Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in exponential cultures.
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ABSTRACT:
SUBMITTER: Goormaghtigh F
PROVIDER: S-EPMC6584399 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundA fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate.ResultsWe monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP) upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency.ConclusionIn principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular organisms other than E. coli.
| S-EPMC2377270 | biostudies-literature
Project description:To cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such as Escherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of the relA gene, the major (p)ppGpp synthetase in E. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starved E. coli synthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCE Bacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.
| S-EPMC6349093 | biostudies-literature
Project description:Persisters comprise a group of phenotypically heterogeneous metabolically quiescent bacteria with multidrug tolerance and contribute to the recalcitrance of chronic infections. Although recent work has shown that toxin-antitoxin (TA) system HipAB depends on stringent response effector (p)ppGppin persister formation, whether other persister pathways are also dependent on stringent response has not been explored. Here we examined the relationship of (p)ppGpp with 15 common persister genes (dnaK, clpB, rpoS, pspF, tnaA, sucB, ssrA, smpB, recA, umuD, uvrA, hipA, mqsR, relE, dinJ) using Escherichia coli as a model. By comparing the persister levels of wild type with their single gene knockout and double knockout mutants with relA, we divided their interactions into five types, namely A "dependent" (dnaK, recA), B "positive reinforcement" (rpoS, pspF, ssrA, recA), C "antagonistic" (clpB, sucB, umuD, uvrA, hipA, mqsR, relE, dinJ), D "epistasis" (clpB, rpoS, tnaA, ssrA, smpB, hipA), and E "irrelevant" (dnaK, clpB, rpoS, tnaA, sucB, smpB, umuD, uvrA, hipA, mqsR, relE, dinJ). We found that the persister gene interactions are intimately dependent on bacterial culture age, cell concentrations (diluted versus undiluted culture), and drug classifications, where the same gene may belong to different groups with varying antibiotics, culture age or cell concentrations. Together, this study represents the first attempt to systematically characterize the intricate relationships among the different mechanisms of persistence and as such provide new insights into the complexity of the persistence phenomenon at the level of persister gene network interactions.
| S-EPMC5611423 | biostudies-literature
Project description:Persisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance in Escherichia coli. We constructed a highly saturated transposon library that covered the majority of E. coli genes and promoter regions and exposed a stationary-phase culture to a lethal dose of gentamicin. Tn-Seq was performed to evaluate the survival of each mutant to gentamicin exposure. We found that the disruption of several distinct pathways affected gentamicin tolerance. We identified 105 disrupted gene/promoter regions with a more than 5-fold reduction in gentamicin tolerance and 37 genes with a more than 5-fold increased tolerance. Functional cluster analysis suggests that deficiency in motility and amino acid synthesis significantly diminished persisters tolerant to gentamicin, without changing the MIC. Amino acid auxotrophs, including serine, threonine, glutamine, and tryptophan auxotrophs, exhibit strongly decreased tolerance to gentamicin, which cannot be restored by supplying the corresponding amino acids to the culture. Interestingly, supplying these amino acids to wild-type E. coli sensitizes stationary-phase cells to gentamicin, possibly through the inhibition of amino acid synthesis. In addition, we found that the deletion of amino acid synthesis genes significantly increases gentamicin uptake in stationary phase, while the deletion of flagellar genes does not affect gentamicin uptake. We conclude that activation of motility and amino acid biosynthesis contributes to the formation of persisters tolerant to gentamicin. Persisters are responsible for the recalcitrance of chronic infections to antibiotics. The pathways of persister formation in E. coli are redundant, and our understanding of the mechanism of persister formation is incomplete. Using a highly saturated transposon insertion library, we systematically analyzed the contribution of different cellular processes to the formation of persisters tolerant to aminoglycosides. Unexpectedly, we found that activation of amino acid synthesis and motility strongly contributes to persister formation. The approach used in this study leads to an understanding of aminoglycoside tolerance and provides a general method to identify genes affecting persister formation.
| S-EPMC4453570 | biostudies-literature
Project description:Magnesium hydroxide nanoparticles are widely used in medicinal and hygiene products because of their low toxicity, environment-friendliness, and low cost. Here, we studied the effects of three different sizes of magnesium hydroxide nanoparticles on antibacterial activity: NM80, NM300, and NM700. NM80 (D50 = 75.2 nm) showed a higher bactericidal effect against Escherichia coli than larger nanoparticles (D50 = 328 nm (NM300) or 726 nm (NM700)). Moreover, NM80 showed a high bactericidal effect against not only exponential cells but also persister cells, which are difficult to eliminate owing to their high tolerance to antibiotics. NM80 eliminated strains in which magnesium-transport genes were knocked out and exhibited a bactericidal effect similar to that observed in the wild-type strain. The bactericidal action involved physical cell damage, as confirmed using scanning electron microscopy, which showed that E. coli cells treated with NM80 were directly injured.
| S-EPMC8234494 | biostudies-literature
Project description:Bactericidal antibiotics can cause metabolic perturbations that contribute to antibiotic-induced lethality. The molecular mechanism underlying these downstream effects remains unknown. Here, we show that ofloxacin, a fluoroquinolone that poisons DNA gyrase, induces a cascade of metabolic changes that are dependent on an active SOS response. We identified the SOS-regulated TisB protein as the unique molecular determinant responsible for cytoplasmic condensation, proton motive force dissipation, loss of pH homeostasis, and H2O2 accumulation in Escherichia coli cells treated with high doses of ofloxacin. However, TisB is not required for high doses of ofloxacin to interfere with the function of DNA gyrase or the resulting rapid inhibition of DNA replication and lethal DNA damage. Overall, the study sheds light on the molecular mechanisms by which ofloxacin affects bacterial cells and highlights the role of the TisB protein in mediating these effects.
| S-EPMC10971487 | biostudies-literature
Project description:Persister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation.
| S-EPMC5075105 | biostudies-literature
Project description:Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.
| S-EPMC2826370 | biostudies-literature
Project description:Despite the identification of many genes and pathways involved in the persistence phenomenon in bacteria, the mechanisms of persistence are not well understood. Here, using Escherichia coli, we identified polynucleotide phosphorylase (PNPase) as a key regulator of persister formation. We constructed the pnp knockout strain (Δpnp) and its complemented strain and exposed them to antibiotics and stress conditions. The results showed that, compared with the wild-type strain W3110, the Δpnp strain had significant defects in persistence to antibiotics and stresses, and the persistence phenotype was restored upon complementation with the pnp gene. Transcriptome sequencing (RNA-seq) analysis revealed that 242 (166 upregulated and 76 downregulated) genes were differentially expressed in the Δpnp strain compared with the W3110 strain. KEGG analysis of the upregulated genes showed that these genes were mostly mapped to metabolism and virulence pathways, of which most are positively regulated by the global regulator cyclic AMP receptor protein (CRP). Correspondingly, the transcription level of the crp gene in the Δpnp strain increased 3.22-fold in the early stationary phase. We further explored the indicators of cellular metabolism of the Δpnp strain, the phenotype of the pnp and crp double-deletion mutant, and the transcriptional activity of the crp gene. Our results indicate that PNPase controls cellular metabolism by negatively regulating the crp operon via targeting the 5'-untranslated region of the crp transcript. This study reveals a persister mechanism and provides novel targets for the development of drugs against persisters for more effective treatment. IMPORTANCE Persisters pose significant challenges for a more effective treatment of persistent infections. An improved understanding of mechanisms of persistence will provide therapeutic targets important for the development of better treatments. Since recent studies with the key tuberculosis persister drug pyrazinamide have implicated polynucleotide phosphorylase (PNPase) as a drug target, in this study, we addressed the possibility that PNPase might be involved in persistence in Escherichia coli. Our study demonstrates PNPase indeed being involved in persistence, provides a mechanism by which PNPase controls persister formation, and suggests a new therapeutic target for treating persistent bacterial infections.
| S-EPMC9927094 | biostudies-literature
Project description:When bacteria are challenged with antimicrobials, small numbers of cells may survive treatment. These so-called persister cells are not mutants, but are a set of phenotypic variants which, after re-growth exhibit the same susceptibility properties as their progenitor populations. The presence of persisters may determine the effective therapeutic dose of antibiotics and account for the intractability of biofilm-related infections. In the current investigation, Escherichia coli persister cells were isolated without prior exposure to antimicrobials, using fluorescence-assisted cell sorting of a strain, labelled with a chromosomal green fluorescent protein marker. Levels of persistence were inversely proportional to fluorescence intensity in the gfp-tagged E. coli population, and separated persister cells showed enhanced expression of two groups of genes located in the e14 and CP4-6 cryptic prophage regions when analysed by DNA microarray.
2013-10-13 | E-MEXP-3511 | biostudies-arrayexpress