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Orthopaedic Proceedings

Vol. 101-B, Issue SUPP_14 | Pages 88 - 88

1 Dec 2019

EFFICACY OF SB-1 BACTERIOPHAGE IN TREATING AND PREVENTING METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS IN A GALLERIA MELLONELLA MODEL OF IMPLANT-ASSOCIATED INFECTION

Luca MD Materazzi A Klatt A Bottai D Tavanti A Trampuz A

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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 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.

Orthopaedic Proceedings

Vol. 101-B, Issue SUPP_14 | Pages 17 - 17

1 Dec 2019

IN VITRO ACTIVITY OF FOSFOMYCIN, CIPROFLOXACIN, GENTAMICIN AND THEIR COMBINATIONS AGAINST ESCHERICHIA COLI AND PSEUDOMONAS AERUGINOSA BIOFILMS

Wang L Luca MD Tkhilaishvili T Gonzalez-Moreno M Trampuz A

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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).

Orthopaedic Proceedings

Vol. 101-B, Issue SUPP_14 | Pages 24 - 24

1 Dec 2019

ISOTHERMAL MICROCALORIMETRY DETECTS THE PRESENCE OF PERSISTER CELLS IN A STAPHYLOCOCCUS AUREUS BIOFILM AFTER VANCOMYCIN TREATMENT

Butini ME Abbandonato G Rienzo CD Trampuz A Luca MD

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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.

Orthopaedic Proceedings

Vol. 99-B, Issue SUPP_22 | Pages 36 - 36

1 Dec 2017

ISOLATION OF NEW LYTIC BACTERIOPHAGES FOR TREATMENT OF PROSTHETIC JOINT INFECTION

Trampuz A Klatt A Luca MD

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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.

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