included methicillin-resistant Staphylococcus aureus (MRSA) and epidermidis (MRSE). Vancomycin was tested at the minimum-inhibitory-concentration for each strain (1 µg/ml for MRSA; 2 μg/ml for MRSE). To investigate the antibiofilm effect of BAG alone or combined with vancomycin, 3 hour-old MRSA or MRSE biofilms were formed on porous glass beads and exposed to BAG ± vancomycin for 24h, 72h and 168h. After co-incubation, biofilm-beads were deep-washed in phosphate-buffered saline and placed in glass vials containing fresh medium. Recovering biofilm bacteria were detected by measuring growth-related heat production at 37°C for 24h by isothermal microcalorimetry. Changes in pH and osmotic pressure over time were assessed after co-incubation of each BAG formulation in Müller-Hinton-Broth for 0h, 24h, 72h and 168h. Results. All BAG formulations showed antibiofilm activity against MRSA and MRSE in a time-dependent manner, where longer incubation times revealed higher antibiofilm activity. BAG-powder and BAG-putty-B were the most effective formulations suppressing biofilm, followed by BAG-granules, BAG-scaffold and finally BAG-putty-A. The addition of vancomycin had no substantial impact on biofilm suppression. An increase in pH and osmotic pressure over time could be observed for all BAG formulations. BAG-powder reached the highest pH value of 12.5, whereas BAG-putty-A resulted in the lowest pH of 9. Both BAG-putty formulations displayed the greatest increase on osmotic pressure. Conclusions. BAG-S53P4 has demonstrated efficient biofilm suppression against MRSA and MRSE, especially in powder-containing formulations. Our data indicates no additional antibiofilm improvement with addition of vancomycin. Moreover, high pH appears to have a larger antimicrobial impact than high osmolarity. Acknowledgements. This work was supported by PRO-IMPLANT Foundation (Berlin, Germany). The tested materials were provided by Bonalive Biomaterials Ltd (Turku, Finland)

The incidence of PJI in knee replacements is 2.8% and slightly lower with hip replacement surgery. PJI make up 15% (or even more) of knee revisions. To combat PJI, antibiotic laden bone cement has been used for many decades, but antibiotic stewardship dictates more prudent management of antimicrobials. Projected increase in infection rate, due to increased surgery and latent infection to be almost 5-fold up to 2035. Biofilm is a complex structure of bacteria and polysaccharide matrix and, is recognised as a major component in PJI and other orthopaedic infections. Biofilm is responsible for high incidence of resistance to antimicrobials and ineffective host immune response. Method. Stabilized hypochlorous acid has been reported to have a rapid kill rate on all pathogens, including MDR pathogens associated with chronic and acute wound infections. It destroys biofilm on contact, is not cytotoxic, reduces inflammation and stimulates wound healing. 0,038% of Hypochlorous acid was used as prophylaxis against infection and to treat PJI. We report on our experience with hypochlorous acid as a wound irrigation as prophylaxis against infection (more than 600 cases) and for PJI. We also report on a University study where a head to head analysis was done on the anti-biofilm efficacy between hypochlorous acid 0,038% (Trifectiv Surgical Wound Irrigation) and Product X (an industry-standard product for the prevention and treatment of biofilm infection. Hypochlorous acid offers a valuable addition to the armamentarium of wound antiseptics, with added anti-inflammatory value. An in vitro study demonstrated superior efficacy against biofilm when compared to Product X

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

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

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

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. Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. Herewith we introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide the biodistribution and specificity in a mouse implant infection model. Methods. 4497-IgG1targeting S. aureus Wall Teichoic Acid was labeled to Indium-111 using “CHXA” as a chelator. SPECT-CT scans were performed at 24, 72 and 120 hours after administration in Balb/cAnNCrl mice with a subcutaneous implant pre-colonized with biofilm of S. aureus. Biodistribution over the various organs of this labelled antibody was visualized and quantified using SPECT-CT imaging and compared to uptake at the target tissue with implant infection. Results. Uptake of the . 111. In-4497 mAbs (half-life 59 hours) at the infected implant gradually increased from 8.34%ID/g at 24 hours to 9.22%ID/g at 120 hours. Uptake at the heart/blood pool decreased over time from 11.60 to 7.58%ID/g whereas the uptake in other organs decreased from 7.26 to less than 4.66%ID/g at 120 hours. Conclusion. 111. In-4497 mAbs was found to specifically detect S. aureus and its biofilm with excellent and prolonged accumulation at the colonized implant site. Therefore, it holds great promise as a drug delivery system for diagnostic and bactericidal treatment of biofilm. However, high activity in the blood pool must be considered as it could pose a risk to healthy tissue

