2026-04-29T02:37:42Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/233902024-11-29T06:01:18Zcom_20.500.12105_15322com_20.500.12105_2051col_20.500.12105_16967col_20.500.12105_16983

00925njm 22002777a 4500 dc Puig-Collderram, Berta author Domene-Ochoa, Sandra author Salvà-Comas, Maria author Montero, Maria Milagro author Duran, Xavier author González, Juan R author Grau, Santiago author Oliver, Antonio author Horcajada, Juan P author Padilla, Eduardo author Segura, Concepción author Prim, Núria author 2022-08-31 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. IMPORTANCE: 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. Puig-Collderram B, Domene-Ochoa S, Salvà-Comas M, Montero MM, Duran X, González JR, et al. ATP Bioluminescence Assay To Evaluate Antibiotic Combinations against Extensively Drug-Resistant (XDR) Pseudomonas aeruginosa. Papp-Wallace KM, editor. Microbiol Spectr. 2022 Jul 25 http://hdl.handle.net/20.500.13003/18598 https://hdl.handle.net/20.500.12105/23390 35876574 10.1128/spectrum.00651-22 2165-0497 Microbiology spectrum 2-s2.0-85137137928 830550700002 L2020047207 ATP Bioluminescence Assay To Evaluate Antibiotic Combinations against Extensively Drug-Resistant (XDR) Pseudomonas aeruginosa