Mechanisms that Trigger Persister Resuscitation

Persister cells were discovered by Gladys Hobby in 1942, but the term “persisters” was coined by Joseph Bigger in 1944, who simply referred to “a small subpopulation in an isogenic bacterial culture that is temporarily tolerant to antibiotics” [1,2]. Despite its definition as a non-growing cell state, persistence is not equivalent to dormancy; persister cells exhibit diverse, but poorly characterized metabolic and gene expression activities. Whereas some persisters can grow in the presence of antibiotics, these cells often survive drug treatments by activating drug efflux systems [3] or bypassing drug-targeted pathways [4]. In contrast, the most abundant persister type does not grow in the presence of antibiotics [5]. These variants are largely formed by passage through the stationary phase, survive lethal concentrations of antibiotics due to their transient non-growing state, and become resuscitated after the conclusion of antibiotic treatment [5-7]. Persisters are an important health problem, because they are thought to underlie the propensity of recurrent infections to relapse and serve as a reservoir from which drug-resistant mutants can emerge [8]. Recurrent and chronic infections are generally associated with biofilms where persister cells are significantly enriched and evade the host immune system [6]. Biofilm infections account for 65% of hospital-treated infections [6], and in the U.S. alone, it is estimated that they cost the healthcare system approximately $94 billion and cause more than half a million deaths per year [9]. Persister eradication holds the potential to cure chronic infections, but effective sterilization methods remain scarce.

Persister cells, which can tolerate lethal concentrations of drugs, are detected in antibiotic-treated microbial cultures by clonogenic survival assays. The presence of persister cells in a population is demonstrated by a bi-phasic kill curve during the antibiotic treatment, in which the rapid-killing regime represents the death of normal cells, and the slow-killing regime indicates the presence of persisters [10]. Once the antibiotic is removed and the persisters are re-cultured, they form cell populations that exhibit antibiotic sensitivity identical to the original culture, which distinguishes them from antibiotic-resistant mutants. Persister cells have been identified in many microorganisms, some of which are highly pathogenic, such as Mycobacterium tuberculosis, Helicobacter pylori, Salmonella enterica and Serovar Typhimurium, as well as the yeast, Candida albicans [7]. Efforts to characterize the molecular mechanisms underlying persistence have identified a number of persister formation pathways; these are generally associated with SOS response, ppGpp-dependent stringent response, toxin/anti-toxin systems, and reactive oxygen species (ROS) and intracellular-degradation [6-8,10,11]. Both stochastic and deterministic factors, pertaining to aforementioned mechanisms, are known to facilitate persister formation [7]. Despite significant advances in understanding the molecular mechanisms underlying persister formation, the phenotypic features that allow persister resuscitation, a hallmark of persistence, remain relatively unexplored [8].

Although intracellular cAMP levels [12], the abundance of ribosomes [13] and membrane repolarization [14] were shown to be critical in persister resuscitation, we still don’t know what mechanisms trigger these phenotypic variants to exit from the persistent state, to repopulate upon cessation of antibiotic treatment, and to regain antimicrobial sensitivity. Due to their non-proliferating state, persisters are largely assumed to be globally dormant cells with depressed physiological activities. In contrast to this common assumption, persisters may have diverse physiological activities that enable them to exit from persistence upon antibiotic removal. On the basis of published studies, two cellular processes seem to play significant roles in persister resuscitation: repair mechanisms and energy metabolism. Two independent groups demonstrated that persister cells experience antibiotic-induced damage indistinguishable from that seen in antibiotic-sensitive subpopulations, with repair mechanisms essential for the recovery of persisters [15,16]. Resuscitation necessitates reactivation of replication together with the production of energy molecules necessary for cellular repair functions. Recent evidence shows that persisters can harbor electron transport chain (ETC) activities [17,18] and consume certain carbon sources to generate proton motive force (PMF) [19,20].

Interestingly, most of the persister-related genes identified thus far are either directly or indirectly involved in cell metabolism [21], indicating that active metabolism is likely essential for the resuscitation of persister cells following antibiotic removal. The physiology of persister phenotypes still remains unclear. The assumption that persisters are globally dormant cells has discouraged many scientists from characterizing their physiology. Moreover, direct assessments of persister physiology using conventional approaches, such as mass spectrometry (LC-MS) or transcriptomic profiling (RNA-seq), are currently hindered by limitations in the isolation procedures. Despite their notable contributions to persister research, existing strategies 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) [19,22]. Although 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 in such media is rarely possible [19,23,24].

