Abstract
BackgroundCurrent in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.Methods100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (105 CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (TRLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of TRLU values was determined. External validation was further done using 50 additional CR-GNB.ResultsPredictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (TRLU = 0.81, UA = 91%), and lowest for AB (TRLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.ConclusionWe developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.
Highlights
In the past decade, the prescription of effective antimicrobial therapy has been challenged by the rising prevalence of extensively-drug resistant (XDR) and pan-drug resistant (PDR) Gram negative bacteria (GNB) [1]
Predictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, unweighted accuracy (UA) = 89%)
Predictive accuracy was highest for Klebsiella pneumoniae (KP) (TRLU = 0.81, UA = 91%), and lowest for Acinetobacter baumannii (AB) (TRLU = 0.83, UA = 87%)
Summary
The prescription of effective antimicrobial therapy has been challenged by the rising prevalence of extensively-drug resistant (XDR) and pan-drug resistant (PDR) Gram negative bacteria (GNB) [1]. Traditional single-antibiotic susceptibility testing methods have limited utility when predicting the efficacy of antibiotic combinations against XDR- or PDR-GNB[4]. While other in vitro combination testing methods such as the time-kill studies have been employed to predict effective combinations, these methods require enumeration using viable plate count and are cumbersome, time-consuming and labor-intensive, and are unlikely to provide results in a timely manner for routine clinical use. A rapid susceptibility testing method that can identify effective antibiotic combinations with a sufficiently rapid turnaround time is urgently needed. Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
