3种青霉素类抗生素对大肠杆菌的时间毒性微板分析法建立及其联合抑菌作用
Establishment of Time-dependent Microplate Toxicity Analysis Method for Combined Antibacterial Effects of Three Penicillin Antibiotics to Escherichia coli
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摘要: 除了浓度外，时间也是影响污染物对生物毒性作用的重要因素。为了考察持续暴露下青霉素类抗生素对大肠杆菌(Escherichia coli, E. coli)的联合抑菌作用，以哌拉西林钠(piperacillin sodium, PIP)、青霉素G钾(penicillin G potassium, PEN)和青霉素V钾盐(penicillin V potassium salt, PHE)为研究对象，以96孔微板为实验载体，通过调整培养基成分比例、培养条件、测试时间、毒性指标计算以及微板设计等进行优化，建立了基于E. coli的时间毒性微板分析法(time-dependent microplate toxicity analysis, t-MTA)，系统考察了3种抗生素以及均匀设计射线法设计的三元混合物体系(PIP-PEN-PHE)对E. coli在不同暴露时间0.25、2、4、8和12 h的抑菌作用，运用浓度加和(concentration addition, CA)模型分析了抗生素联合抑菌作用类型。结果表明，建立的t-MTA可以测定3种青霉素类抗生素及其混合物对E. coli在不同暴露时间、不同暴露浓度的抑菌作用数据，且3种抗生素及其三元混合物对E. coli的抑菌作用均具有明显的时间依赖性，浓度-效应曲线均为“S”型；以半数效应浓度的负对数(pEC50)为毒性大小指标，在暴露8~12 h时，3种抗生素对E. coli抑菌活性为PIP(pEC50=4.46~5.21)> PEN(pEC50=4.16~4.39)>PHE(pEC50=4.01~4.39)，三元混合体系的5条射线对E. coli的抑菌作用为R4>R5>R3>R2>R1，且与混合物组分中的PIP浓度比正相关(R=0.9189, RMSE=0.0703)，即混合物毒性具有时间和组分浓度比依赖抑菌性；3种抗生素及其三元混合物对E. coli的抑菌作用，在同一暴露时间与药物的浓度呈正相关，暴露浓度相同时，与暴露时间呈正相关；依据CA模型，三元混合体系整体表现为典型的加和作用。Abstract: In addition to concentration, time is also an important factor affecting toxicity. In order to investigate the combined antibacterial effects of penicillin antibiotics on Escherichia coli (E. coli) under continuous exposure time, piperacillin sodium (PIP), penicillin G potassium (PEN) and penicillin V potassium salt (PHE) were used as the research objects, and 96 well microplates were used as the experimental carriers. By adjusting the proportion of medium components, culture conditions, test time, toxicity index calculation and microplate design, a time-dependent microplate toxicity analysis method (t-MTA) based on E. coli was established. The antibacterial effects of three antibiotics and their ternary mixture system (PIP-PEN-PHE) designed by the uniform design ray (UD-Ray) method on E. coli at different exposure times of 0.25, 2, 4, 8 and 12 h were systematically investigated. The types of combined antibacterial action of antibiotics were analyzed by concentration addition model (CA). The results showed that the established method t-MTA could determine the bacteriostatic effects of three penicillin antibiotics and their mixtures on E. coli at different exposure times and different exposure concentrations, and the bacteriostatic effects of three antibiotics and their ternary mixtures on E. coli were obviously time-dependent, and the concentration-effect curves were sigmoid. The bacteriostatic activity order of three antibiotics to E. coli was PIP (pEC50=4.46~5.21)>PEN (pEC50=4.16~4.39)>PHE (pEC50=4.01~4.39) within 8~12 h by using the negative logarithm of the half effective concentration (pEC50) as toxicity index. The bacteriostatic effects of the five rays in the ternary mixture system on E. coli was R4>R5>R3>R2>R1, and positively correlated with the concentration ratios of PIP in the mixture (R=0.9189, RMSE=0.0703), i.e. the toxicity of the mixture was time- and component-concentration dependent. The bacteriostatic effects of three antibiotics and their ternary mixtures on E. coli were positively correlated with the medicines’ concentration at the same exposure time, and positively correlated with the exposure time at the same exposure concentration. According to CA model, the ternary mixture system as a whole exhibited a typical addition.
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