Biosynth AG, Staad, Switzerland.
Faculty of Biology, Department of Biochemistry and Molecular Biology, Institute of Physiology and Biochemistry, University of Belgrade, Belgrade, Serbia.
PLoS One. 2021 Jan 22;16(1):e0244200. doi: 10.1371/journal.pone.0244200. eCollection 2021.
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.
三磷酸腺苷（ATP）是细胞活力的最重要指标之一。真核细胞和原核细胞的培养物中通常都能检测到细胞外三磷酸腺苷（eATP），但它不是当前科学研究的重点。尽管 ATP 的释放传统上被认为主要是由于细胞破坏而发生，但目前的证据表明，在多种细菌物种的生长阶段也会发生 ATP 泄漏，并且可能在细菌生理学中发挥重要作用。可以在基于荧光素酶的生物发光测定中方便地测量具有高灵敏度的 ATP。但是，野生型荧光素酶稳定性差，限制了其使用。在这里，我们证明了一种工程化的、热稳定的荧光素酶适合实时监测细菌中 ATP 的释放，无论是在肉汤培养物中还是在琼脂表面上。不同的细菌物种显示出不同的 eATP 积累和下降模式。通过将发光起始时间与初始细胞浓度相关联，可以实时监测 eATP 来估计活菌数。此外，该方法能够快速检测抗生素对细菌培养物的影响，因为用β内酰胺类抗生素挑战氨苄青霉素敏感株时，即使在没有生长的情况下，也会导致 eATP 的积累明显增加，这可以通过光密度来确定。通过实时发光测量确定的 eATP 模式可用于推断氨苄青霉素的最小抑菌浓度。与传统的抗生素药敏试验相比，本方法更快、更灵敏，这对于更好的治疗效果和降低诱导抗生素耐药性的风险至关重要。实时 eATP 生物发光测定适用于不同的细胞类型，无论是原核细胞还是真核细胞，因此，允许将其应用于研究的多个领域。例如，它可用于研究 eATP 在生理学和病理生理学中的作用，用于监测微生物污染或用于临床诊断中的抗生素药敏试验。
