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

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

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

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

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

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

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

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

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

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

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

Aim. 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. Irrigation and debridement with an irrigation solution are essential components of the surgical management of acute and chronic periprosthetic joint infection (PJI). Nevertheless, there is a lack of agreement regarding the most effective solution to use. The aim of the study was to perform a systematic review and meta-analysis of the current literature concerning the efficacy of different irrigation solutions over bacterial biofilm. Method. This study was conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analysis extension for Network meta-analysis (PRISMA-NMA) checklist for systematic reviews and meta-analyses. A comprehensive literature search of PubMed, Cochrane Library, Web of Science and Scopus databases from inception to September 1, 2023. We combined terms related to PJI, biofilm and irrigation solutions studied in vitro. We performed a network meta-analysis to analyze which irrigation solution achieved a higher reduction of colony forming units (CFU) after specific exposure times, always with a maximum of five minutes, replicating intraoperative conditions. Effect-size was summarized with logarithmic response ratio (logRR) and 95% confidence intervals (95% CI). The rank probability for each treatment was calculated using the p-scores. Results. We screened 233 potential sources. Following deduplication, screening and full-text review, four studies with ten irrigation solutions for different duration of exposures were included, always less than five minutes, replicating intraoperative conditions. Solutions were studied over mature biofilms of most frequent bacteria grown over metal, bone cement or polyethylene surfaces. The highest effect was achieved with povidone iodine 10% during 5 minutes (logRR: −12.02; 95% CI: −14.04, −9.99). The best ranked solutions were povidone iodine 10% during five, three and one minute (respective p-scores: 0.977, 0.932, 0.887) and its combination with hydrogen peroxide for 3 minutes (p-score: 0.836). Povidone iodine 0.3% acting for 5 minutes completed the top 5 best ranked solutions in this study (p-score: 0.761). We assumed that there were no inconsistencies in our network because after examining both scenarios, with and without inconsistencies, the results were not significantly different. Conclusions. Our results show that 10% povidone-iodine is the best antiseptic solution when studied in vitro in the context of prosthetic joint infection. However, the included studies did not evaluate the possible cytotoxic effects of these solutions. This should also be taken into account before choosing the most appropriate antiseptic solution

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

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

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

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

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

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

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