Rapid antibiotic combination testing for carbapenem-resistant Gram negative bacteria within 6h using adenosine triphosphate bioluminescence [PublishAheadOfPrint]

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

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