# Single-cell analysis reveals critical toxin/antitoxin ratio triggering persister resuscitation

## TL;DR

Researchers used single-cell analysis to discover how bacteria switch between dormant and active states using toxin and antitoxin levels, revealing a critical ratio for antibiotic resistance.

## Contribution

The study introduces nano-flow cytometry methods to quantify toxin-antitoxin dynamics and identifies a critical T/A ratio threshold for persister resuscitation.

## Key findings

- Bacteria use two TA expression modes to increase T/A ratio and survive antibiotics, with T/A = 1.0 as a critical threshold.
- Subinhibitory antibiotic exposure increases T/A ratio, pushing cells into deeper dormancy and highlighting risks of under-dosing.
- Resuscitation involves a triphasic detoxification process lowering T/A ratio to allow cell proliferation.

## Abstract

Persisters represent a transient, antibiotic-tolerant subpopulation within isogenic bacterial populations, contributing to infection relapses. However, the mechanisms driving persister formation and resuscitation remain elusive. Here, we developed nano-flow cytometry (nFCM)-based methods for single-cell quantification of toxin (T) RelE and antitoxin (A) RelB levels, as well as for monitoring persister states through cell wall growth. We demonstrate that bacteria elevate the T/A ratio through two distinct TA expression modalities to withstand bacteriostatic antibiotic challenge, with T/A = 1.0 as a critical threshold. Intriguingly, single-cell resuscitation dynamics revealed that subinhibitory antibiotic exposure promotes entry into a deeper dormant state characterized by elevated T/A ratios, underscoring the importance of maximizing therapeutic antibiotic concentrations. Crucially, we uncovered a triphasic detoxification process during resuscitation where progressive toxin depletion drives T/A ratio reduction to a critical proliferation-permissive threshold. Proteomic profiling unveiled that persisters with high RelE production have increased transmembrane transporter levels linked to stress response and drug efflux. Our findings offer pivotal molecular insights underlying persister transitions and underscore the need for high-throughput, single-cell analysis of these heterogeneity phenotypes.

Nano-flow cytometry-based methods were developed to quantify toxin-antitoxin dynamics and monitor persister states, revealing the mechanism driving persister transitions and underscoring the critical role of toxin/antitoxin ratio in bacterial dormancy and resuscitation.

Bacteria modulate the toxin/antitoxin (T/A) ratio via two distinct expression modalities to withstand bacteriostatic antibiotics, with T/A = 1.0 as a critical threshold.Subinhibitory antibiotic exposure drives cells into a deeper dormant state by elevating the T/A ratio, highlighting the risk of under-dosing antibiotics.Resuscitation is governed by a triphasic detoxification process where progressive toxin depletion lowers the T/A ratio to a level permissive for proliferation.Persisters with high RelE production upregulate transmembrane transporters, linking toxin-mediated dormancy to stress response and drug efflux mechanisms.

Bacteria modulate the toxin/antitoxin (T/A) ratio via two distinct expression modalities to withstand bacteriostatic antibiotics, with T/A = 1.0 as a critical threshold.

Subinhibitory antibiotic exposure drives cells into a deeper dormant state by elevating the T/A ratio, highlighting the risk of under-dosing antibiotics.

Resuscitation is governed by a triphasic detoxification process where progressive toxin depletion lowers the T/A ratio to a level permissive for proliferation.

Persisters with high RelE production upregulate transmembrane transporters, linking toxin-mediated dormancy to stress response and drug efflux mechanisms.

Nano-flow cytometry-based methods were developed to quantify toxin-antitoxin dynamics and monitor persister states, revealing the mechanism driving persister transitions and underscoring the critical role of toxin/antitoxin ratio in bacterial dormancy and resuscitation.