Effects Of Low-frequency And Low-density Ultrasound Combined With Antibiotics On Extended-Spectrum Beta-Lactamases-Producing Escherichia Coli

Currently, the formation of biofilm is an important reason leading to clinical difficulties in diagnosis and treatment. In the 2014 biofilm infection diagnosis and treatment guidelines, it is mentioned that ultrasound combined with antibiotics is a therapeutic method against biofilm infection which needs deeper development urgently. In this study we explored the effects of low-frequency and low-intensity ultrasound combined with antibiotics on ESBLs E.coli biofilm and its bacteria suspensionOBJECTIVES To explore the effects of low-frequency and low-intensity ultrasound combined with antibiotics on vitality, morphology and antibiotic penetration of Extended-spectrum Beta-lactamases(ESBLs)- producing Escherichia coli biofilm. To study the effect of low-frequency and low-intensity ultrasound with antibiotics on permeability of the outer membrane and the synergistic effect against the vitality of ESBLs-producing Escherichia coli. Check the cavitation and thermal effect of this device.MATERIALS AND METHODS 1.Effects of low-frequency and low-density ultrasound combined with antibiotics on extended-spectrum beta-lactamases(ESBLs)-producing Escherichia coli(E.coli) biofilmsThe biofilms were distributed into 6 groups: control group(C), gentamicin group(G, the working concentration of gentamicin was 200 μg/m L), tobramycin group(T, the working concentration of tobramycin was 200 μg/m L), ultrasound group(U), ultrasound combined with gentamicin group(U+G, the working concentration of gentamicin was 200 μg/m L), ultrasound combined with tobramycin group(U+T, the working concentration of tobramycin was 200 μg/m L). ESBLs E.coli biofilm placed in the 6-well plate on the ultrasound probe within 5 m L PBS was treated with ultrasonic irradiation 42 k Hz and 0.66 W/cm2 for 1.5 h. The morphology and vitality of bacteria were measured before and after the treatments. A ciprofloxacin penetration test was also conducted.2.The study of synergistic effect of low-frequency and low-intensity ultrasound with antibiotics against extended-spectrum beta-lactamases-producing Escherichia coliCulture of ESBLs-producing Escherichia coli was maintained in Luria–Beritani(LB) broth and then the standard the suspension were randomly divided into 4 groups: the control group, the drug group, the ultrasonic irradiation group and the combination of drug and ultrasonic irradiation group. Gentamicin and tobramycin were the therapeutic drug. The frequency and the intensity of ultrasonic irradiation were respectively 42 k Hz and 0.66 W/cm2. The temperature, the vitality and the permeability of the bacteria of each groups were measured before and after the experiment processing.RESULTS 1.The viability of biofilm experienced a significant reduce after treatment especially the amount of ultrasound+gentamicin(U+G)group and ultrasound+tobramycin(U+T) group decrease from(9.198 ±0.022)log10CFU/cm2 to(5.243 ± 0.007)log10CFU/cm2,(3.509 ±0.020)log10CFU/cm2 respectively. Additionally, the combination above could also enhance the permeability of biofilms to ciprofloxacin. Meanwhile, SEM was conducted to observe the changes in biofilm morphology caused by ultrasound, and from the result of the confocal laser scanning microscope, the intermediate layer of ESBLs E.coli biofilm was affected least and the outer layer most.2.The vitality of the suspension experienced a significant reducing after treatment, especially(97.73±0.12)% decrease in the ultrasonic irradiation with tobramycin group. The fluorescence intensity of Propidium Iodide(PI) showed a causal relationship between the mortality rate and the permeability of the bacteria. Transmission electron microscopy showed that ultrasound could destroy the structure of ESBLs-producing Escherichia coli, thereby changing the permeability of the bacteria. Regard to the temperature and cavitation measured before and after ultrasonic irradiation, acoustic cavitation may play a predominant role in the synergistic effect.CONCLUSION 1.In conclusion, ultrasound can remove ESBLs E.coli biofilm, cause injury of bacteria within biofilm, and enhance biofilm permeability to antibiotics. Ultrasound combined with antibiotics can enhance its effects on biofilm mentioned above and produce different synergistic bactericidal effect due to different MIC of combined antibiotic. This found could potentially be a therapeutic method for bacterial biofilms generated by ESBLs E.coli in vitro. Evaluation of other multi-drug resistant organisms and the mechanism of the two antibiotics have little difference in their effects on biofilm remain for future studies.2.A certain dose of ultrasound may change the permeability of ESBLs-producing Escherichia coli and it’s synergistic effect of ultrasound with antibiotics against ESBLs-producing Escherichia coli is also conspicuous.