The Formation of Persister Cells in Stationary-Phase Cultures of Escherichia Coli Is Associated with the Aggregation of Endogenous Proteins

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.     

Persister control by leveraging dormancy associated reduction of antibiotic efflux

Persistent bacterial infections do not respond to current antibiotic treatments and thus present a great medical challenge. These conditions have been linked to the formation of dormant subpopulations of bacteria, known as persister cells, that are growth-arrested and highly tolerant to conventional antibiotics. Here, we report a new strategy of persister control and demonstrate that minocycline, an amphiphilic antibiotic that does not require active transport to penetrate bacterial membranes, is effective in killing Escherichia coli persister cells [by 70.8 ± 5.9% (0.53 log) at 100 μg/mL], while being ineffective in killing normal cells. Further mechanistic studies revealed that persister cells have reduced drug efflux and accumulate more minocycline than normal cells, leading to effective killing of this dormant subpopulation upon wake-up. Consistently, eravacycline, which also targets the ribosome but has a stronger binding affinity than minocycline, kills persister cells by 3 logs when treated at 100 μg/mL. In summary, the findings of this study reveal that while dormancy is a well-known cause of antibiotic tolerance, it also provides an Achilles’ heel for controlling persister cells by leveraging dormancy associated reduction of drug efflux.     

Biofilm induced tolerance towards antimicrobial peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.     

Biofilms and Planktonic Cells of Pseudomonas aeruginosa Have Similar Resistance to Killing by Antimicrobials

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.     

(Z)-4-Bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one sensitizes Escherichia coli persister cells to antibiotics

Persisters are a small subpopulation of bacterial cells that are dormant and extremely tolerant to antibiotics. The intrinsic antibiotic tolerance of persisters also facilitates the development of multidrug resistance through acquired mechanisms based on drug resistance genes. In this study, we demonstrate that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can reduce persistence during Escherichia coli growth and revert the antibiotic tolerance of its persister cells. The effects of BF8 were more profound when the pH was increased from 6 to 8.5. Although BF8 is a quorum sensing (QS) inhibitor, similar effects were observed for the wild-type E. coli RP437 and its ΔluxS mutant, suggesting that these effects did not occur solely through inhibition of AI-2-mediated QS. In addition to its effects on planktonic persisters, BF8 was also found to disperse RP437 biofilms and to render associated cells more sensitive to ofloxacin. At the doses that are effective against E. coli persister cells, BF8 appeared to be safe to the tested normal mammalian cells in vitro and exhibited no long-term cytotoxicity to normal mouse tissues in vivo. These findings broadened the activities of brominated furanones and shed new light on persister control.     

毒素-抗毒素系统介导滞留菌形成机制

滞留菌是一类处于低代谢休眠状态的抗生素耐受细菌亚群，能够在致死性压力应激后存活下来，是抗生素治疗失败和复发性感染的主要原因之一。毒素-抗毒素系统(toxin-antitoxin system, TA)作为压力应激模块普遍存在于各种细菌中，由稳定的毒素和不稳定但可以中和毒素的同源抗毒素组成。压力情况下，第二信使(p)ppGpp激活Lon,随后大多数II型TA系统被激活，诱导滞留菌形成。同样在(p)ppGpp存在的情况下，Obg刺激hokB转录，使毒素积累，抑制细菌DNA复制、转录、翻译等重要的生理过程，驱动细菌形成滞留菌。SOS反应是激活TA系统的另一个主要途径，解除了对tisB转录的抑制，使其在细胞内积累并插入细胞膜，破坏质子动力势，降低胞内ATP水平，诱使休眠和滞留菌形成。讨论TA系统介导滞留菌形成的机制有助于提出新型抗菌策略。     

A Quorum Sensing Small Volatile Molecule Promotes Antibiotic Tolerance in Bacteria

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant “persister” trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2’ Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.     

Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell’s bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.     

Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins

Bacterial populations contain persisters, cells which survive exposure to bactericidal antibiotics and other lethal factors. Persisters do not have a genetic resistance mechanism, and their means to tolerate killing remain unknown. In exponentially growing populations of Escherichia coli the frequency of persister formation usually is 10⁻⁷ to 10⁻⁵. It has been shown that cells overexpressing either of the toxic proteins HipA and RelE, both members of the bacterial toxin-antitoxin (TA) modules, have the ability to form more persisters, suggesting a specific role for these toxins in the mechanism of persistence. However, here we show that cells expressing proteins that are unrelated to TA modules but which become toxic when ectopically expressed, chaperone DnaJ and protein PmrC of Salmonella enterica, also form 100- to 1,000-fold more persisters. Thus, persistence is linked not only to toxicity caused by expression of HipA or dedicated toxins but also to expression of other unrelated proteins.     

Formation and Properties of Persister Cells of Staphylococcus capitis and Staphylococcus epidermidis,Bacteria Inhabiting Human Skin

Microbiology - Persisters (P) are the cells surviving, but not reproducing in the presence of lethal doses of antibiotics (AB). These are the cells of a scarce (0.001–1.0%) subpopulation,...     

Bacterial behavior in human blood reveals complement evaders with some persister-like features

Bacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins (ExoS, ExoU or ExlA) and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named “evaders”. Evaders shared some features with bacterial persisters, which tolerate antibiotic treatment. Notably, in bi-phasic killing curves, the evaders represented 0.1–0.001% of the initial bacterial load and displayed transient tolerance. However, the evaders are not dormant and require active metabolism to persist in blood. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, in particular in the absence of effective antibiotic treatments.     

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.     

Physiologic Stresses Reveal a Salmonella Persister State and TA Family Toxins Modulate Tolerance to These Stresses

Bacterial persister cells are considered a basis for chronic infections and relapse caused by bacterial pathogens. Persisters are phenotypic variants characterized by low metabolic activity and slow or no replication. This low metabolic state increases pathogen tolerance to antibiotics and host immune defenses that target actively growing cells. In this study we demonstrate that within a population of Salmonella enterica serotype Typhimurium, a small percentage of bacteria are reversibly tolerant to specific stressors that mimic the macrophage host environment. Numerous studies show that Toxin-Antitoxin (TA) systems contribute to persister states, based on toxin inhibition of bacterial metabolism or growth. To identify toxins that may promote a persister state in response to host-associated stressors, we analyzed the six TA loci specific to S. enterica serotypes that cause systemic infection in mammals, including five RelBE family members and one VapBC member. Deletion of TA loci increased or decreased tolerance depending on the stress conditions. Similarly, exogenous expression of toxins had mixed effects on bacterial survival in response to stress. In macrophages, S. Typhimurium induced expression of three of the toxins examined. These observations indicate that distinct toxin family members have protective capabilities for specific stressors but also suggest that TA loci have both positive and negative effects on tolerance.     

Signaling-mediated bacterial persister formation

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 subpopulation against antibiotics by activating stress responses, leading to persister formation.     

Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes

Bacteria living as biofilm are frequently reported to exhibit inherent tolerance to antimicrobial compounds, and might therefore contribute to the persistence of infections. Antimicrobial peptides are attracting increasing interest as new potential antimicrobial therapeutics; however, little is known about potential mechanisms, which might contribute to resistance or tolerance development towards these compounds in biofilms. Here we provide evidence that a spatially distinct subpopulation of metabolically active cells in Pseudomonas aeruginosa biofilms is able to develop tolerance to the antimicrobial peptide colistin. On the contrary, biofilm cells exhibiting low metabolic activity were killed by colistin. We demonstrate that the subpopulation of metabolically active cells is able to adapt to colistin by inducing a specific adaptation mechanism mediated by the pmr operon, as well as an unspecific adaptation mechanism mediated by the mexAB-oprM genes. Mutants defective in either pmr -mediated lipopolysaccharide modification or in mexAB-oprM -mediated antimicrobial efflux were not able to develop a tolerant subpopulation in biofilms. In contrast to the observed pattern of colistin-mediated killing in biofilms, conventional antimicrobial compounds such as ciprofloxacin and tetracycline were found to specifically kill the subpopulation of metabolically active biofilm cells, whereas the subpopulation exhibiting low metabolic activity survived the treatment. Consequently, targeting the two physiologically distinct subpopulations by combined antimicrobial treatment with either ciprofloxacin and colistin or tetracycline and colistin almost completely eradicated all biofilm cells.     

