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Published on: August 29, 2025
Flow-cytometry analysis reveals persister resuscitation characteristics
Sayed Golam Mohiuddin1, Pouria Kavousi1, Mehmet A Orman2
1Department of Chemical and Biomolecular Engineering, University of Houston, S222 Engineering Bldg 1, 4726 Calhoun Rd, Houston, TX, 77204, USA.
Background:
Persisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during antibiotic treatment, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics. Despite considerable progress in the characterization of persister formation mechanisms, their resuscitation mechanisms remain unclear due to technical limitations in detecting and isolating these cell types in culture environments that are highly heterogeneous.
Results:
In this study, we used a methodology integrating flow cytometry, fluorescent protein expression systems and ampicillin-mediated cell lysing technique to monitor persister resuscitation at the single-cell level. With this method, we were able to investigate the effects of various culture conditions (e.g., antibiotic treatment time, the length of the stationary phase in overnight pre-cultures, or pretreatment of cells with a metabolic inhibitor) on persister resuscitation. Although we observed long-term pre-cultures have many more VBNC cells compared to short-term pre-cultures, only a small fraction of non-lysed cells was able to resuscitate in all conditions tested. Regardless of pre-culturing and ampicillin treatment times, these persister cells started to resuscitate within 1 hour, after they were transferred to fresh liquid media, with the same doubling time that normal cells have. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, a metabolic inhibitor commonly used to increase bacterial persistence. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and a reduction in persister levels despite the significant decrease in ATP levels in the cells.
Conclusions:
The strategy presented in this study helps us monitor persister resuscitation at the single-cell level, and simultaneously quantify persister, VBNC and dead cell subpopulations in ampicillin-treated cultures. Our results indicate that the characterization of persister resuscitation with flow cytometry will enhance the current molecular-level understanding of persistence and its evolution.
Insights
This study reveals that persister cells, a type of antibiotic-tolerant bacteria, can rapidly resuscitate within an hour in fresh media. This finding advances our understanding of bacterial persistence and antibiotic tolerance mechanisms.
Area of Science:
- Microbiology
- Cell Biology
- Antibiotic Resistance
Background:
- Persister cells and viable but non-culturable (VBNC) cells exhibit high antibiotic tolerance.
- Distinguishing persisters from VBNC cells relies on their ability to recolonize after antibiotic treatment.
- Resuscitation mechanisms of persisters are poorly understood due to detection challenges.
Purpose of the Study:
- To develop and apply a single-cell methodology for monitoring persister cell resuscitation.
- To investigate factors influencing persister resuscitation under various culture conditions.
- To quantify persister, VBNC, and dead cell subpopulations.
Main Methods:
- Integration of flow cytometry, fluorescent protein systems, and ampicillin-mediated cell lysis.
- Monitoring persister resuscitation at the single-cell level.
- Analysis of culture conditions including antibiotic exposure time and metabolic inhibitors.
Main Results:
- Persister cells initiated resuscitation within 1 hour of transfer to fresh media, exhibiting normal cell doubling times.
- Arsenate, a metabolic inhibitor, prevented ampicillin-mediated cell lysis, increasing persister levels.
- Removal of arsenate led to cell lysis and reduced persister populations despite decreased ATP levels.
Conclusions:
- The developed strategy enables precise monitoring and quantification of bacterial subpopulations (persisters, VBNC, dead cells).
- Flow cytometry characterization of persister resuscitation enhances understanding of bacterial persistence.
- This research contributes to the molecular-level comprehension of persistence and its evolutionary implications.
