Pressure- and frequency-dependent acoustic behavior of second-generation acoustic reporter genes-expressing bacteria...

Acoustic reporter genes (ARGs) enable genetically engineered bacteria to express gas vesicles (GVs), which function as intracellular acoustic scatterers for noninvasive ultrasound imaging. While second-generation ARGs (bARG_Ser) exhibit enhanced nonlinear acoustic properties, their signal-level acoustic characteristics remain incompletely understood. Moreover, how basic nonlinear pulse schemes and imaging parameters should be selected based on these acoustic characteristics for bARG_Ser-expressing bacteria has not been systematically evaluated. In this study, we investigated the pressure- and frequency-dependent acoustic characteristics of Escherichia coli BL21 expressing bARG_Ser, and compared with purified gas vesicles (GV_Ser) isolated from bacteria under identical conditions. Collapse behavior and nonlinear acoustic responses were characterized at selected imaging frequencies (5, 8, and 10 MHz) under varying acoustic pressures, while attenuation properties were measured over a broader frequency range (4-20 MHz). Several basic nonlinear pulse schemes were evaluated to assess their contrast-to-tissue ratio (CTR) performance for bARG_Ser-expressing bacteria under acoustically characterized pressure conditions. Engineered bacteria exhibited progressive vesicle collapse at acoustic pressures exceeding 1.5 MPa across all tested frequencies, indicating minimal frequency dependence of the collapse threshold within the investigated range. Below the collapse threshold, pressure-dependent nonlinear scattering was observed above 0.7 MPa, with harmonic amplitudes exceeding those of linear scatterers. Higher transmission frequencies resulted in increased harmonic-to-fundamental ratios, although detection bandwidth limitations influenced spectral observations. Attenuation measurements revealed frequency- and concentration-dependent increases in acoustic attenuation, while pressure-dependent attenuation exhibited a non-monotonic trend, increasing prior to collapse and decreasing thereafter due to vesicle destruction. In addition, compared with intracellular GVs, purified GV_Ser demonstrated slightly lower collapse thresholds and stronger nonlinear responses, and lower acoustic attenuation. Phantom experiments comparing multi-pulse imaging schemes revealed that, among the evaluated schemes, triple-pulse amplitude modulation (AM3) at 1.81 MPa provided the highest CTR under the tested conditions, where partial GV collapse began to occur. In vivo tumor imaging further confirmed enhanced visibility of the bacterial region using this optimized AM3 condition. These findings provide a basis for optimizing imaging frequency, pressure, and basic pulse-scheme selection in ultrasound imaging of second-generation ARGs-expressing bacteria, and may inform future development of GV-specific nonlinear imaging methods.