Aim. Antibiotics have limited activity in the treatment of multidrug-resistant or chronic biofilm-associated infections, in particular when implants cannot be removed. Lytic bacteriophages can rapidly and selectively kill bacteria, and can be combined with antibiotics. However, clinical experience in patients with surgical infections is limited. We investigated the outcome and safety of local application of bacteriophages in addition to antimicrobial therapy. Method. 8 patients (2 female and 6 male) with complex orthopedic and cardiovascular infections were included, in whom standard treatment was not feasible or impossible. The treatment was performed in agreement with the Article 37 of the Declaration of Helsinki. Commercial or individually prepared bacteriophages were provided by ELIAVA Institute in Tbilisi, Georgia. Bacteriophages were applied during surgery and continued through drains placed during surgery three times per day for the following 5–14 days. Follow-up ranged from 1 to 28 months. Results. Median age was 57 years, range 33–75 years. Two patients were diagnosed with a persistent knee arthrodesis infection, one chronic periprosthetic joint infection (PJI), one cardiovascular implantable electronic device (CIED) infection and four patients with left ventricular assist device (LVAD) infection. The isolated pathogens were multi-drug-resistant Pseudomonas aeruginosa (n=3), methicillin-sensitive Staphylococcus aureus (n=4), methicillin-resistant Staphylococcus aureus (MRSA) (n=1) and methicillin-resistant Staphylococcus epidermidis (MRSE) (n=1). 4 infections were polymicrobial. 5 patients underwent surgical debridement with retention of the implant, 1 patient with PJI underwent the exchange of the prosthesis and one patient with LVAD infection was treated conservatively. All patients received intravenous and oral antibiotic therapy and local application of bacteriophages. At follow-up of 12 month, 5 patients were without signs or symptoms of infection, whereas in one patient with LVAD infection, a relapse was observed with emergence of phage-resistant Pseudomonas aeruginosa. In this patient, no surgical revision was performed. Conclusions. Bacteriophage therapy may represent a valid additional approach, when standard antimicrobial and surgical treatment is not possible or feasible, including in difficult-to-treat infections. In our case series, 5 of 6 patients were infection free after 1 year. Further studies need to address the optimal bacteriophage administration route, concentration, duration of treatment and combination with antimicrobials

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

Background. Currently, the gold standard for the microbiological diagnosis remains the culturing of preoperative aspirated joint fluid and intraoperative periprosthetic tissue samples, which give false negative results in about 7 % of cases. Lytic bacteriophages are viruses that specifically infect and lyse bacteria within their replication cycle. Aim. The aim of our study was to explore possibilities for the use of bacteriophage K for the detection of live Staphylococcus spp. bacteria in sonicate fluid of infected prosthetic joints, to possibly contribute to the development of a faster, more sensitive, specific and at the same time economical and handy method for the establishment of the right diagnosis. Material and methods. Sonicate fluid samples obtained from 104 patients with revision arthroplasty were analysed. After the optimisation two indirect phage-based methods were used: a) bioluminescence detection of bacterial intracellular ATP released by bacteriophage K mediated lysis and b) q-PCR with primers specific for bacteriophage K DNA. The results were compared with classical microbiological cultivation methods. Results. With both methods the analysis of sonicate fluid and the analysis of its over-night culture achieved 100 % specificity and predictive value, as there were no false positive results. The sensitivity of the methods was lower when analysing sonicate fluid samples directly, without cultivation. The sensitivity of qPCR detection was higher (81.25 %) compared to the sensitivity of ATP detection (62.5 %) in sonicate fluid directly as a result of 3 false negative results with the qPCR method compared to 6 false negatives with the ATP detection method. The sensitivity of the methods was significantly improved (to 94.12 %) with overnight cultivation of sonicate fluid samples prior to analysis, with no difference in detection between the methods. With both methods, with pre-cultivation of sonicate fluid samples, only one of the tested samples resulted in a false negative result. However, the same sample was negative even when tested with standard microbiological methods. In this patient, only the microbiological cultivation of the periprosthetic tissue sample was positive. The bioluminescence method took 3h with a limit of detection (LOD) in the bacterial concentration range of 10. 3. CFU/mL. The method with qPCR took 4h and had a LOD of 10. 2. CFU/mL. Conclusion. Detection of staphylococci within sonicate fluid with bacteriophage K based methods is a rapid, sensitive and specific approach

