header advert
Results 1 - 3 of 3
Results per page:
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
Full Access
View article

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. 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
Full Access
View article

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. 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
Full Access
View article

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.