(91d) FLOW-Cytometry Analysis Reveals Persister Resuscitation Characteristics | AIChE

(91d) FLOW-Cytometry Analysis Reveals Persister Resuscitation Characteristics


Mohiuddin, S. G. - Presenter, University of Houston
Kavousi, P., University of Houston
Orman, M., University of Houston

Persisters and viable but non-culturable cells (VBNCs) are two distinct phenotypes that are known to tolerate high levels of antibiotics temporarily (1). These variants are often formed by stochastic and deterministic factors in isogenic bacterial cultures and appear as non-growing during the antibiotic treatment. Since the resuscitation of VBNCs is rarely possible, these phenotypic variants can only be discriminated after persisters exit their persistence state and start to replicate following antibiotic removal (2-3). Despite the fact that persistence is a very complex, crucial adaptation strategy evolved in many bacteria, the current molecular-level understanding of their resuscitation mechanisms remains limited. Unfortunately, detecting and isolating resuscitating persisters in a highly heterogeneous environment is challenging. In this study, we explored ampicillin-mediated cell lysing techniques, fluorescent protein expression systems and flow cytometry analysis to establish an integrated approach to study persister resuscitation at single cell level (Figure 1).


Our approach enabled us to investigate the effects of various environmental conditions (e.g., antibiotic treatment time; pre-culture length; or pretreatment of cells with metabolic inhibitors, such as arsenate) on persister resuscitation in Escherichia coli cultures. Although, long-term (9-day) pre-cultures have many more VBNC cells compared to short-term (1-day) pre-cultures, only a small fraction of non-lysed cells was able to resuscitate after the removal of ampicillin in all conditions tested. Regardless of pre-culturing lengths and antibiotic treatment times, the same amount of persister cells started to resuscitate within one hour after transferred to fresh liquid media, with a doubling time similar to that of normal cells. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, an inhibitor commonly used to study bacterial persistence. This chemical is known to increase the persister levels in bacteria by depleting ATP levels in the cells. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and reduction in persister levels despite the observed ATP depletion, indicating arsenate-induced persistence is potentially due to the bacteriostatic effect of the inhibitor in co-treated cultures.


In summary, we presented here a strategy that enables us to quantify both persister and VBNC cells in antibiotic-treated cultures, and monitor their resuscitation abilities after the removal of antibiotics. Our results demonstrate that monitoring persister resuscitation with a flow cytometer can enhance the current molecular-level understanding of persistence and its evolution.


  1. Van den Bergh, B., Fauvart, M. and Michiels, J., 2017. Formation, physiology, ecology, evolution and clinical importance of bacterial persisters. FEMS microbiology reviews, 41(3), pp.219-251.
  2. Orman, M.A. and Brynildsen, M.P., 2013. Establishment of a method to rapidly assay bacterial persister metabolism. Antimicrobial agents and chemotherapy, 57(9), pp.4398-4409.
  3. Ayrapetyan, M., Williams, T.C. and Oliver, J.D., 2015. Bridging the gap between viable but non-culturable and antibiotic persistent bacteria. Trends in microbiology, 23(1), pp.7-13.