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Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 9 - 9
1 Dec 2018
AN ANTISTAPHYLOCOCCAL BACTERIOPHAGE LYSATE TO TREAT DRUG-RESISTANT STAPHYLOCOCCUS AUREUS INVOLVED IN PROSTHETIC JOINT INFECTIONS: AN ALTERNATIVE STRATEGY
Di Luca M Klatt A Trampuz A
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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.

Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 66 - 66
1 Dec 2018
COMPARISON OF SONICATION AND CHEMICAL METHODS FOR THE BIOFILM DETECTION, INCLUDING CHELATING AND REDUCING AGENTS
Karbysheva S Di Luca M Butini ME Trampuz A
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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/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.

Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 67 - 67
1 Dec 2018
SPECIFIC ANTIBIOFILM PROPERTIES OF BACTERIOPHAGE SB-1 MAKE IT SUITABLE FOR THE THERAPY OF PROSTHETIC JOINT INFECTIONS DUE TO STAPHYLOCOCCUS AUREUS: BIOFILM MATRIX DEGRADATION AND PERSISTER CELLS KILLING
Di Luca M Tkhilaishvili T Trampuz A
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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.

Method

MRSA ATCC 43300 24h-old biofilm was treated for 18h with Sb-1 titers (from 104 to 106 pfu/ml). Biofilm matrix was evaluated by confocal laser scanning microscopy after staining with wheat germ agglutinin conjugate with Alexafluor488 (WGA488) to label exopolysaccharide matrix and Syto 85 to label bacterial cells. Persister status was induced using two different protocols: i) by exposing stationary phase S. aureus to 400 µg/ml carbonyl cyanide m-chlorophenylhydrazone (CCCP) in PBS for 3h at 37°C and ii) by treatment of 24h old biofilm with 512 µg/ml ciprofloxacin for further 24h at 37°C. Then, induced persister cells and non-induced controls (106 CFU/ml) were treated with 104 PFU/ml and 107 PFU/ml Sb-1 for 3h, followed by CFU counting. Alternatively, bacteria were washed and incubated in fresh BHI medium for the resumption of normal growth and the bacterial growth assessed after further 24 hours.

Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 65 - 65
1 Dec 2018
SIMULTANEOUS AND SEQUENTIAL APPLICATIONS OF PHAGES AND CIPROFLOXACIN IN KILLING MIXED-SPECIES BIOFILM OF PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS AUREUS
Tkhilaishvili T Di Luca M Trampuz A
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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 (106 CFU/ml) and P.aeruginosa (103 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 × MBECbiofilm), 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

Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_22 | Pages 8 - 8
1 Dec 2017
SYNERGISTIC ACTIVITY OF LYTIC BACTERIOPHAGE AND ANTIBIOTICS AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS BIOFILM
Tkhilaishvili T Di Luca M Trampuz A Gaudias J
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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.

Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 16 - 16
1 Dec 2016
IN VITRO EVALUATION OF LYTIC BACTERIOPHAGE ACTIVITY AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) BIOFILM
Tkhilaishvili T Javakhadze M Trampuz A Di Luca M
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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 (5×106 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).

Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 75 - 75
1 Dec 2016
ACTIVITY OF A GENTAMICIN-LOADED BONE GRAFT SUBSTITUTE AGAINST DIFFERENT BACTERIAL BIOFILM BY MICROCALORIMETRY
Butini ME Trampuz A Di Luca M
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Aim

To investigate the antimicrobial activity of a gentamicin-loaded bone graft substitute (GLBGS) in the prevention and eradication of bacterial biofilms associated with prosthetic joint infections (PJI).

Method

The GLBGS (17,5 mg gentamicin/ml paste) with 40% hydroxyapatite/60% calcium sulfate1 was tested against biofilms of methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, methicillin-susceptible S. aureus (MSSA) ATCC 29213, Escherichia coli Bj HDE-1, S. epidermidis ATCC 12228 and Enterococcus faecalis ATCC 19433. For prevention studies, glass beads and different combinations of GLBGS were co-incubated for 24h at 37°C in CAMH broth with 1–5 × 106 CFU/mL of bacteria. For eradication, biofilms were formed on glass beads for 24h at 37°C in CAMH broth. Then, beads were incubated with different combinations of GLBGS in medium at 37°C for 24h. For microcalorimetric measurements, beads were placed in ampoules and heat flow (µW) and total heat (J) were measured at 37°C for 24h. The minimal heat inhibitory concentration (MHIC) was defined as the lowest gentamicin concentration reducing the heat flow peak by ≥90% at 24h.

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