Aim. “Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material. Biofilms are bacterial communities adhered to a surface with a self-made extracellular polymeric substance that surrounds resident bacteria. In contrast to planktonic bacteria, bacteria in a biofilm are in an adherent, dormant state and are insensitive to most antibiotics. In addition, bacteria in a biofilm are protected from phagocytic cells of the immune system. Therefore, complete surgical removal and replacement of the prosthetic implant is often necessary to treat this type of infections. Neutrophils play a crucial role in clearing bacterial pathogens. They recognize planktonic bacteria via immunoglobulin (Ig) and complement opsonisation. In this project, we aim to evaluate the role of IgG and complement in the recognition and clearance of staphylococcal biofilms by human neutrophils. Furthermore, we evaluate if monoclonal antibodies (mAbs) targeting biofilm structures can enhance recognition and clearance of staphylococcal biofilms by the human immune system.”. Method. “We produced a set of 20 recombinant mAbs specific for staphylococcal antigens. Using flow cytometry and ELISA-based methods we determined the binding of these mAbs to planktonic staphylococci and in vitro staphylococcal biofilms. Following incubation with IgG/IgM depleted human serum we determined whether mAbs can react with the human complement system after binding to biofilm. Confocal microscopy was used to visualize the location of antibody binding in the biofilm 3D structure.”. Results. “We show that mAbs directed against several staphylococcal surface targets such as wall teichoic acid (a glycopolymer on the S. aureus/S. epidermidis cell wall) and polymeric-N-acetyl-glucosamine (major constituent of the S. epidermidis biofilm extracellular matrix) bind biofilms in a dose-dependent manner. This interaction was specific since no binding was observed for control antibodies (recognizing the hapten DNP). Furthermore we show that these antibodies can penetrate the complete 3D structure of an in vitro biofilm. Products of complement activation via the classical pathway were detected upon incubation with human serum and the biofilm binding mAbs.”. Conclusions. “Having established that our mAbs can bind biofilms and induce complement opsonisation via C3b deposition, we will now study if we can engineer these antibodies to enhance complement deposition. A combination of enhanced complement and antibody opsonisation may improve recognition and clearance of biofilms by phagocytic immune cells. These mAbs could be used to boost the immune system to clear implant associated infections, without the need to replace the implant via invasive surgical procedures.”

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. Ciprofloxacin is recommended as anti-biofilm therapy for gram-negative periprosthetic joint infection. With ciprofloxacin monotherapy, resistance in gram-negative bacteria was observed. Therefore, we evaluated in vitro synergistic activity of fosfomycin, ciprofloxacin and gentamicin combinations against biofilms formed by E. coli and P. aeruginosa strains. 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). Results. Table 1 summarizes the antimicrobial susceptibility of planktonic (MIC), biofilm bacteria (MBEC) and synergism. Among 9 E. coli isolates, the synergism was observed in 78% of isolates treated with fosfomycin/gentamicin, 44% treated with gentamicin/ciprofloxacin and 22% treated with fosfomycin/ciprofloxacin. Among 8 P. aeruginosa isolates, the synergism was observed 75% of isolates treated with gentamicin/ciprofloxacin, 63% treated with fosfomycin/gentamicin and 50% treated with fosfomycin/ciprofloxacin. Conclusions. Based on our results, fosfomycin in combination with gentamicin seems to be a promising therapeutic approach against E. coli biofilm related infections. Combination of gentamicin with ciprofloxacin represent the most optimal treatment option for P. aeruginosa biofilm. For any figures or tables, please contact the authors directly

The aim of the present study was to assess the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL100 (EUD) microparticles against mature biofilms of polysaccharide intercellular adhesin-positive S. epidermidis. The effect of plain, daptomycin- and vancomycin-loaded PMMA and PMMA-EUD microparticles on S. epidermidis biofilms was assessed by isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). Biofilms were grown for 48h onto poly-urethane pieces of fixed dimensions. Each sample was washed with PBS in order to remove planktonic bacteria and incubated for 24h with different concentrations of acrylic microparticles (20–1.25 mg/mL). The minimal biofilm inhibitory concentration (MBIC) of the antibiotic-loaded particles was defined as the lowest concentration of particles that was able to prevent heat flow associated to the recovery of the biofilms. After incubation with the microparticles, sessile cocci were hybridized with the pan-bacterial EUB338-FITC and the staphylococci-specific STAPHY-FICT probes and stained with DAPI. Biofilm structure and metabolic state were characterized by fluorescence microscopy. According to the IMC results, plain PMMA-particles showed no effect on S. epidermidis biofilms, whereas PMMA-EUD-microparticles negatively influenced the recovery of the biofilm probably due to the highly positive charge of these particles. The MBIC of daptomycin-loaded PMMA-microparticles was 20 mg/mL, whereas vancomycin-loaded PMMA microparticles were not able to inhibit biofilm recovery. Adding EUD to the formulation reduced the MBIC of daptomycin-loaded microparticles to 1.25 mg/mL, corresponding to a 16-fold reduction. Regarding the vancomycin-loaded microparticles, EUD caused a further decrease of their antibiofilm activity. The FISH micrographs corroborated the IMC results and provided additional insights on the antibiofilm effect of these carriers. According to FISH, daptomycin-loaded PMMA-EUD microparticles were responsible for the most pronounced reduction in biofilm mass. In addition, FISH showed that both PMMA and PMMA-EUD microparticles were able to attach to the biofilms. Adding EUD to the formulations proved to be a powerful strategy to improve daptomycin-loaded microparticles antibiofilm activity. In addition, the combination of IMC and FISH was essential in order to fully assess the effect of polymeric microparticles on sessile S. epidermidis. Although the present study enabled gaining further insights on this subject, the nature of these interactions remains unclear. However, this may be a crucial aspect for the enhancement of antibiofilm activity of antibiotic-loaded polymeric microcarriers against mature biofilms. This work was supported by the Portuguese government (Fundação para a Ciência e a Tecnologia) and FEDER (grant SFRH/BD/69260/2010 and research project EXCL/CTM-NAN/0166/2012) and strategic project PEst-OE/SAU/UI4013/2011

