Laboratori de Referència de Catalunya, Barcelona, Spain.
Infectious Diseases Service, Hospital del Mar, Infectious Pathology and Antimicrobials Research Group (IPAR), Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain.
Microbiol Spectr. 2022 Aug 31;10(4):e0065122. doi: 10.1128/spectrum.00651-22. Epub 2022 Jul 25.
Time-kill curves are used to study antibiotic combinations, but the colony count method to obtain the results is time-consuming. The aim of the study was to validate an ATP assay as an alternative to the conventional colony count method in studies of antibiotic combinations. The cutoff point for synergy and bactericidal effect to categorize the results using this alternative method were determined in Pseudomonas aeruginosa. The ATP assay was performed using the GloMax 96 microplate luminometer (Promega), which measures bioluminescence in relative light units (RLU). To standardize this assay, background, linearity, and the detection limit were determined with one strain each of multidrug-resistant P. aeruginosa and Klebsiella pneumoniae. Twenty-four-hour time-kill curves were performed in parallel by both methods with 12 strains of P. aeruginosa. The conventional method was used as a "gold" standard to establish the pharmacodynamic cutoff points in the ATP method. Normal saline solution was established as washing/dilution medium. RLU signal correlated with CFU when the assay was performed within the linear range. The categorization of the pharmacodynamic parameters using the ATP assay was equivalent to that of the colony count method. The bactericidal effect and synergy cutoff points were 1.348 (93% sensitivity, 81% specificity) and 1.065 (95% sensitivity, 89% specificity) log RLU/mL, respectively. The ATP assay was useful to determine the effectiveness of antibiotic combinations in time-kill curves. This method, less laborious and faster than the colony count method, could be implemented in the clinical laboratory workflow. Combining antibiotics is one of the few strategies available to overcome infections caused by multidrug-resistant bacteria. Time-kill curves are usually performed to evaluate antibiotic combinations, but obtaining results is too laborious to be routinely performed in a clinical laboratory. Our results support the utility of an ATP measurement assay using bioluminescence to determine the effectiveness of antibiotic combinations in time-kill curves. This method may be implemented in the clinical laboratory workflow as it is less laborious and faster than the conventional colony count method. Shortening the obtention of results to 24 h would also allow an earlier guided combined antibiotic treatment.
时间杀菌曲线用于研究抗生素组合，但使用菌落计数法获得结果很耗时。本研究旨在验证 ATP 测定法作为替代传统菌落计数法在抗生素组合研究中的可行性。在铜绿假单胞菌中确定了协同作用和杀菌效果的分类结果的截断点，使用这种替代方法。ATP 测定使用 GloMax 96 微孔板发光计（Promega）进行，该测定以相对光单位（RLU）测量生物发光。为了标准化该测定，使用每种多药耐药铜绿假单胞菌和肺炎克雷伯菌各一株确定了背景、线性和检测限。通过两种方法同时对 12 株铜绿假单胞菌进行 24 小时时间杀菌曲线。传统方法用作 ATP 方法中建立药效学截断点的“金标准”。生理盐水溶液被建立为洗涤/稀释介质。当测定在线性范围内进行时，RLU 信号与 CFU 相关。使用 ATP 测定对药效学参数的分类与菌落计数法相当。杀菌效果和协同作用的截断点分别为 1.348（93%敏感性，81%特异性）和 1.065（95%敏感性，89%特异性）log RLU/mL。ATP 测定可用于确定时间杀菌曲线中抗生素组合的有效性。与菌落计数法相比，该方法更省力、更快，可在临床实验室工作流程中实施。 联合使用抗生素是克服多药耐药菌引起的感染的少数策略之一。通常进行时间杀菌曲线以评估抗生素组合，但获得结果过于繁琐，无法在临床实验室中常规进行。我们的结果支持使用生物发光的 ATP 测量测定来确定时间杀菌曲线中抗生素组合的有效性。与传统的菌落计数法相比，该方法在临床实验室工作流程中实施起来更省力、更快。将结果的获得缩短至 24 小时也可以更早地指导联合抗生素治疗。
