Comparative Study of Histotripsy Pulse Parameters Used to Inactivate Escherichia coli in Suspension

Abstract

Objective: Bacterial loads can be effectively reduced using cavitation-mediated focused ultrasound, or histotripsy. In this study, gram-negative bacteria (Escherichia coli) in suspension were used as model bacteria to evaluate the effectiveness of two regimens of histotripsy treatments: cavitation histotripsy (CH) and boiling histotripsy (BH).

Methods: Ten-milliliter volumes of Escherichia coli were treated at different negative focal pressure amplitudes and over time periods up to 40 min. Cavitation activity was characterized with coaxial passive cavitation detection (PCD) and synchronized plane wave B-mode imaging.

Results: CH treatments exhibited a threshold behavior that was consistent with PCD metrics of cavitation. Above the threshold, bacterial inactivation followed a monotonically increasing log-linear relationship that indicated an exponential inactivation rate. BH exhibited no threshold, but instead followed a different monotonically increasing inactivation rate. Inactivation rates were larger for BH at or below the CH threshold, and larger for CH substantially above the threshold. CH studies performed at different pulse lengths at the same duty cycle had similar inactivation rates, suggesting that at any given pressure amplitude, the "on time" was the most important variable for inactivating E. coli. The maximum inactivation was produced by CH at the highest pressure amplitudes used, leading to a log reduction >4.2 for a 40 min treatment.

Conclusion: The results of this study suggest that both CH and BH can be used to inactivate E. coli in suspension, with the optimal regimen depending on the attainable peak negative focal pressure at the target.

Keywords: Bactericide; Boiling histotripsy; Cavitation histotripsy; Focused ultrasound; Histotripsy; Inactivation; Shock-scattering histotripsy.

Figure 1.

Custom experimental apparatus. A 1 MHz transducer with integrated inline P4/2 imaging probe was attached to the bottom of a degassed water bath. The apparatus was designed so that the transducer’s focus was at the center of a sample vial containing 10 ml of E. coli bacteria (~ 1 x 10⁹ cells/ml). A thin acoustically transparent membrane was placed at the bottom of the vial for improved acoustic energy transmission. The vial was aligned to the transducer using two pairs of magnets to allow for fast reproducible alignment of replicate samples.

Figure 2.

Transducer characterization. (a) Peak positive and negative amplitudes vs. input voltage. Most studies were performed between peak negative pressure amplitudes 12 MPa to 23 MPa. The red line indicates the output level corresponding to fully developed shock formation. (b) Representative waveforms corresponding to the output levels below (black line), and above (blue and red lines) shock formation.

Figure 3.

Mean log reduction (± SD) in bacterial load with increasing peak negative pressure amplitude. (a) The CH threshold for inactivating E. coli depended on pulse parameters. 5 cycle pulses at 2 kHz (1% duty cycle) had a higher threshold than 10 cycle pulses (2% duty cycle). Above the threshold, the bacterial log reduction increased linearly with amplitude (R²=0.93 for both curves). (b) With BH (10,000 cycles at 1 Hz PRF), there was no measured threshold. However, the log reduction in bacteria also increased linearly with amplitude (R²=0.90). The open circles in each figure have the same log reduction over 10 minutes (0.9) at the same pressure amplitude (17.8 MPa).

Figure 4.

Representative B-mode ultrasound images observed during CH (top) and BH (bottom). The hyperechoic CH bubble cloud was generally confined to the focal area; it slightly elongated and shifted pre-focally with the increase of P⁻ from 14.5 to 23 MPa (see supplementary videos V2-4, respectively). Conversely, following each BH pulse hyperechoic bubbles filled the entire sample volume through visible streaming-induced mixing (see supplementary video V1) and persisted until the next pulse arrived. Note the HIFU reverberation artifact in the image immediately following the BH pulse (40msec).

Figure 5.

Passive cavitation detection metrics obtained from the 5-cycle CH exposures in Fig. 3a: (a) Doppler power associated with spatio-temporal changes in the bubble cloud. The red circles help identify the change in slope, where cavitation occurs each pulse. (b) Average amplitude of broadband noise emissions over the 10-minute exposure. Both metrics exhibit a threshold-like behavior at the focal pressure of 16 MPa that also corresponds to the threshold for inactivating E.coli and indicates the onset of consistent cavitation. The error bars represent standard deviation. Trendlines are used to aid the eye.

Figure 6.

A comparison between CH (5 cycles, 2000 Hz PRF; red circles) and BH (10,000 cycle pulses and 1 Hz PRF; open triangles) bacterial load reduction at a pressure of 17.8 MPa and 1% duty cycle. R² = 0.99 (CH) and 0.97 (BH). See Table I for converting treatment time to pulse number.

Figure 7.

Mean log reduction (± SD.) in bacterial load over time at different peak negative pressures. Dashed lines represent best fit linear trendlines. (a) CH treatments used 5 cycle bursts at 2,000 Hz PRF. R² values for 23 MPa and 17.8 MPa = 0.95 and 0.99, respectively. The slope of the trendline for 23 MPa and 17.8 MPa = 0.11 and 0.07 respectively. (b) BH treatments used 10,000 cycle bursts at 1 Hz PRF. R² values for 17.8 MPa and 14.5 MPa = 0.98 and 0.99, respectively. The slope of the trendlines for 17.8 MPa and 14.5 MPa = 0.075 and 0.054, respectively. See Table II for converting treatment time to pulse number.

Figure 7.

Mean log reduction (± SD.) in bacterial load over time at different peak negative pressures. Dashed lines represent best fit linear trendlines. (a) CH treatments used 5 cycle bursts at 2,000 Hz PRF. R² values for 23 MPa and 17.8 MPa = 0.95 and 0.99, respectively. The slope of the trendline for 23 MPa and 17.8 MPa = 0.11 and 0.07 respectively. (b) BH treatments used 10,000 cycle bursts at 1 Hz PRF. R² values for 17.8 MPa and 14.5 MPa = 0.98 and 0.99, respectively. The slope of the trendlines for 17.8 MPa and 14.5 MPa = 0.075 and 0.054, respectively. See Table II for converting treatment time to pulse number.

Figure 8.

Mean log reduction (± SD.) in bacterial load over time. Dashed lines represent best fit linear trendlines. CH pulse parameters: Black triangles: 10 cycles, 1000 Hz PRF. Red circles: 5 cycles, 2000 Hz PRF. P^--= 16 MPa for both. R² values for 5 cycles and 10 cycles = 0.93 and 0.96, respectively. See Table III for converting treatment time to pulse number.