Method

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm was grown on porous glass beads for 24 h in vitro. After the biofilm formation, beads were exposed to 0.9% saline, then sonication. Quantitative and qualitative biofilm analyses were performed by colony counting, scanning electron microscopy and isothermal microcalorimetry. A rat model of total knee arthroplasty infected with the bioluminescent MRSA strain was developed as the PJI model to evaluate the efficacy of Lip-RIF@Phage anti-biofilm therapy in vivo, then the creatinine, alanine transaminase, and aspartate transaminase values were evaluated throughout the entire treatment process.

Material and Methods

We conducted a prospective study at our center between March 2022 and December 2023. Out of the 56 patients from Ukraine, 21 met the inclusion criteria who had severe war injuries were included in the study. Each of these patients presented with multiple injuries to both bones and soft tissues, having initially undergone treatment in Ukraine involving multiple surgeries. The diagnosis of infection was established based on the EBJIS criteria. Prior to our treatment patients had undergone multiple revision surgeries, including debridement, biopsies, implant and fixator replacement. Additionally, soft tissue management required previously VAC therapy and flap reconstruction for successful treatment.

Method

This restrospective study analyzed 41 patients with concomitant PJI and PF during the PJI treatment period from 2013 to 2022 involving THA. Patients were divided in two cohorts termed success and failure and were statistically compared. The median follow-up time was 66 months (>12 months). All patients were considered individually and treated according to their individual needs in fracture and infection treatment. Re-arthroplasty survival was analyzed using the Kaplan-Meier method. Relevant risk factors were analyzed using the Mann-Whitney test or Chi-square, depending on the variable's scale.

Method

Our prospective study includes 89 patients who underwent revision surgery after THA and TKA with application of MPH and were compared to 102 patients who did not receive MPH and underwent revision surgery after THA and TKA. Five grams of MPH¹ were applied periarticular before fascia closure and to the subcutaneous soft tissue. The follow-up was conducted in daily clinical visits during the inpatient stay and three months postoperatively in our outpatient clinic. Repeated revision surgery was performed in case of prolonged secretion (>10 days) or clinical suspicion of infection. After matching the cohorts the outcomes were statistically analyzed using paired methods.

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 (10⁷ 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.

Method

Consecutive patients with streptococcal PJI (defined by EBJIS criteria) treated 2009–2021 were prospectively included and allocated into standard and suppression (> 6 months) treatment group. Infection-free survival was assessed with Kaplan-Meier-method and compared between the groups with log rank test. Rates of infection-free, streptococcal infection-free and relapse-free status as well as tolerability of suppression were assessed.

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.

Method

Consecutive patients with suspicion of septic arthritis were prospectively included. They underwent joint aspiration and synovial fluid was collected for culture, leukocyte count and D-lactate concentration (by spectrophotometry). Youden's J statistic was used for determining optimal D-lactate cut-off value on the receiver operating characteristic (ROC) curve by maximizing sensitivity and specificity.

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.

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.

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 (10⁷ 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.

Method

Consecutive patients with PJI caused by at least one of the following isolates were prospectively included: staphylococci (MIC ≤32 mg/l), streptococci (MIC ≤128 mg/l), enterococci (MIC ≤128 mg/l), Enterobacteriaceae (MIC ≤32 mg/l) and Pseudomonas spp. (MIC ≤128 mg/l). PJI was defined by the proposed European Bone and Joint Infection Society (EBJIS) criteria. Follow up with clinical (joint function and quality of life scores), laboratory and radiological evaluation at 3, 12 and 24 months after last surgery is performed. Infection outcome was assessed as the proportion of infection-free patients. The probability of infection-free survival was estimated using the Kaplan-Meier survival method.

Method

The stability of Sb-1 in G. mellonella larvae was investigated by injecting a phage titer of 10⁸ 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 (1×10⁵ 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 (10⁷ 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.

Method

Consecutive patients undergoing diagnostic joint aspiration of prosthetic joint were prospectively included. PJI was diagnosed according to the proposed European Bone and Joint Infection Society (EBJIS) definition criteria. Synovial fluid was collected for culture, D-lactate measurement (by spectrophotometry, λ = 570 nm) and leukocyte count and differential (by flow cytometry). The receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of D-lactate and leukocyte count.

Method

E. coli ATCC 25922, P. aeruginosa ATCC 27853 and 15 clinical isolates were used for this study. MIC values were determined by Etest. Biofilms were formed on porous sintered glass beads for 24h and exposed to antibiotics for further 24h. Viability of bacteria on the glass beads after antibiotic treatment was detected by cfu counting of the sonicated beads. The minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotic required to kill biofilm cells. Synergistic activity against biofilm was evaluated by calculation of the fractional inhibitory concentration index (FICI).

Method

Culture-positive PJI cases treated at our institution from 02/2011 to 07/2018, for which culture results from preoperative (synovial fluid) and intraoperative samples (periprosthetic tissue, synovial or sonication fluid) were available, were retrospectively assessed. For organisms belonging to the resident skin flora (coagulase-negative staphylococci, cutibacteria and corynebacteria) significant growth was considered, if the identical pathogen grew in ≥2 samples or >50 cfu/ml sonication fluid. For other pathogens (S. aureus, streptococci, enterococci, fungi and gram-negative rods) or patients under antimicrobials, any growth was considered positive. We determined the pathogen detection rate in preoperative and intraoperative cultures and compared it in different subgroups using Fisher's exact test. Furthermore, we assessed the concordance of preoperative and intraoperative cultures.

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.

Method

Consecutive patients with IIF diagnosed at our institution from 01/2010-10/2017 were retrospectively included. Infection was defined as visible purulence, sinus tract, microbial growth in ≥2 independent samples or positive histopathology. The outcome was compared before and after implementation of a comprehensive surgical and antimicrobial treatment algorithm in 04/2013.

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.

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/cm²), 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.