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Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_5 | Pages 7 - 7
1 Mar 2021
IN VITRO EVALUATION OF STAPHYLOCOCCAL BACTERIOPHAGE SB-1 AS AN ADJUNCTIVE AGENT TO ANTIBIOTICS AGAINST RIFAMPIN-RESISTANT STAPHYLOCOCCUS AUREUS BIOFILMS
Wang L Tkhilaishvili T Trampuz A Gonzalez-Moreno M
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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 (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.

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. 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).

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