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

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

Aim. Rifampicin plays an important role in the treatment of staphylococcal prosthetic joint infection, as rifampicin-containing combinations have shown a high efficacy against S. aureus biofilm infections. However, the emergence of rifampin-resistant strains is a feared complication and the use of rifampicin in those cases seems unwarranted. Therefore, we evaluated the activity of bacteriophage Sb1 in combination with different antibiotics against the biofilm of four rifampicin-resistant MRSA strains as alternative therapeutic approach. Method. Four rifampicin-resistant MRSA strains were used in this study. The MIC for all tested antibiotics was determined by Etest. Biofilms were formed on porous glass beads for 24h and exposed to Sb1 (10. 7. PFU/mL) for 24h followed by exposure to antibiotic for 24h. Viability of bacteria after antimicrobial treatment was detected by beads sonication and plating of the sonication fluids. The minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotic required to kill all cells resulting in the appearance of no colony after plating of the sonication fluid (detection limit <20 CFU/mL). The synergistic effects were observed when Sb1 combined with antibiotics used at least 2 log-reduction lower concentrations. Results. All strains were susceptible to the three antibiotics except for MRSA3, resistant to fosfomycin, according to the EUCAST breakpoints. All tested antibiotics presented high MBEC values (ranging from 64 to >1024 µg/mL) when tested alone against biofilm (Table 1). A sequential administration of Sb1 followed by vancomycin (VAN) showed no synergistic effect against any of the tested strains, whereas the combination with fosfomycin (FOF) showed synergism in 50% of the strains with improvement of the eradication activity. The combination of Sb1 with daptomycin (DAP) showed the highest synergistic effects in 100% of the strains, with a 2 or 3-log reduction in the MBEC. Conclusions. Sb1 bacteriophage in combination with daptomycin seems a promising alternative for the treatment of rifampicin-resistant MRSA biofilm infections. Table 1 (not included). Antimicrobial activities against rifampicin-resistant MRSA strains. For the table, please contact authors directly

Aim. Bacteriophages are remerging as alternative and adjunctive therapy for fracture-related infection (FRI). However, current administration protocols involve prolonged retention of a percutaneous draining tube with potential risk of developing superinfection. In this study, we applied a cocktail of in vitro evolved biofilm-targeting phages for Methicillin-resistant Staphylococcus aureus (MRSA) in a hydrogel platform co-delivering vancomycin. In vitro synergy and antibiofilm activity was assessed and a subsequent in vivo study was performed in a mouse FRI model with MRSA. Method. Two evolved bacteriophages (MRSA-R14 and COL-R23) with improved antibiofilm activity against a clinical isolate (MRSA3) were tested in combination with vancomycin and a carboxymethylcellulose (CMC) hydrogel in vitro and in vivo. MRSA3 bacterial biofilms were formed on sterile 4 mm sintered porous glass beads at 37 °C for 24 h. Biofilms were exposed to i-phage cocktail (10. 7. PFU/ml), ii-vancomycin at concentrations of 0.5, 1, 10 and 100 times the MIC, or iii-combination of phage cocktail and vancomycin. Recovered biofilm cells, were quantified by colony counting. The stability and release profiles of phage cocktail and vancomycin in co-delivery hydrogel were assessed in vitro for 8 days and 72 hrs, respectively, and subsequently tested in the treatment of 5-day-old MRSA3 infection of a femoral plate osteotomy in mice. Results. In vitro: The cocktail of evolved phages (10. 7. PFU/ml, 1:1) combined with 0.5 MIC vancomycin achieved 99.72% reduction in MRSA3 biofilm in vitro compared to the growth control. This combination was stable in the co-delivery hydrogel over 8 days. The release profile showed that 57% of phages and 80% of vancomycin were released after 72hrs, which was identical to the performance for gels loaded with phage or antibiotic alone. In the in vivo study, the bacterial load from animals that received co-delivery hydrogel and systemic vancomycin was significantly reduced compared to controls, animals that received systemic vancomycin and animals that received co-delivery hydrogel alone (p<0.05). Conclusions. Our study demonstrates the potential of using evolved phages in combination with vancomycin and hydrogel delivery systems for the treatment of MRSA-related infections. Further research in this area may lead to the development of specific therapies for biofilm-related infection

