Flow-cytometry analysis reveals persister resuscitation characteristics.

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.