Elimination of Extracellular Adenosine Triphosphate for the Rapid Prediction of Quantitative Plate Counts in 24 h Time-Kill Studies against Carbapenem-Resistant Gram-Negative Bacteria

Yiying Cai,Tse-Hsien Koh,Winnie Lee,Jonathan J Ng,Hui Leck,Jocelyn Q Teo,Thuan-Tong Tan,Tze-Peng Lim,Andrea L Kwa,Jia-Xuan Goh

doi:10.3390/microorganisms8101489

Abstract

Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint (0, 2, 4, 8, 24 h), with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models.

Highlights

Infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) is a major public health problem worldwide [1]
We described the relationship between bioluminescence (RLU) and colony-forming units (CFU) with and without apyrase treatment in the absence of antibiotics using the four American Type Culture Collection (ATCC) strains
No interpretative standards are provided by the Clinical and Laboratory Standards Institute (CLSI) for Enterobacterales or P. aeruginosa against tigecycline; tigecycline Minimum Inhibitory Concentrations (MICs) for the clinical isolates ranged from 0.25–≥ 32 mg/L [17]

Summary

Introduction

Infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) is a major public health problem worldwide [1]. Adenosine triphosphate (ATP) bioluminescence is a rapid bacterial enumeration method which uses firefly luciferase to convert chemical energy in the form of ATP into light energy that can be detected by a luminometer within a rapid turn-around time of 15–30 min [5]. In the field of rapid antimicrobial susceptibility testing, the use of ATP bioluminescence has been limited to semi-quantitative prediction of bacterial counts (i.e., inhibitory versus non-inhibitory) [9,10,11,12]. In previous susceptibility testing studies that attempted to use ATP bioluminescence to quantitatively predict bacterial counts upon exposure to antibiotics, correlation of ATP levels to bacterial counts was poor [13,14]. One postulation for the lack of correlation is that extracellular ATP (EC-ATP) is released during bacteria growth and upon bacterial cell lysis when exposed to antibiotics, leading to an overestimation of viable bacterial counts [15,16]

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