It is possible that persistence represents a transitory phase leading to the VBNC state and contributes to accumulation of VBNC cells due to the accumulation of stress-induced intracellular damage. Given their ability to reawaken, persisters likely repair damages and produce precursors (e.g., energy molecules) to support growth upon removal of stress. However, the underlying mechanisms remain to be elucidated. Despite their non-proliferating state, persister cells must maintain their viability by sustaining a minimum adenylate energy charge; synthesizing spontaneously denatured or degraded proteins and RNAs; and repairing antibiotic-induced DNA/protein/lipid damage [17-21]. Given that persistence is a very complex, crucial adaptation strategy evolved in many organisms, elucidating persister resuscitation mechanisms will complement the current molecular-level understanding of persistence and its evolution. This will also enable us to answer the question why only a small fraction of non-growing cells (persisters) resuscitate while the majority of them (VBNCs) do not. Moreover, many of the persister mechanisms identified so far may be directly related to persister resuscitation, which remain to be clarified.

1. Hobby GL, Meyer K, Chaffee E (1942) Observations on the mechanism of action of penicillin. Proceedings of the Society for Experimental Biology and Medicine 50(2): 281-285.
2. Bigger JW (1944) Treatment of staphylococcal infections with penicillin by intermittent sterilization. Lancet Oct 244(6320): 497-500.
3. Pu Y, Zhao Z, Li Y, Zou J, Ma Q, et al. (2016) Enhanced efflux activity facilitates drug tolerance in dormant bacterial cells. Molecular cell 62(2): 284-294.
4. Wakamoto Y, Dhar N, Chait R, Schneider K, et al. (2013) Dynamic persistence of antibiotic-stressed mycobacteria. Science 339(6115): 91-95.
5. Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S (2004) Bacterial persistence as a phenotypic switch. Science 305(5690): 1622-1625.
6. Lewis K (2007) Persister cells, dormancy and infectious disease. Nature Reviews Microbiology 5(1): 48-56.
7. Vanden Bergh B, Fauvart M, Michiels J (2017) Formation, physiology, ecology, evolution and clinical importance of bacterial persisters. FEMS Microbiology Reviews 41(3): 219-251.
8. Wilmaerts D, Windels EM, Verstraeten N, Michiels J (2019) General mechanisms leading to persister formation and awakening. Trends in Genetics 35(6): 401-411.
9. Römling U, Kjelleberg S, Normark S, Nyman L, Uhlin BE, et al. (2014) Microbial biofilm formation: a need to act. Journal of internal medicine 276(2): 98-110.
10. Maisonneuve E, Gerdes K (2014) Molecular mechanisms underlying bacterial persisters. Cell 157(3): 539-548.
11. Fisher RA, Gollan B, Helaine S (2017) Persistent bacterial infections and persister cells. Nature Reviews Microbiology 15(8): 453-464.
12. Yamasaki R, Song S, Benedik M, Wood T (2019) Persister cells resuscitate using membrane sensors that activate chemotaxis, lower camp levels, and revive ribosomes. Cell Reports, pp. 1-72.
13. Kim JS, Yamasaki R, Song S, Zhang W, Wood TK (2018) Single cell observations show persister cells wake based on ribosome content. Environmental Microbiology 20(6): 2085-2098.
14. Wilmaerts D, Dewachter L, Loose PJ, Bollen C, Verstraeten N, et al. (2019) HokB monomerization and membrane repolarization control persister awakening. Molecular Cell 1097-2765(19): 30471.
15. Völzing KG, Brynildsen MP (2015) Stationary-phase persisters to ofloxacin sustain DNA damage and require repair systems only during recovery. MBio 6(5): e00731-15.
16. Goormaghtigh F, Van Melderen L (2019) Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in exponential cultures. Science advances 5(6): 9462.
17. Orman MA, Brynildsen MP (2015) Inhibition of stationary phase respiration impairs persister formation in Escherichia coli Nature communications 6: 7983.
18. Mohiuddin SG, Hoang T, Saba A, Karki P, Orman MA (2019) Therapeutically exploring persister metabolism in bacteria. Cold Spring Harbor Laboratory, USA.
19. Allison KR, Brynildsen MP, Collins JJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473(7346): 216.
20. Orman MA, Brynildsen MP (2013) Establishment of a method to rapidly assay bacterial persister metabolism. Antimicrobial agents and Chemotherapy 57(9): 4398-4409.
21. Amato SM, Fazen CH, Henry TC, Mok WW, Orman MA, et al. (2014) The role of metabolism in bacterial persistence. Frontiers in microbiology 5: 70.
22. Roostalu J, Jõers A, Luidalepp H, Kaldalu N, Tenson T (2008) Cell division in Escherichia coli cultures monitored at single cell resolution. BMC microbiology 8(1): 68.
23. Oliver JD (2010) Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS microbiology reviews 34(4): 415-425.
24. Ayrapetyan M, Williams TC, Oliver JD (2015) Bridging the gap between viable but non-culturable and antibiotic persistent bacteria. Trends in microbiology 23(1): 7-13.