Effects Of Intensity High-Intensity Focused Ultrasound On Pseudomonas Aeruginosa Biofilm

【Objective】To investigate the effect of High-Intensity Focused Ultrasound ( HIFU ) on bactericidal action and the structure of Pseudomonas aeruginosa biofilm in vitro.【Methods】Models of 3 d, 6 d PAO1 biofilm were established in vitro, divided into control group(sham exposure group) and intervention group(HIFU group).The treatment effect was accessed by determining the number of colony forming units (CFU) in the bacteria biofilm. The biofilm was stained by FITC-ConA, observed by fluorescence microscope. The effects of sham exposure and HIFU intervention on biofilm were monitored by Confocal Laser Scanning Microscope (CLSM) respectively, based on fluorophores from PAO1 with nucleic acid SYTO9/PI stained. The bacterial mortality rate under different conditions was analyzed by Image Pro Plus 6.0. Quantification of the effect could be calculated by Image Structural Analyzer (ISA) software.【Results】(1) Colony forming units (CFU)of 3d model of the intervention group and control group were(4.72±1.06)Lg CFU/ml and (7.64±0.16)Lg CFU/ml, t=7.35, P<0.05, while 6d model were(5.02±0.96)Lg CFU/ml and(7.10±1.20)Lg CFU/ml , t=3.84, P<0.05.( 2 )Exopolysaccharides (EPS) of biofilm intervened by HIFU reduced in some way .(3)Image Pro Plus 6.0 (IPP):Bactercial mortality rate of 3d model of the intervention group and control group were(55.66±7.28)% and ( 39.74±7.54 )% , t=8.99, P<0.05, while 6d model were (60.05±4.00)% and (43.36±6.56)% , t=12.85, P<0.05.(4)Parameters of Image Structural Analyzer ( ISA):3d model: biofilm thickness of the intervention group and control group wer(e5.11±0.92)μm and (8.06±0.79)μm, t=6.89, P<0.05; the areal porosity (AP) were 0.89±0.26 and 0.50±0.17 ,t=3.51, P<0.05; the average diffusion distance (ADD) were 1.57±0.03 and 3.12±1.69 , t=-2.60, P<0.05; the textual entropy(TE) were 3.82±0.63 and 5.57±0.45 , t=-6.749, P<0.05.6d model: biofilm thickness of the intervention group and control group were(7.77±0.48)μm and(21.33±4.76)μm ,t=7.94, P< 0.05;AP were 0.97±0.02 and 0.35±0.14 ,t=-6.75, P< 0.05 ; ADD were 1.56±0.09 and 2.28±0.57 , t=3.53, P<0.05 ; TE were 4.31±1.16 and 6.17±0.65 ,t=3.96, P<0.05 .【Conclusions】The bacterial mortality rate increases in some way and the structure of biofilm could be destroyed by HIFU in vitro. 【Objective】To investigate the effects of different intensity High-Intensity Focused Ultrasound on the three-dimensional structure of Pseudomonas aeruginosa biofilm in vitro.【Methods】Models of 3 d Pseudomonas aeruginosa PAO1 biofilm were established in vitro. Output powers of HIFU were respectively 40 W and 60 W. Changes of the number of colony forming units (CFU) were calculated by MTT assay. Confocal Laser Scanning Microscope (CLSM) was used, based on fluorophores from PAO1 with nucleic acid SYTO9/PI stained. Quantification of the effects could be calculated by Image Structural Analyzer (ISA) software.【Results】Colony forming units (CFU) of control group , 40W group and 60W group were 5.10±0.87, 3.13±0.29 and 1.30±0.36 (*10^7 CFU/ml) . There were statistically significant differences in each two groups. Parameters of Image Structural Analyzer (ISA): after intervention biofilm thickness and the textual entropy (TE) were reduced and there were statistically significant differences in each two groups. The areal porosity (AP) was increased and the average diffusion distance (ADD) was reduced, and ADD and AP in 40 W group and 60 W group compared with the control group were significantly different, but there is no significant difference between the two groups.【Conclusions】Higher power of the HIFU shows more powerful effects in the killing of bacteria and the destruction of BF structure in vitro.