New-found fundamentals of bacterial persistence (original) (raw)
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Persister cells and tolerance to antimicrobials
FEMS Microbiology Letters, 2000
Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics. Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology. Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters. The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase. Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin). This shows that production of persisters depends on growth stage. Maintaining a culture of E. coli at early exponential phase by reinoculation eliminated persisters. This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics. Our data indicate that persisters are specialized survivor cells. ß
Molecular Mechanisms Underlying Bacterial Persisters
Cell, 2014
All bacteria form persisters, cells that are multidrug tolerant and therefore able to survive antibiotic treatment. Due to the low frequencies of persisters in growing bacterial cultures and the complex underlying molecular mechanisms, the phenomenon has been challenging to study. However, recent technological advances in microfluidics and reporter genes have improved this scenario. Here, we summarize recent progress in the field, revealing the ubiquitous bacterial stress alarmone ppGpp as an emerging central regulator of multidrug tolerance and persistence, both in stochastically and environmentally induced persistence. In several different organisms, toxin-antitoxin modules function as effectors of ppGpp-induced persistence.
Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells
bioRxiv (Cold Spring Harbor Laboratory), 2017
Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules in E. coli persister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field. controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters. protease Lon (8, 9). Though similar findings have been made, e.g., in Salmonella Typhimurium (17), our model has also been met with skepticism by other researchers in the field (5, 13, 14, 18). In the present study we therefore carefully re-evaluated our previous conclusions as well as the underlying methodology and also probed common experimental procedures for sources of the inconsistencies frequently observed between studies in the field. In short, we discovered that E. coli mutant strains used in our previous work had been inadvertently infected with several different lysogenic bacteriophages and that prophage carriage strongly affected persistence measurements. However, we also show that already the resident cryptic prophages of the E. coli K-12 MG1655 wild type strain distort the results of persister assays under conditions that are commonly used in the field. Importantly, we show that the common practice of determining persister levels at only a single growth time-point is often inappropriate to account for shifted dynamics of growth and persister formation in mutant strains. We finally tested the key components of our previously proposed model of persister formation using new mutant strains and a refined methodology. Crucially, we could confirm a role of (p)ppGpp, polyphosphate, and Lon in bacterial persister formation and / or survival, but did not find strong evidence for the involvement of TA modules or the connection of these components in a single pathway of persister formation as we had proposed earlier. Results Classical persister assays suffer from technical and biological drawbacks The formation of persister cells is typically measured by determining the fraction of antibiotic-tolerant cells in bacterial cultures that are considered to be exponentially growing some hours after inoculation from dense overnight cultures (Figure 1A). A biphasic kinetic of antibiotic killing reveals the presence of persister cells, because these are killed slowly and can be detected after the regular cells have been rapidly eliminated in a first phase of killing (19). Despite the apparent simplicity of this experimental setup, persister assays are
Signaling-mediated bacterial persister formation
Nature Chemical Biology, 2012
Here we show that bacterial communication through indole signaling induces persistence, a phenomenon in which a subset of an isogenic bacterial population tolerates antibiotic treatment. We monitor indole-induced persister formation using microfluidics, and identify the role of oxidative stress and phage-shock pathways in this phenomenon. We propose a model in which indole signaling "inoculates" a bacterial sub-population against antibiotics by activating stress responses, leading to persister formation. Bacterial persisters are dormant cells 1 within an isogenic bacterial population that tolerate antibiotic treatment 2 and have been implicated in chronic and recurrent infections 3-5. Persister formation occurs heterogeneously within an antibiotic-susceptible population, predominantly at the transition to stationary phase 6,7. Though numerous genes have been associated with persistence 8-10 , a complete understanding of persister formation remains elusive.
Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells
mBio
Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection ...
Nature Precedings, 2007
Persisters are a small subpopulation of bacteria that survive a lethal concentration of antibiotic without antibiotic resistance genes. Isolation of persisters from normally dividing population is considered difficult due to their slow growth, low numbers and phenotypic shift i.e. when re-grown in antibiotic free medium, they revert to parent population. Inability to isolate persisters is a major hindrance in this field of research. Here we reject the ‘phenotypic shift’ phenomenon exhibited by persisters. Persisters, on the other hand, exhibit a heritable phenotype and can be easily isolated from a normally dividing population that allows their selective growth. Rather than a single subset, they comprise many distinct subgroups each exhibiting different growth rates, colony sizes, antibiotic tolerance and protein expression levels. Clearly, they are one of the sources of bacterial heterogeneity and noise in protein expression. Existence of persisters in normally dividing population ...
Mechanisms of bacterial persistence during stress and antibiotic exposure
BACKGROUND: The escalating crisis of mul-tidrug resistance is raising fears of untreatable infections caused by bacterial " superbugs. " However , many patients already suffer from infections that are effectively untreatable due to innate bacterial mechanisms for persistence. This phenomenon is caused by the formation of specialized persister cells that evade antibiotic killing and other stresses by entering a physiologically dormant state, irrespective of whether they possess genes enabling antibiotic resistance. The recalcitrance of persister cells is a major cause of prolonged and recurrent courses of infection that can eventually lead to complete antibiotic treatment failure. Regularly growing bacteria differentiate into persister cells stochastically at a basal rate, but this phenotypic conversion can also be induced by environmental cues indicative of imminent threats for the bacteria. Size and composition of the persister subpopulation in bacterial communities are largely controlled by stress signal
In Vitro Studies of Persister Cells
Microbiology and Molecular Biology Reviews, 2020
Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. Research into antibiotic persistence has uncovered large intrapopulation heterogeneity of bacterial growth and regrowth but has not identified essential, dedicated molecular mechanisms of antibiotic persistence. Diverse factors and stresses that inhibit bacterial growth reduce killing of the bulk population and may also increase the persister subpopulation, implying that an array of mechanisms are present. Hopefully, further studies under conditions that simulate the key aspects of persistent infections will lead to identifying target mechanisms for effective therapeutic solutions.
The EMBO journal, 2018
Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing...
2022
Failure of antibiotic therapies causes > 700,000 deaths yearly and involves both bacterial resistance and persistence. Persistence results in the relapse of infections by producing a tiny fraction of pathogen survivors that stay dormant during antibiotic exposure. From an evolutionary perspective, persistence is either a 'bet-hedging strategy' that helps to cope with stochastically changing environments or an unavoidable minimal rate of 'cellular errors' that lock the cells in a low activity state. Here, we analyzed the evolution of persistence over 50,000 bacterial generations in a stable environment by improving a published method that estimates the number of persister cells based on the growth of the reviving population. Our results challenged our understanding of the factors underlying persistence evolution. In one case, we observed a substantial decrease in persistence proportion, suggesting that the naturally observed persistence level is not an unavoidable ...