Introduction. Major trauma during military conflicts involve heavily contaminated open fractures. Staphylococcus aureus (S. aureus) commonly causes infection within a protective biofilm. Lactoferrin (Lf), a natural milk glycoprotein, chelates iron and releases bacteria from biofilms, complimenting antibiotics. This research developed a periprosthetic biofilm infection model in rodents to test an Lf based lavage/sustained local release formulation embedded in Stimulin beads. Method. Surgery was performed on adult rats and received systemic Flucloxacillin (Flu). The craniomedial tibia was exposed, drilled, then inoculated with S. aureus biofilm. A metal pin was placed within the medullary cavity and treatments conducted. Lf in lavage solutions: The defect was subject to 2× 50 mL lavage with 4 treatment groups (saline only, Lf only, Bactisure with Lf, Bactisure with saline). Lf embedded in Stimulin beads: 4 bead types were introduced (Stimulin only, Lf only, Flu only, Lf with Flu). At day 7, rats are processed for bioluminescent and X-ray imaging, and tibial explants/pins collected for bacterial enumeration (CFU). Results. Rats without treatments established a mean infection of 2×106 CFU/tibia. 4 treatment groups with a day 0, one-off lavage demonstrated >95% reduction in bacterial load 7 days post-op, with a reduction in CFU from 1×106/tibia down to 1×104/tibia. There was no statistically significant difference between each group (p = 0.55 with one way ANOVA). The stimulin bead experiments are ongoing and complete results will be obtained in the end of July. Conclusions. This research demonstrated a clinically relevant animal model of implanted metalware that establishes infection. No additional benefit was observed with a one-off, adjuvant Lf lavage during the initial decontamination of the surgical wound, compared with saline alone, and in combination with the antiseptic Bactisure. This animal model provides the foundation for future antibiofilm therapies

We developed a novel silorane-based biomaterial (SBB) for use as an orthopedic cement. SBB is comprised of non-toxic silicon-based monomers, undergoes non-exothermic polymerization, and has weight-bearing strength required of orthopedic cements. We sought to compare the antibiotic release kinetics of this new cement to that of commercially available PMMA bone cement. We also evaluated each material's inherent propensity to support the attachment of bacteria under both static and dynamic conditions. One gram of either rifampin or vancomycin was added to 40g batches of PMMA and SBB. Pellets were individually soaked in PBS. Eluate was collected and tested daily for 14 days using HPLC. Compressive strength and modulus were tested over 21 days. Bioassays were used to confirm the bioactivity of the antibiotics eluted. We measured the growth and maturation of staphylococcus aureus (SA) biofilm on the surface of both PMMA and SBB disks over the course of 72 hours in a static well plate and in a dynamic biofilm reactor (CDC Biofilm Reactor). N=4 at 24, 48, and 72 hours. A luminescent strain of SA (Xen 29) was employed allowing imaging of bacteria on the discs. SBB eluted higher concentrations of vancomycin than did PMMA over the course of 14 days (p<0.001). A significant 55.1% greater day 1 elution was observed from SBB. Silorane cement was able to deliver rifampin in clinically favorable concentrations over 14 days. On the contrary, PMMA was unable to deliver rifampin past day 1. The incorporation of rifampin into PMMA severely reduced its mechanical strength (p<0.001) and modulus (p<0.001). Surface bacterial radiance of PMMA specimens was significantly greater than that of SBB specimens at all time points (p<0.05). The novel silorane-based cement demonstrated superior antibiotic release and, even without antibiotic incorporation, demonstrated an innate inhabitation to bacterial attachment and biofilm