Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase.

Julian Ihssen,Teja Sirec,Urs Spitz,Nina Jovanovic,Yung-Fu Chang

doi:10.1371/journal.pone.0244200

Abstract

Adenosine triphosphate (ATP) is one of the most important indicators of cell viability. Extracellular ATP (eATP) is commonly detected in cultures of both eukaryotic and prokaryotic cells but is not the focus of current scientific research. Although ATP release has traditionally been considered to mainly occur as a consequence of cell destruction, current evidence indicates that ATP leakage also occurs during the growth phase of diverse bacterial species and may play an important role in bacterial physiology. ATP can be conveniently measured with high sensitivity in luciferase-based bioluminescence assays. However, wild-type luciferases suffer from low stability, which limit their use. Here we demonstrate that an engineered, thermostable luciferase is suitable for real-time monitoring of ATP release by bacteria, both in broth culture and on agar surfaces. Different bacterial species show distinct patterns of eATP accumulation and decline. Real-time monitoring of eATP allows for the estimation of viable cell number by relating luminescence onset time to initial cell concentration. Furthermore, the method is able to rapidly detect the effect of antibiotics on bacterial cultures as Ampicillin sensitive strains challenged with beta lactam antibiotics showed strongly increased accumulation of eATP even in the absence of growth, as determined by optical density. Patterns of eATP determined by real-time luminescence measurement could be used to infer the minimal inhibitory concentration of Ampicillin. Compared to conventional antibiotic susceptibility testing, the method presented here is faster and more sensitive, which is essential for better treatment outcomes and reducing the risk of inducing antibiotic resistance. Real-time eATP bioluminescence assays are suitable for different cell types, either prokaryotic or eukaryotic, thus, permitting their application in diverse fields of research. It can be used for example in the study of the role of eATP in physiology and pathophysiology, for monitoring microbial contamination or for antimicrobial susceptibility testing in clinical diagnostics.

Highlights

Role of extracellular ATP in prokaryotic and eukaryotic cellsAdenosine triphosphate (ATP) is the universal energy carrier in all living systems mediating intracellular energy transfer, and enabling cells to store and transport chemical energy through its high-energy phosphate bonds [1]
We tested whether the stability of a commercially available thermostable luciferase (X-ShiningTM luciferase, product code BX174908, Biosynth Carbosynth) in standard growth medium for bacteria is sufficiently high in order to allow real-time monitoring of extracellular ATP in bacterial cultures for up to 24 h at 37 ̊C
The thermostable luciferase was shown to be suitable for assays in liquid and on solid media, giving opportunity to study extracellular ATP (eATP) dynamics with a luminometer, and using different techniques, such as time-lapse microscopy experiments, where bacteria are grown on small agar pads [33]

Summary

Introduction

Role of extracellular ATP (eATP) in prokaryotic and eukaryotic cellsAdenosine triphosphate (ATP) is the universal energy carrier in all living systems mediating intracellular energy transfer, and enabling cells to store and transport chemical energy through its high-energy phosphate bonds [1]. Several studies have reported that eukaryotic cells release ATP via exocytosis, ATP-containing granules, plasma membrane carriers or large conductance channels [8,9,10,11]. These findings suggest that eATP plays important roles in both bacterial and human physiology, and operates as a regulatory signal in cross-talk between bacteria and host, which in turn regulates the bacterial community and stabilizes the gut ecosystem [6]. Addition of eATP to cultures was found to improve survival of Escherichia coli and Salmonella spp. in the stationary phase [2] supporting the claim for such altruistic actions

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Conclusion