Aim

The management of PJIs is slowed down by the presence of bacteria forming biofilms where they may withstand antibiotic therapy. The use of adjuvant strategies, such as hydrolytic enzymes cocktail targeting biofilm matrices and facilitating their dispersion, is a promising option to limit impact of biofilms. Our aim was to evaluate the effect of enzymes cocktail combined with antibiotic dual therapy of rifampicin and vancomycin in a relevant in-vitro 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.

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, with cumulative treatment costs for all prosthetic joint infections estimated to be ∼ $1 billion per annum (UK and North America). Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Low intensity pulsed ultrasound is already licensed for clinical use in fracture management and thus could be translated quickly into a clinical treatment

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged with gentamicin +/− low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 180 minutes and 20 minutes, respectively. The primary outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. Assessment of cellular metabolism was conducted using a liquid-chromatography-mass spectrometry, as well as a triphenyltetrazolium chloride assay coupled with spectrophotometry.

There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was reduced to 64mg/L. Metabolic activity of biofilm-associated S. aureus was increased by 25% following ultrasound therapy (p < 0 .0001), with identification of key biosynthetic pathways activated by non-lethal dispersal.

Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin, bringing the MBEC of gentamicin to within clinically achievable concentrations. The mechanism of action was due to partial disruption of the extracellular matrix which led to an increase of nutrient availability and oxygen tension within the biofilm. This metabolic stimulus was responsible for the reversal of gentamicin tolerance in the biofilm-associated S. aureus.

Aims

Prosthetic joint infection (PJI) remains the most severe complication of arthroplasty. Failure of intensive, long-term antibiotic treatment for PJI often requires removal of the implant. Antibiotic failure is thought to be caused by biofilm and persister formation. Novel anti-biofilm and anti-persister strategies are urgently needed. Here, we investigated the effects of several antimicrobial peptides on the bacteria within antibiotic-treated biofilms in an in vitro mature biofilm model on abiotic surfaces.

Aim

Irrigation is a major step during debridement surgery in the context of Prosthetic Joint Infections (PJI), but its effects on biofilms are poorly described.

The present study aims at evaluating the effect of PW alone or followed by antibiotics on MSSA and MRSA biofilms grown on Ti6Al4V coupons in-vitro.

Peri-prosthetic joint infection (PJI) is one the most devastating complications of joint arthroplasty. Although PJI is an infrequent complication (the reported incidence is 1%-2% in the United States), it is the most common indication for revision total knee arthroplasty in the Medicare population and the third most frequent indication for revision total hip arthroplasty. Moreover, the prevalence of PJI appears to be on the rise, with a projected number exceeding 60,000 to 70,000 cases in the United States by 2020.

It is estimated that more than 25% of revision procedures annually are attributed to PJI and this number is expected to increase in the upcoming years. The increase in the prevalence of obesity, diabetes, and other comorbidities among the patient population and the emergence of resistant infecting organisms are some of the reasons for the expected rise in the number of infections that medical community will witness.

The challenges that PJI present to the orthopaedic community are on many fronts. Prevention of PJI has proven to be a difficult task indeed. Effective strategies for prevention of PJI are being refined. The Center for Disease Control will be publishing its updated Surgical Site Prevention Guidelines in the next few months that consists of specific recommendations for prevention of PJI. In recent years, strides are made in introducing novel molecular techniques for diagnosis of PJI, which may stand to change our practices. The current surgical technique for management of PJI, besides the immense cost, fall short of delivering high success to the patients. The major problem in eradication of infection relates to formation of biofilm on the implant surface and internalization of the organisms by affected cells. Biofilm is a sophisticated structure comprising of organisms embedded in multiple layers of glycoccalyx that allows the organisms to evade host immunity and is impenetrable to antibiotics. These organisms are capable of communicating through molecular mechanisms such as quorum sensing that affords them advantage for survival in the host environment. In recent years strategies to prevent colonization of the implant surface, an essential first step in formation of biofilm, or biofilm disruption techniques have been introduced. A recent International Consensus meeting on PJI that assembled more than 350 experts identified some of the best practices in this field and identified areas in need of future research. Moving into the future, the field of orthopaedics in general and PJI in particular stand to benefit from the discoveries in the field of molecular diagnostics, metabolomics and epigenetics.

Aim

Determine the time concentration profile required to achieve vancomycin-mediated eradication of Staphylococcus aureus biofilm. This is critical for the identification of performance targets for local antibiotic delivery, yet has not been described.

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