In Vitro Study Of The Low-frequency Low-intensity Ultrasound On The Permeability Of The Cell Wall Of Mycobacterium Smegmatis And The Synergistic Effect With Antibiotic

Objective Perform an in vitro experimental study of the possible effects on the permeability of mycobacterium smegmatis’ cell wall by low-frequency(42 k Hz) ultrasound(US) of different irradiation of spatially and temporally averaged low intens ities(hereinafter called intensity) and exposure times. Then study the synergistic effect of the low-frequency and low intensities ultrasound(LFLIU) with antibiotic levofloxacin.Methods The frequency of the low-frequency and low-intensity ultrasound equipment is 42 k Hz experimently, the acoustic irradiation intens ity is continuous ly adjustable(range 0.13-0.34 W/cm2), and the transducer diameter is 45 mm. Selection of ultrasound equipment 0.14 W/cm2, 0.33 W/cm2 intensities are irradiated Mycobacterium smegmatis for 5 min and 20 min respectively to detect the impact of the device on the viability of mycobacterium smegmatis. The fluorescence intens ity of FDA and PI and the structure of the bacteria were measured by flow cytometer and electron microscopy after US(intens ity/exposure time) irradiation respectively. And then a suitable ultrasonic irradiation intens ity was chosen according to the above mentioned results. An amount of bacteria suspension was added into the well of a 6--well cell culture plate. Then they were randomly divided into 4 groups, each group included samples of 3 wells, with Group 1 as the control group, Group 2 as ultrasound alone treatment group, Group 3 as levofloxacin group, and Group 4 as US + levofloxacin group. After US irradiation, the ratio of damage was examined. The cavitation effect of the device was detected by the method of passive cavitation detection(PCD).Results 1. By 0.14 W/cm2, 0.33 W/cm2 irradiated Mycobacterium smegmatis for 5 min and 20 min later, there are no significant differences(P> 0.05) between the survival rate of the control group and the experimental groups of bacteria, namely, the US equipment in this range radiation dose could not kill the Mycobacterium smegmatis. 2. After 0 W/cm2, 0.14 W/cm2, 0.19 W/cm2, 0.33 W/cm2 sound intens ity ultrasonic irradiation for 5 min, the fluorescence intens ity of FDA in the vivo of the M. smegmatis was 2299.95 ± 143.07, 1384.33±85.23, 1051.00 ± 106.0, 970.33±58.39 respectively, that is, with an increase in the sound intens ity the fluorescence intensity of FDA in the vivo of the bacteria gradually weakened(P<0.01); by 0 W/cm2, 0.14 W/cm2, 0.19 W/cm2, 0.33 W/cm2 sound intensity ultrasonic irradiation for 5 min, the fluorescence intens ity of PI in the vivo of M. smegmatis was respectively 31.33 ± 1.15, 40.33 ± 1.5, 49.33 ± 2.5, 63.00 ± 1.00, the fluorescence intens ity of PI as a function of irradiation dose enhancement and gradually increased(P<0.05); 3. The fluorescence intensity of FDA and PI in the bac teria were gradually decrease and increase respectively(P<0.05) with the increase of radiation exposure time after by 0.14 W/cm2, 0.33 W/cm2 sound intens ity radiation 0 min, 5 min, 15 min, 20 min respectively. 4. The electron microscopy study results showed that the structures of mycobacterium smegmatis were destroyed by low-frequency and low-intens ity US. 5. The ratio of bacteria damage tested by flow cytometer showed that suspensions of the US + levofloxacin group were significantly higher than the other groups(P<0.05); 6. A stable(non-inertial) cavitation was detected by passive cavitation detection system under the experimental conditions.Conclusions Low-frequency and low-intensity US can increase the permeability of the cell wall effectively, and the degree of enhancement is closely correlated to the US dose applied. The Synergistic interaction of Low-frequency and low-intensity US with levofloxacin is noteworthy.