Abstract
BackgroundPersisters 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Persisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics
Persister and VBNC cells are concurrently present in cell cultures, appear as non-growing and stay alive during antibiotic treatments, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics whereas the resuscitation of VBNCs on such media is rarely possible [12, 13]
All cells exhibited a high level of mCherry fluorescence, which declined as the cells divided, except in a small subpopulation (~ 4% of the entire population at t = 150 min, Optical density at 600 nm wavelength (OD600) = 0.25) whose fluorescence remained constant due to the lack of division (Fig. 1, subpopulations highlighted with red circles)
Summary
Persisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics 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. Direct assessments of persister physiology using conventional approaches, such as mass spectrometry or transcriptomic profiling, are currently hindered by limitations in the isolation procedures Despite their notable contributions to persister research, existing strategies [7,8,9,10,11] often yield samples that are enriched in persisters but are highly contaminated with other cell types, such as viable but non-culturable cells (VBNCs), which are generally more abundant than persisters (~ 2-log-fold more) [8, 9]. It is possible that persistence represents a transitory phase leading to the VBNC state and contributes to the accumulation of VBNC cells due to the accumulation of stress-induced intracellular damage
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