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. 2015 Oct 13;10(10):e0140446.
doi: 10.1371/journal.pone.0140446. eCollection 2015.

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Yiying Cai  1 , Hui Leck  1 , Tze Peng Lim  1 , Jocelyn Teo  1 , Winnie Lee  1 , Li Yang Hsu  2 , Tse Hsien Koh  3 , Thuan Tong Tan  4 , Thean-Yen Tan  5 , Andrea Lay-Hoon Kwa  6
Affiliations

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Yiying Cai et al. PLoS One. .

Abstract

Background: Current 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.

Methods: 100 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.

Results: Predictive 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.

Conclusion: We 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.

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Conflict of interest statement

Competing Interests: Dr. Andrea Lay-Hoon Kwa, Ms. Winnie Lee and Mr. Tze Peng Lim have received funding for research from Janssen-Cilag, Pfizer. Inc and Merck Sharp and Dohme (I.A) Corp. Dr. Li Yang Hsu has received funding and speaker’s honoraria from AstraZeneca, Janssen-Cilag, Pfizer Inc and Merck Sharp and Dohme (I.A) Corp. Other authors have declared that no competing interests exist. None of the above companies provided any funding for above study. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Relationship between bioluminescence (in RLU) and number of organisms determined by viable plate count method.
A linear log10 RLU/100μl to log10 CFU/ml relationship was observed for all six strains, including the carbapenem-resistant GNB strains (r2 range: 0.96–0.99). Abbreviations used: AB = A. baumannii, ATCC = American Type Culture Collection, CFU = colony forming units, GNB = Gram negative bacteria, KP = K. pneumoniae, PA = P. aeruginosa, RLU = relative light units.
Fig 2
Fig 2. Receiver operator characteristic (ROC) curve for (A) all GNB organisms and antibiotic combinations, (B) A. baumannii, (C) P. aeruginosa, and (D) K. pneumoniae, for single and 2-drug combinations.
High area under the ROC curves (0.92–0.96) was observed, signifying high predictive accuracy when bioluminescent assay was compared to the conventional viable plate count method. Abbreviations used: AUC = area under curve, CI = confidence interval, GNB = Gram negative bacteria, ROC = Receiver operator characteristic
See this image and copyright information in PMC

References

    1. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis. 2009; 48: 1–12. 10.1086/595011 - DOI - PubMed
    1. Nordmann P, Dortet L, Poirel L Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med. 2012; 18: 263–272. 10.1016/j.molmed.2012.03.003 - DOI - PubMed
    1. Zavascki AP, Bulitta JB, Landersdorfer CB Combination therapy for carbapenem-resistant Gram-negative bacteria. Expert Rev Anti Infect Ther. 2013; 11: 1333–1353. 10.1586/14787210.2013.845523 - DOI - PubMed
    1. van Belkum A, Durand G, Peyret M, Chatellier S, Zambardi G, Schrenzel J, et al. Rapid clinical bacteriology and its future impact. Ann Lab Med. 2013; 33: 14–27. 10.3343/alm.2013.33.1.14 - DOI - PMC - PubMed
    1. Hanberger H, Svensson E, Nilsson M, Nilsson LE, Hornsten EG, Maller R Effects of imipenem on Escherichia coli studied using bioluminescence, viable counting and microscopy. J Antimicrob Chemother. 1993; 31: 245–260. - PubMed

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