Rapid Antibiotic Combination Testing for Carbapenem-Resistant Gram-Negative Bacteria within Six Hours Using ATP Bioluminescence

Antimicrob Agents Chemother. 2018 Aug 27;62(9):e00183-18. doi: 10.1128/AAC.00183-18. Print 2018 Sep.

Abstract

To guide the timely selection of antibiotic combinations against carbapenem-resistant Gram-negative bacteria (CR-GNB), an in vitro test with a short turnaround time is essential. We developed an in vitro ATP bioluminescence assay to determine effective antibiotic combinations against CR-GNB within 6 h. We tested 42 clinical CR-GNB strains (14 Acinetobacter baumannii, 14 Pseudomonas aeruginosa, and 14 Klebsiella pneumoniae strains) against 74 single antibiotics and two-antibiotic combinations. Bacteria (approximately 5 log10 CFU/ml) were incubated with an antibiotic(s) at 35°C; ATP bioluminescence was measured at 6 h and 24 h; and the measurements were compared to viable counts at 24 h. Receiver operating characteristic (ROC) curves were used to determine the optimal luminescence thresholds (TRLU) for distinguishing between inhibitory and noninhibitory combinations. The areas under the 6-h and 24-h ROC curves were compared using the DeLong method. Prospective validation of the established thresholds was conducted using 18 additional CR-GNB. The predictive accuracy of TRLU for the 6-h ATP bioluminescence assay was 77.5% when all species were analyzed collectively. Predictive accuracies ranged from 73.7% to 82.7% when each species was analyzed individually. Upon comparison of the areas under the 6-h and 24-h ROC curves, the 6-h assay performed significantly better than the 24-h assay (P < 0.01). Predictive accuracy remained high upon prospective validation of the 6-h ATP assay (predictive accuracy, 79.8%; 95% confidence interval [CI], 77.6 to 81.9%), confirming the external validity of the assay. Our findings indicate that our 6-h ATP bioluminescence assay can provide guidance for prospective selection of antibiotic combinations against CR-GNB in a timely manner and may be useful in the management of CR-GNB infections.

Keywords: in vitro combination testing; luciferin-luciferase reaction; multidrug resistance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Anti-Bacterial Agents / pharmacology*
  • Carbapenems / pharmacology*
  • Drug Resistance, Multiple, Bacterial / drug effects*
  • Gram-Negative Bacteria / drug effects*
  • Gram-Negative Bacteria / metabolism
  • Gram-Negative Bacterial Infections / drug therapy*
  • Gram-Negative Bacterial Infections / metabolism
  • Humans
  • Luminescent Measurements / methods
  • Prospective Studies

Substances

  • Anti-Bacterial Agents
  • Carbapenems
  • Adenosine Triphosphate