Abstract

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.

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop 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 explores, but preliminary work has shown potential benefit, especially when combined with existing antibiotics.

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, bacterial biofilms were challenged by gentamicin +/− low-intensity ultrasound (1.5MHz, 30W/cm2, pulse duration 200µs/1KHz) for 20 minutes. The 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. 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 64mg/L.

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

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. 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.

Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged by a low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 20 minutes and gentamicin. The 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. 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 64mg/L. Further studies confirmed that the mechanism of action was due to incomplete disruption of the extracellular matrix with subsequent metabolic stimulation of the dormant biofilm-associated bacteria due to increased nutrient availability and oxygen tension.

Low intensity pulsed ultrasound was associated with a 4-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations.