(p)ppGpp Controls Bacterial Persistence by Stochastic Induction of Toxin-Antitoxin Activity
TL;DR: It is shown that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate.
About: This article is published in Cell.The article was published on 2013-08-29 and is currently open access. It has received 447 citations till now. The article focuses on the topics: Population.
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TL;DR: This Opinion article describes recent studies of tolerance, resistance and persistence, outlining how a clear and distinct definition for each phenotype can be developed from these findings and proposes a framework for classifying the drug response of bacterial strains according to these definitions that is based on the measurement of the minimum inhibitory concentration.
Abstract: Antibiotic tolerance is associated with the failure of antibiotic treatment and the relapse of many bacterial infections. However, unlike resistance, which is commonly measured using the minimum inhibitory concentration (MIC) metric, tolerance is poorly characterized, owing to the lack of a similar quantitative indicator. This may lead to the misclassification of tolerant strains as resistant, or vice versa, and result in ineffective treatments. In this Opinion article, we describe recent studies of tolerance, resistance and persistence, outlining how a clear and distinct definition for each phenotype can be developed from these findings. We propose a framework for classifying the drug response of bacterial strains according to these definitions that is based on the measurement of the MIC together with a recently defined quantitative indicator of tolerance, the minimum duration for killing (MDK). Finally, we discuss genes that are associated with increased tolerance - the 'tolerome' - as targets for treating tolerant bacterial strains.
1,019 citations
TL;DR: This review presents the current understanding of the molecular mechanisms of biofilm recalcitrance toward antibiotics and describes how recent progress has improved the capacity to design original and efficient strategies to prevent or eradicate biofilm-related infections.
Abstract: Surface-associated microbial communities, called biofilms, are present in all environments. Although biofilms play an important positive role in a variety of ecosystems, they also have many negative effects, including biofilm-related infections in medical settings. The ability of pathogenic biofilms to survive in the presence of high concentrations of antibiotics is called "recalcitrance" and is a characteristic property of the biofilm lifestyle, leading to treatment failure and infection recurrence. This review presents our current understanding of the molecular mechanisms of biofilm recalcitrance toward antibiotics and describes how recent progress has improved our capacity to design original and efficient strategies to prevent or eradicate biofilm-related infections.
862 citations
TL;DR: The central regulatory role of quorum sensing and signaling systems by nucleotide-based second messengers resulting in different lifestyles of P. aeruginosa is reviewed and various regulatory proteins will be discussed which form a plethora of controlling systems acting at transcriptional level for timely expression of genes enabling rapid responses to external stimuli and unfavorable conditions.
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen affecting immunocompromised patients. It is known as the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and as one of the leading causes of nosocomial infections. Due to a range of mechanisms for adaptation, survival and resistance to multiple classes of antibiotics, infections by P. aeruginosa strains can be life-threatening and it is emerging worldwide as public health threat. This review highlights the diversity of mechanisms by which P. aeruginosa promotes its survival and persistence in various environments and particularly at different stages of pathogenesis. We will review the importance and complexity of regulatory networks and genotypic-phenotypic variations known as adaptive radiation by which P. aeruginosa adjusts physiological processes for adaptation and survival in response to environmental cues and stresses. Accordingly, we will review the central regulatory role of quorum sensing and signaling systems by nucleotide-based second messengers resulting in different lifestyles of P. aeruginosa. Furthermore, various regulatory proteins will be discussed which form a plethora of controlling systems acting at transcriptional level for timely expression of genes enabling rapid responses to external stimuli and unfavorable conditions. Antibiotic resistance is a natural trait for P. aeruginosa and multiple mechanisms underlying different forms of antibiotic resistance will be discussed here. The importance of each mechanism in conferring resistance to various antipseudomonal antibiotics and their prevalence in clinical strains will be described. The underlying principles for acquiring resistance leading pan-drug resistant strains will be summarized. A future outlook emphasizes the need for collaborative international multidisciplinary efforts to translate current knowledge into strategies to prevent and treat P. aeruginosa infections while reducing the rate of antibiotic resistance and avoiding the spreading of resistant strains.
849 citations
TL;DR: Recent developments in the understanding of bacterial persister cells are discussed and their potential implications for the treatment of persistent infections are discussed.
Abstract: Many bacteria can infect and persist inside their hosts for long periods of time In this Review, Fisher, Gollan and Helaine discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections Many bacteria can infect and persist inside their hosts for long periods of time This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by antibiotics Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections
722 citations
TL;DR: This Review summarizes recent insights on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p)ppGpp.
Abstract: The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p) ppGpp) are involved in regulating growth and several different stress responses in bacteria. In ...
