ATP bioluminescence assay to evaluate antibiotic combinations against extensively drug-resistant (XDR) Pseudomonas aeruginosa
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- dc.contributor.author Puig-Collderram, Berta
- dc.contributor.author Domene Ochoa, Sandra
- dc.contributor.author Salvà-Comas, Maria
- dc.contributor.author Milagro Montero, Maria
- dc.contributor.author Duran Jordà, Xavier, 1974-
- dc.contributor.author González, Juan Ramón
- dc.contributor.author Grau Cerrato, Santiago
- dc.contributor.author Oliver, Antonio
- dc.contributor.author Horcajada Gallego, Juan Pablo
- dc.contributor.author Padilla, Eduardo
- dc.contributor.author Segura, Concepción
- dc.contributor.author Prim, Núria
- dc.date.accessioned 2022-12-14T07:41:17Z
- dc.date.available 2022-12-14T07:41:17Z
- dc.date.issued 2022
- dc.description.abstract 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.
- dc.format.mimetype application/pdf
- dc.identifier.citation Puig-Collderram B, Domene-Ochoa S, Salvà-Comas M, Montero MM, Duran X, González JR, Grau S, Oliver A, Horcajada JP, Padilla E, Segura C, Prim N. ATP bioluminescence assay to evaluate antibiotic combinations against extensively drug-resistant (XDR) Pseudomonas aeruginosa. Microbiol Spectr. 2022 Aug 31;10(4):e0065122. DOI: 10.1128/spectrum.00651-22
- dc.identifier.doi http://dx.doi.org/10.1128/spectrum.00651-22
- dc.identifier.issn 2165-0497
- dc.identifier.uri http://hdl.handle.net/10230/55132
- dc.language.iso eng
- dc.publisher American Society for Microbiology
- dc.relation.ispartof Microbiol Spectr. 2022 Aug 31;10(4):e0065122
- dc.rights © 2022 Puig-Collderram et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
- dc.rights.accessRights info:eu-repo/semantics/openAccess
- dc.rights.uri https://creativecommons.org/licenses/by/4.0/
- dc.subject.keyword ATP
- dc.subject.keyword Pseudomonas aeruginosa
- dc.subject.keyword Antibiotic combination
- dc.subject.keyword Bioluminescence
- dc.subject.keyword Luminometer
- dc.subject.keyword Synergy
- dc.title ATP bioluminescence assay to evaluate antibiotic combinations against extensively drug-resistant (XDR) Pseudomonas aeruginosa
- dc.type info:eu-repo/semantics/article
- dc.type.version info:eu-repo/semantics/publishedVersion