Effect of low-intensity ultrasound upon biofilm structure from confocal scanning laser microscopy observation

Ultrasonic irradiation at 500 kHz and 10 mW cm-2 of a 24 h old biofilm of P. aeruginosa enhanced the killing of bacteria by gentamicin. To determine whether this bioacoustic effect was caused by ultrasonic-induced changes in the biofilm morphology (biofilm breakup or disruption), the biofilms were examined by confocal scanning laser microscopy (CSLM). Such disruption would be undesirable in the possible ultrasonic treatment of implant infections. The CSLM results showed that the biofilm is a partial monolayer of cells with occasional aggregates of cells, non-cellular materials and extracellular spaces. The aggregates contained large amounts of exopolysaccharide. The structure of biofilm was not changed when the biofilm was exposed to continuous ultrasound at 500 kHz and 10 mW cm-2, the same irradiation parameters that increased cell killing by nearly two orders of magnitude. The observation that low-intensity ultrasound does not disrupt biofilm or disperse the bacteria has significance in the possible use of ultrasound to enhance the action of antibiotics against biofilms.