Aim. To investigate the ability of the bacteriophage Sb-1 to treat and prevent implant-associated infections due to methicillin-resistant Staphylococcus aureus (MRSA) in Galleria mellonella larvae implanted with a K-wire. Method. The stability of Sb-1 in G. mellonella larvae was investigated by injecting a phage titer of 10. 8. PFU and evaluating the presence of Sb-1 in hemolymph at different time points. For infection experiments, sterile stainless-steel K-wires (4 mm, 0.6 mm Ø) were implanted into larvae. Two days after implant, larvae were infected with MRSA ATCC 43300 (1×10. 5. CFU) and incubated at 37°C for further 2 days. Implanted-infected larvae were thus treated for 2 days (3×/day) with 10µL of: i) PBS; ii) Sb-1 (10. 7. PFU); iii) Daptomycin (4mg/kg), iv) PBS (24h)/Daptomycin(24h); v) Sb-1(24h)/Daptomycin(24h). To evaluate the prophylactic efficacy of Sb-1, an experiment based on phages or vancomycin (10mg/kg) administration, followed by MRSA infection of implanted larvae was performed. Both two days post-infection and post-treatment, K-wires were explanted, and the material was sonicated and plated for MRSA colony counting. Results. Sb-1 titer resulted stable in hemolymph of G. mellonella larvae for 6–8 h post-administration. Two days post-infection of K-wire implanted larvae, ≈5×10. 7. CFU/ml MRSA were found on the material. K-wires from larvae treated with Sb-1 or Daptomycin showed a MRSA CFU/ml reduction of ≈1 log compared to the CFU/ml values of the untreated control. The staggered administration Sb-1/Daptomycin determined higher CFU reduction (≈ 3.5 log). Prophylaxis with Sb-1 prevented MRSA infection of 7out of 10 larvae similarly to vancomycin. Conclusions. G. mellonella larvae implanted with K-wires are a suitable model to test antibiofilm formulations in vivo. Sb-1 phage is able to prevent implant-associated infection due to MRSA in larvae. Sequential combination of Sb-1 and Daptomycin strongly reduces the MRSA load on implanted K-wires

Aim. Virulent bacteriophages are known to be an effective therapy against various human bacterial infections. The aims of the study are to evaluate i) the killing activity of an antistaphylococcal phage lysate (ASPL), available in the Czech Republic for topical application, against Staphylococcal aureus (Sa) strains isolated in orthopedic infections; ii) the antimicrobial activity of ASPL against biofilm-embedded cells of a methicillin-resistant Sa (MRSA) standard strain. Method. The susceptibility of 25 MRSA and 18 methicillin-sensitive Sa (MSSA) strains to the ASPL was evaluated by spot assay. In addition, susceptibility of four laboratory MRSA strains, including ATCC 43300, ATCC 33591, Mu3 (MRSA/hetero vancomycin intermediate resistant Sa) and Mu50 (MRSA/vancomycin-resistant Sa) was also tested. The activity of ASPL against planktonic and biofilm-embedded MRSA ATCC 43300 was evaluated in real-time by isothermal microcalorimetry. The minimum heat inhibitory concentrations (MHIC) was defined as the lowest antimicrobial concentration leading to the lack of heat flow production after 24h for both planktonic and biofilm-embedded cells. The viability of bacterial cells was assessed by plating and colony counting. The minimum bactericidal concentration (MBC) was defined as the lowest antimicrobial concentration leading the reduction of 3 log CFU compared to the untreated control. Results. Around 34 out of 43 (79%) Sa strains were susceptible to the ASPL, including 17 MRSA and 17 MSSA strains. Both Mu3 and Mu50 (vancomycin intermediate and resistant MRSA, respectively) strains were also susceptible. Microcalorimetric evaluation of the activity of ASPL against planktonic cells of MRSA ATCC 43300 revealed the MHIC and the MBC were 10. 4. PFU/ml and 10. 5. PFU/ml, respectively. ASPL tested at 10. 5. PFU/ml was able to suppress the heat produced by biofilm bacterial cells, although this titer was not able to completely eradicate MRSA biofilm. Conclusions. ASPL showed a broad host spectrum among MRSA and MSSA strains associated with infections on implants, including strains that are resistant to vancomycin as well. ASPL exhibits a lytic activity against planktonic and biofilm MRSA and a titer of phage higher than 10. 5. PFU/ml is needed in order to achieve a complete eradication of MRSA biofilm. In conclusion, the antistaphylococcal phage lysate shows an excellent potential treatment of implant-related infections caused by Sa strains

Background

Bacteriophages are natural viruses of interest in the field of PJI. A paper previously reported the PhagoDAIR procedure (use of phages during DAIR) in three patients with PJI for whom explantation was not desirable. As the need to isolate the pathogen before surgery to perform phage susceptibility testing is a strong hindrance for the development of this procedure, we developed post-operative phage injections using ultrasound, in patients infected with S. aureus and/or P. aeruginosa who were eligible for the PhagoDAIR procedure, but for whom phages were not available at the time of surgery.

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.

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

Aim. To describe the management of PJI due to S. aureus in CRIOAcs in 2019 and to particularly focus on the evaluation of the efficacy of DAIR regarding control of infection and risk factors for failure up to 12 months. Method. Thirteen CRIOAcs were selected to participate to the study. Data concerning the management of all the PJI in the year 2019 were retrospectively collected and registered in eCRFs. Inclusion criteria were: ≥ 18 years old patients with S. aureus ± other bacteria (in per surgical procedure sample); knee or hip PJI and with clinical signs of infection. Patients treated with bacteriophages were excluded. All eligible patients were notified by an information letter. Patients treated by the DAIR procedure were selected, and rate of control of infection (no inflammatory local signs or no new surgical procedure or no S. aureus in case of puncture) was analyzed using Kaplan Meier method and risk factors for failure at 12 months were assessed using Cox regression model. Results. A total of 978 PJI were managed in the 9 CRIOAcs, including 238 hip and knee PJI due to S. aureus and 79 to S. aureus plus another bacteria. Among all of them, 154 were managed with DAIR, and 100 fulfilled inclusion criteria, notifying no opposition to their data collection. The median age was 73.0 years; 57% were male, the median Charlson score was 4.0; 66% had hip PJI. A total of 45 failure were observed during the period studied. At 12 months, the control rate was 58. 7% [36.5–75.4], 49.3% [34.3–62.7] in in early and late PJI respectively according to Tsukuyama classification and 49.6% [30.5–66.1], 54.1% [37.7 – 68.0] in early and delayed/late PJI respectively according to Zimmerli classification, 56.6% [39.5–70.5] in case of mobile part exchange, 53.4% [35.3–68.5] for MRSA PJI and 63.4% [50.5–73. 8] in patients treated with rifampicin. No rifampicin intake was the only significative risk factor for failure in univariate analysis (HR=0.31 (0.17–0.57), p=0.0002), and remained significant after adjustment on Charlson score (aHR=0.34 (0.18–0.64), p=0.0008). Conclusions. The DAIR procedure is frequently performed in patients with acute and late PJI, and is associated with a high rate of failure, especially for patients who cannot receive rifampin. There is a strong rational to assess the use of bacteriophages during the DAIR, as bacteriophages have antibiofilm activity in vitro, and could improve the efficacy of the DAIR to control the disease

