基于刃天青的电化学方波伏安法快速鉴别大肠杆菌的耐药性

Rapid identification of drug resistance of Escherichia coli based on electrochemical square wave voltammetry of resazurin

目的:评价5株食源性大肠杆菌临床分离株对环丙沙星、庆大霉素和氨苄西林的抗生素耐药性。方法:基于细菌能量代谢过程中的电子传递理论,引入刃天青作为氧化还原探针,建立了一种基于刃天青介导的大肠杆菌耐药性快速鉴别的电化学方波伏安法(square wave voltammetry,SWV)。结果:在1.69×10^(2)~1.69×10^(8)CFU/mL范围内,大肠杆菌浓度与刃天青氧化峰电流(I)之间存在较好的线性关系,回归方程为Y=-0.1560 X-2.9655,R^(2)=0.9775;在进行大肠杆菌耐药性测定时,参考美国临床和实验室标准协会制定的药敏试验标准,以刃天青的氧化峰电流(I_(0))和大肠杆菌直接与刃天青作用后得到的氧化峰电流(I_(-a))作为对照,测定大肠杆菌用抗生素处理后与刃天青作用得到氧化峰电流(I_(+a)),引入细菌活性值V_(ETA)作为电化学方波伏安法鉴别细菌耐药性的指标,建立了刃天青介导的大肠杆菌电化学快速药敏试验方法;运用建立的方法检测了5种食源性大肠杆菌临床分离株对3种抗生素的耐药性,以V ETA=50作为判断细菌敏感和耐药的临界值,均得到与纸片扩散法结果相符的药敏试验结果,而纸片扩散法报告结果需18 h,该法仅需2 h。结论:建立的基于刃天青介导的大肠杆菌耐药性快速鉴别的电化学方波伏安法能够准确且快速检测5株食源性大肠杆菌临床分离株对环丙沙星、庆大霉素和氨苄西林的抗生素耐药性。

Objective:To evaluate the antibiotic resistance of five foodborne Escherichia coli clinical isolates to ciprofloxacin,gentamicin and ampicillin.Methods:Based on the theory of electron transfer during bacterial energy metabolism and the introduction of resazurin as a redox probe,an electrochemical square wave voltammetry(SWV)method based on bladed tensin-mediated rapid identification of E.coli drug resistance was established.Results:The electrochemical square wave voltammetry values of E.coli at different concentrations mediated by resazurin were determined,and there was a good linear relationship between the E.coli concentration and the oxidized peak current(I)of resazurin in the range of 1.69×10^(2)~1.69×10^(8)CFU/mL,with the regression equation of Y=-0.1560 X-2.9655,and R^(2)=0.9775;When performing the determination of E.coli drug resistance,the drug sensitivity test standard was developed by Clinical and Laboratory Standards Institute(CLSI).The oxidized peak current(I_(0))of resazurin and the oxidized peak current(I_(-a))obtained by E.coli after directly interacting with resazurin were used as the control,and the oxidized peak current(I_(+a))obtained by E.coli after treating it with antibiotics and interacting with resazurin was determined.The value of bacterial activity V ETA was introduced as V ETA as an indicator for the identification of bacterial resistance by electrochemical square wave voltammetry,and established a rapid electrochemical drug sensitivity test method for E.coli mediated by resazurin.The established method was used to detect the resistance of five clinical isolates of foodborne E.coli to three antibiotics,and V_(ETA)=50 was used as the critical value for determining bacterial sensitivity and resistance,and the results of the drug sensitivity tests were all in accordance with the results of the paper diffusion method.The V ETA=50 was used as the critical value for determining bacterial susceptibility and resistance,and all the results were consistent with the results of the paper diffusion method,and the paper diffusion method required 18 h to report the results,whereas this method required only 2 h.Conclusion:Our electrochemical square-wave voltammetry method based on the resazurin-mediated rapid discrimination of E.coli drug resistance is able to accurately and rapidly detect the antibiotics resistance of the five foodborne clinical isolates of E.coli to ciprofloxacin,gentamicin,and ampicillin.