The Effects of Low-frequency Ultrasound (35 kHz) on Methicillin-resistant Staphylococcus aureus (MRSA) in vitro

Scanning electron microscopy study results support an in vitro bactericidal effect of low-frequency ultrasound (LFU) delivered at 40 kHz on bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The purpose of this in vitro study was to determine the effects of LFU delivered at 35 kHz on bacterial viability, cell wall structure, and colony characteristics, including antibiotic resistance on vegetative forms of MRSA. A known MRSA isolate from a patient lower extremity wound was subcultured, plated, and grown on sheep blood agar (SBA). Serial dilutions of the organisms were made and treated with LFU for 30, 60, and 180 seconds. One hundred microliters (mu L) of control (untreated organisms) and treated samples were inoculated to SBA in triplicate and three separate experiments were conducted. Using standard microbiological techniques, a reduction in MRSA from 1 million colony forming units (CFU) at baseline to 6 CFU after 30 seconds of treatment with 35 kHz was observed. MRSA plated at 10(8) CFU and treated with 35 kHz showed a 44.1% viability with flow cytometry, compared to 92.5% viability of untreated control MRSA. Changes in pigmentation, odor, colony size, and hemolysis pattern also were observed in the LFU-treated bacteria. The effect of LFU on methicillin resistance was dose-dependent; the zone of inhibition increased from 6 mL at baseline to 14.3 mL after 30, 16.7 after 60, and 20.3 after 180 seconds of treatment. The results suggest that, in this in vitro model, 35-kHz LFU reduces CFU of bacteria, punctures and fractures cell walls, and alters colonial characteristics of MRSA, including resistance to the oral form of methicillin. Studies to elucidate the observed effects of LFU on MRSA and evaluate its effect in vivo are warranted.