Aim. Phage therapy has attracted attention as a promising alternative treatment option for biofilm infections. To establish a successful phage therapy, a comprehensive stock of different phages covering a broad bacterial spectrum is crucial. We screened human and environmental sources for presence of lytic phages against selected bacteria. Methods. Saliva collected from 10 volunteers and 500 ml of sewage water were screened for the presence of lytic phages active against 20 clinical strains of Staphylococcus aureus and 10 of Escherichia coli, both isolated from patients with prosthetic joint infection. Laboratory strains of methicillin-resistant S. aureus (MRSA)*1 and E. coli*2 were also tested. Screening was performed plaque-assay to detect phages for different strains. Isolated plaques were collected and phages were enriched to determine their activity against their bacterial host strains. The activity of bacteriophages against adherent E. coli and MRSA was evaluated by crystal violet, staining bacterial biofilms grown on glass beads. Results. Six bacteriophages specific for MRSA were isolated from saliva. Bacteriophages for E. coli strains were isolated from sewage water (n=3) and saliva (n=1). All bacteriophages tested against biofilms of their bacterial host showed a reduction of the total biomass (ranging from 19% to 84%). Conclusions. Both sewage and saliva samples provided bacteriophages specific against selected bacterial strains. 24h phage treatment of E. coli and S. aureus biofilms lead to a reduction but not to a complete eradication of biofilm

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. Many bone and joint infections, in spite of appropriated antibiotics therapy and surgery, lead to a therapeutic dead end. We are then faced with a chronic infection with or without continuous antibiotic treatment, with daily local care, and an exhorbitant economic and social cost. Pami the incriminated factors: the presence of foreign implant material, the poor diffusion of antibiotics at the infectious site, the presence of biofilm. The bacteriophages, biological drug, natural environmental viruses possess the properties to meet these difficulties: well diffusion to the infectious focus with possibilities of local use, destruction of the biofilm allowing a release of the bacteria and a synergistic effect with the antibiotics, antibiofilm effect for the restoration of osteoblastosis. Method and results. We report a cohort of phage - treated patients with or without antibiotics in bone and joint infections in a therapeutic dead end. Without disponibility of therapeutic phages available in the European Union, commercial cocktails of phages, antistaphylococcal or polyvalent, of Russian* or Georgian** origin were used. Ten patients have benefited since 2008 from phages, alone or in combination with an adapted antibiotic therapy. Patients were 40 to 89 years old and had chronic bone and joint infections except for one case with acute MRSA infection on femoral implant. Bacteria were Staphylococcus aureus 7 times, Pseudomonas aeruginosa 3 times, Klebsiella 2 times. In 4 cases implant was left in place (knee prosthesis, femoral screw plate) or introduced (1 screw in 1 case) during the procedure. In all cases except 1 patient, the phages were applied in per-operative. With a follow-up of up to 9 years for some patients, the initial bacteria were eradicated and in 2 cases replaced by another bacterium (Pseudomonas in place of S. aureus in one case and Enterococcus in place of P. aeruginosa for an elderly patient with a knee prosthesis without possible surgery. Conclusion. The combination of surgery, phages and antibiotics appear a very efficient option, to treat patients with bone and joint infections in therapeutic dead end. The quick availability of these treatments for these patients is a health emergency. *Microgen® Pharma. **Eliava Institute

The number of arthroplasties being undertaken is expected to grow year on year, and periprosthetic joint infections will be an increasing socioeconomic burden. The challenge to prevent and eradicate these infections has resulted in the emergence of several new strategies, which are discussed in this review.

Cite this article: Bone Joint J 2015;97-B:1162–9.