Coincident Light/ultrasound therapy to treat bacterial biofilms

Bacteria preferentially grow as colonies surrounded by a complex matrix, together called a biofilm. According to the NIH over 80% of microbial infections in the human body consist of biofilms. Biofilms are highly resistant to antibiotics and the immune system, making them difficult to treat at best, and nearly impossible to eradicate at worst. We propose to treat bacterial biofilms by simultaneous exposure to low energy, non-focused ultrasound, combined with blue/violet light (i.e. CLENS™: Coincident Light Energy and Nonfocused ultraSound). Low intensity ultrasound has previously been explored for anti-biofilm activity, however it has generally been applied in conjunction with an antibiotic. Similarly, blue/violet light has been explored as a bactericidal agent for planktonic bacteria. We hypothesized that the simultaneous application of blue/violet light with ultrasound would synergize the beneficial properties of each, and successfully provide a technologically new method to attack biofilms. A prototype laboratory device was built to deliver simultaneous ultrasound and light energy to bacterial biofilm grown in-vitro. Light emitting diodes (405nm) were used to generate 30mW/cm
²
; and a coincident ultrasound source (450kHz) created a pressure field at less than 100mW/cm
²
. Staphylococcus epidermidis, S. aureus, and P. acnes were grown on hanging inserts (i.e. transwells) at 37°C for up to 72 hours before exposure. Following CLENS exposure, bacteria killing was quantified by serial dilution and plating on media agar. After an appropriate growth time on agar, Colony Forming Units (CFUs) were counted. Killing was dose dependent on exposure time, and could be optimized with tuning of either the light or ultrasound energy. There was >1 log
10
reduction after 5 min and >3 log
10
reduction after 30 min treatment (p<0.05). In comparison, 24 hr exposure to erythromycin reduced biofilm CFU by less than 1 log
10
(p<0.05). While a 30 min CLENS treatment induced significant bacterial death, the die off continued to 24 hr after exposure with an additional 2 log
10
reduction (p<0.05). Biofilm regrowth started at 48 hr, but after 4 additional days of growth, the biofilm had not returned to the original CFU levels (p<0.05). Importantly, the two energies must be delivered coincidentally for optimal effect. These initial results validate that the basic CLENS mechanism is effective on relevant biofilm producing bacteria: low intensity ultrasound “activates” the bacteria within the biofilm such that they become susceptible to the antimicrobial effects of blue light.