660 citations
References
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TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
14,389 citations
"(p)ppGpp Controls Bacterial Persist..." refers methods in this paper
...The gfp-frtaphA-frt recombinant allele was then transduced by P1into MG1655 and aphA removed (Datsenko and Wanner, 2000)....
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...The gfp-frt-aphA-frt recombinant allele was then transduced by P1 into a MG1655 strain and aphA removed (Datsenko and Wanner, 2000)....
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...The rpoS::mcherry-frt-aphA-frt recombinant allele was then transduced by P1 into MG1655 and aphA removed (Datsenko and Wanner, 2000)....
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...Using P1, the Lon-gfp-frt-aphA-frt allele was then transduced into MG1655 and the aphA resistance cassette removed as described previously (Datsenko and Wanner, 2000)....
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...Construction of Bacterial Strains and Plasmids Construction of MG1655 lon::gfp A strain containing a chromosomal lon::gfp translational fusion was constructed by lRed recombineering (Datsenko and Wanner, 2000)....
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TL;DR: These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome‐wide testing of mutational effects in a common strain background, E. coli K‐12 BW25113.
Abstract: We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).
7,428 citations
TL;DR: Investigating the persistence of single cells of Escherichia coli with the use of microfluidic devices found phenotypic switching occurred between normally growing cells and persister cells having reduced growth rates, leading to a simple mathematical description of the persistence switch.
Abstract: A fraction of a genetically homogeneous microbial population may survive exposure to stress such as antibiotic treatment. Unlike resistant mutants, cells regrown from such persistent bacteria remain sensitive to the antibiotic. We investigated the persistence of single cells of Escherichia coli with the use of microfluidic devices. Persistence was linked to preexisting heterogeneity in bacterial populations because phenotypic switching occurred between normally growing cells and persister cells having reduced growth rates. Quantitative measurements led to a simple mathematical description of the persistence switch. Inherent heterogeneity of bacterial populations may be important in adaptation to fluctuating environments and in the persistence of bacterial infections.
2,599 citations
"(p)ppGpp Controls Bacterial Persist..." refers background in this paper
...The resulting phenotypic variability revealed bimodality of the growth rate where the slow or nongrowing cells became tolerant to the lethal action of the antibiotics (Balaban et al., 2004)....
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...Work in Escherichia coli on high persister mutants (hip) indicated that persisters constitute a pre-existing subpopulation of cells that is formed stochastically (Balaban et al., 2004)....
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...…strain carrying a mutation in the hipA toxin gene that increased persistence 100-fold (Black et al., 1991; Moyed and Bertrand, 1983), thereby facilitating the microscopic analysis of the persistence phenotype at the single-cell level (Balaban et al., 2004; Gefen et al., 2008; Rotem et al., 2010)....
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TL;DR: The molecular mechanisms that underlie the formation of dormant persister cells are now being unravelled and are the focus of this Review.
Abstract: Several well-recognized puzzles in microbiology have remained unsolved for decades. These include latent bacterial infections, unculturable microorganisms, persister cells and biofilm multidrug tolerance. Accumulating evidence suggests that these seemingly disparate phenomena result from the ability of bacteria to enter into a dormant (non-dividing) state. The molecular mechanisms that underlie the formation of dormant persister cells are now being unravelled and are the focus of this Review.
1,823 citations
"(p)ppGpp Controls Bacterial Persist..." refers background in this paper
...Unsurprisingly, bacterial persistence has been implicated in many recurrent and chronic infections (Allison et al., 2011; Lafleur et al., 2010; Lewis, 2007; Mulcahy et al., 2010)....
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TL;DR: Examples and emerging principles that connect noise, the architecture of the gene circuits in which it is present, and the biological functions it enables are reviewed.
Abstract: The genetic circuits that regulate cellular functions are subject to stochastic fluctuations, or ‘noise’, in the levels of their components. Noise, far from just a nuisance, has begun to be appreciated for its essential role in key cellular activities. Noise functions in both microbial and eukaryotic cells, in multicellular development, and in evolution. It enables coordination of gene expression across large regulons, as well as probabilistic differentiation strategies that function across cell populations. At the longest timescales, noise may facilitate evolutionary transitions. Here we review examples and emerging principles that connect noise, the architecture of the gene circuits in which it is present, and the biological functions it enables. We further indicate some of the important challenges and opportunities going forward.
1,464 citations
"(p)ppGpp Controls Bacterial Persist..." refers background in this paper
...Bacteria display remarkably high degrees of individuality or phenotypic heterogeneity in populations of genetically identical cells (Dubnau and Losick, 2006; Eldar and Elowitz, 2010; Lidstrom and Konopka, 2010)....
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