Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD+ synthesis

Dong Yang; Wenxin Wang; Liang Zhao; Lei Rao; Xiaojun Liao

doi:10.1016/j.jare.2023.08.002

Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD⁺ synthesis

J Adv Res. 2023 Aug 3:S2090-1232(23)00206-0. doi: 10.1016/j.jare.2023.08.002. Online ahead of print.

Authors

Dong Yang¹, Wenxin Wang¹, Liang Zhao¹, Lei Rao², Xiaojun Liao³

Affiliations

¹ College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China; National Engineering Research Center for Fruit & Vegetable Processing, Beijing, China; Key Laboratory of Fruit & Vegetable Processing, Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-thermal Processing, Beijing, China.
² College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China; National Engineering Research Center for Fruit & Vegetable Processing, Beijing, China; Key Laboratory of Fruit & Vegetable Processing, Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-thermal Processing, Beijing, China. Electronic address: rao.lei@cau.edu.cn.
³ College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China; National Engineering Research Center for Fruit & Vegetable Processing, Beijing, China; Key Laboratory of Fruit & Vegetable Processing, Ministry of Agriculture and Rural Affairs, Beijing Key Laboratory for Food Non-thermal Processing, Beijing, China. Electronic address: liaoxjun@cau.edu.cn.

PMID: 37541583
DOI: 10.1016/j.jare.2023.08.002

Abstract

Introduction: Entry into the viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria to survive harsh environment. Although VBNC cells still have metabolic activity, they lose the ability to form colonies on nonselective culture media. Thus, conventional bacterial detection methods, such as plate counting, are unable to detect the presence of VBNC cells. When the environmental conditions are appropriate, VBNC cells can initiate resuscitation, posing a great risk to the safety of public health. The study of the VBNC resuscitation mechanism could provide new insights into the prevention and control of VBNC resuscitation.

Objectives: Uncovering the molecular mechanism of VBNC cell resuscitation by investigating the role of O-antigen ligase (RfaL) in inhibiting the resuscitation of Escherichia coli O157:H7 in the VBNC state.

Methods: RfaL was screened and verified as a resuscitation inhibitor of VBNC Escherichia coli O157:H7 by detecting resuscitation curve and time-lapse microscopy. The mechanism of RfaL impacts VBNC E. coli resuscitation was investigated by detecting the single cell ATP content, metabolomic changes, NAD(H) content and new protein biosynthesis of WT and ΔrfaL at different stage of resuscitation.

Results: Mutation of rfaL, which encoded an O-antigen ligase, markedly shortened the resuscitating lag phase. Further studies indicated that ΔrfaL VBNC cells contained higher ATP levels, and ATP consumption during the resuscitating lag phase was highly correlated with resuscitation efficiency. Metabolomic analysis revealed that ATP was utilized to activate the Handler and salvage pathways to synthesize NAD⁺, balancing redox reactions to recover cell activity and promote cell resuscitation.

Conclusion: Our findings revealed a strategy employed by VBNC cells for revival, that is, using residual ATP to primarily recover metabolic activity, driving cells to exit dormancy. The synthesis pathway of lipopolysaccharide (LPS) in rfaL null mutant was inhibited and could supply more ATP to synthesis NAD⁺ and promote resuscitation.

Keywords: ATP; Bacteria; Molecular mechanism; Resuscitation; RfaL; VBNC.