Bacterial tolerances to metals and antibiotics in metal-contaminated and reference streams

Anthropogenic-derived sources of selection are typically implicated as mechanisms for maintaining antibiotic resistance in the environment. Here we report an additional mechanism for maintaining antibiotic resistance in the environment through bacterial exposure to metals. Using a culture-independent approach, bacteria sampled along a gradient of metal contamination were more tolerant of antibiotics and metals compared to bacteria from a reference site. This evidence supports the hypothesis that metal contamination directly selects for metal tolerant bacteria while co-selecting for antibiotic tolerant bacteria. Additionally, to assess how antibiotic and metal tolerance may be transported through a stream network, we studied antibiotic and metal tolerance patterns over three months in bacteria collected from multiple stream microhabitats including the water column, biofilm, sediment and Corbicula fluminea (Asiatic clam) digestive tracts. Sediment bacteria were the most tolerant to antibiotics and metals, while bacteria from Corbicula were the least tolerant. Differences between microhabitats may be important for identifying reservoirs of resistance and for predicting how these genes are transferred and transported in metal-contaminated streams. Temporal dynamics were not directly correlated to a suite of physicochemical parameters, suggesting that tolerance patterns within microhabitats are linked to a complex interaction of the physicochemical characteristics of the stream.     

Bacterial persisters tolerate antibiotics by not producing hydroxyl radicals

In a phenomenon called persistence, small numbers of bacterial cells survive even after exposure to antibiotics. Recently, bactericidal antibiotics have been demonstrated to kill bacteria by increasing the levels of hydroxyl radicals inside cells. In the present study, we report a direct correlation between intracellular hydroxyl radical formation and bacterial persistence. By conducting flow cytometric analysis in a three-dimensional space, we resolved distinct bacterial populations in terms of intracellular hydroxyl radical levels, morphology and viability. We determined that, upon antibiotic treatment, a small sub-population of Escherichia coli survivors do not overproduce hydroxyl radicals and maintain normal morphology, whereas most bacterial cells were killed by accumulating hydroxyl radicals and displayed filamentous morphology. Our results suggest that bacterial persisters can be formed once they have transient defects in mediating reactions involved in the hydroxyl radical formation pathway. Thus, it is highly probable that persisters do not share a common mechanism but each persister cell respond to antibiotics in different ways, while they all commonly show lowered hydroxyl radical formation and enhanced tolerance to antibiotics.     

Persister cells and the riddle of biofilm survival

This review addresses a long standing puzzle in the life and death of bacterial populations—the existence of a small fraction of essentially invulnerable cells. Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanism of MDT and the nature of persisters, which were discovered in 1944, have remained elusive. Our research has shown that persisters are largely responsible for the recalcitrance of infections caused by bacterial biofilms. The majority of infections in the developed world are caused by biofilms, which sparked a renewed interest in persisters. We developed a method to isolate persister cells, and obtained a gene expression profile of Escherichia coli persisters. The profile indicated an elevated expression of toxin-antitoxin modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function, such as translation. For example, aminoglycosides interrupt translation, producing toxic peptides. Inhibition of translation leads to a shutdown of other cellular functions as well, preventing antibiotics from corrupting their targets, which will give rise to tolerant persister cells. Overproduction of chromosomally-encoded toxins such as RelE, an inhibitor of translation, or HipA, causes a sharp increase in persisters. Deletion of the hipBA module produces a sharp decrease in persisters in both stationary and biofilm cells. HipA is thus the first validated persister/MDT gene. We conclude that the function of toxins is the exact opposite of the term, namely, to protect the cell from lethal damage. It appears that stochastic fluctuations in the levels of MDT proteins lead to formation of rare persister cells. Persisters are essentially altruistic cells that forfeit propagation in order to ensure survival of kin cells in the presence of lethal factors.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 327–336.Original Russian Text Copyright © 2005 by Lewis.This revised version was published online in April 2005 with corrections to the post codes.