title: Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacy, Singapore General Hospital, Singapore, Singapore.
authors.forename: Yiying
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Department of Pharmacy, Singapore General Hospital, Singapore, Singapore.
authors.forename: Hui
authors.initials: H
authors.lastname: Leck
authors.affiliation: Department of Pharmacy, Singapore General Hospital, Singapore, Singapore.
authors.forename: Tze Peng
authors.initials: TP
authors.lastname: Lim
authors.affiliation: Department of Pharmacy, Singapore General Hospital, Singapore, Singapore.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Teo
authors.affiliation: Department of Pharmacy, Singapore General Hospital, Singapore, Singapore.
authors.forename: Winnie
authors.initials: W
authors.lastname: Lee
authors.affiliation: Department of Infectious Diseases, National University Health Systems, Singapore, Singapore.
authors.forename: Li Yang
authors.initials: LY
authors.lastname: Hsu
authors.affiliation: Department of Pathology, Singapore General Hospital, Singapore, Singapore.
authors.forename: Tse Hsien
authors.initials: TH
authors.lastname: Koh
authors.affiliation: Department of Infectious Diseases, Singapore General Hospital, Singapore, Singapore.
authors.forename: Thuan Tong
authors.initials: TT
authors.lastname: Tan
authors.affiliation: Department of Laboratory Medicine, Changi General Hospital, Singapore, Singapore.
authors.forename: Thean-Yen
authors.initials: TY
authors.lastname: Tan
authors.affiliation: Department of Pharmacy, Singapore General Hospital, Singapore, Singapore; Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore, Singapore; Pharmacy, Faculty of Science, National University of Singapore, Singapore, Singapore.
authors.forename: Andrea Lay-Hoon
authors.initials: AL
authors.lastname: Kwa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015780:Carbapenems / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004305:Dose-Response Relationship, Drug; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects*; D006090:Gram-Negative Bacteria / Q000187:drug effects*; D008163:Luminescent Measurements / Q000379:methods*; D008826:Microbial Sensitivity Tests; D012372:ROC Curve; D015203:Reproducibility of Results
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D000255:Adenosine Triphosphate