scholarly journals A robust method for generating, quantifying and testing large amounts of Escherichia coli persisters

2020 ◽  
Author(s):  
Silke R. Vedelaar ◽  
Jakub L. Radzikowski ◽  
Matthias Heinemann
Keyword(s):  
Bacterial Population ◽  
Large Fraction ◽  
Experimental Methods ◽  
Bacterial Cells ◽  
Robust Method ◽  
Persister Cells ◽  
E Coli ◽  
Persister Cell ◽  
Detailed Protocol ◽  
Tolerant State

AbstractBacteria can exhibit phenotypes, which makes them tolerant against antibiotics. However, often only a few cells of a bacterial population show such so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient-shifts generate E. coli populations that consist of almost only antibiotic tolerant persister cells. Such nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this paper, we provide a detailed protocol of how to generate such large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and describe a new way to assess antibiotic tolerance with flow cytometry. We envision that these methods facilitate research into the important and exciting phenotype of bacterial cells.

scholarly journals Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

2019 ◽  
Author(s):  
Sooyeon Song ◽  
Thomas K. Wood
Keyword(s):  
Escherichia Coli ◽  
Small Rna ◽  
23S Rrna ◽  
Bacterial Cells ◽  
Persister Cells ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Dormant Cells ◽  
Wide Range ◽  
Persister Cell

ABSTRACTUpon a wide range of stress conditions (e.g., nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survive by halting metabolism. These cells resuscitate rapidly to reconstitute infections once the stress is removed and nutrients are provided. However, how these dormant cells resuscitate is not understood well but involves reactivating ribosomes. By screening 10,000 compounds directly for stimulating Escherichia coli persister cell resuscitation, we identified that 2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET) stimulates resuscitation. Critically, by screening 4,267 E. coli proteins, we determined that BPOET activates hibernating ribosomes via 23S rRNA pseudouridine synthase RluD, which increases ribosome activity. Corroborating the increased waking with RluD, production of RluD increased the number of active ribosomes in persister cells. Also, inactivating the small RNA RybB which represses rluD led to faster persister resuscitation. Hence, persister cells resuscitate via activation of RluD.

scholarly journals Bacterial Persister Cell Formation and Dormancy

2013 ◽  
Vol 79 (23) ◽  
pp. 7116-7121 ◽  
Author(s):  
Thomas K. Wood ◽  
Stephen J. Knabel ◽  
Brian W. Kwan
Keyword(s):  
Stationary Phase ◽  
Cell Formation ◽  
Genetic Change ◽  
Bacterial Cells ◽  
Mechanistic Studies ◽  
Chronic Infections ◽  
Persister Cells ◽  
Metabolic State ◽  
Dormant State ◽  
Persister Cell

ABSTRACTBacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.

scholarly journals The Fatty Acid Signaling Moleculecis-2-Decenoic Acid Increases Metabolic Activity and Reverts Persister Cells to an Antimicrobial-Susceptible State

2014 ◽  
Vol 80 (22) ◽  
pp. 6976-6991 ◽  
Author(s):  
Cláudia N. H. Marques ◽  
Aleksey Morozov ◽  
Penny Planzos ◽  
Hector M. Zelaya
Keyword(s):  
Fatty Acid ◽  
Cell Number ◽  
Antimicrobial Treatment ◽  
Bacterial Cells ◽  
Chronic Infections ◽  
Persister Cells ◽  
Decenoic Acid ◽  
Signaling Molecule ◽  
Content Type ◽  
Persister Cell

ABSTRACTPersister cells, which are tolerant to antimicrobials, contribute to biofilm recalcitrance to therapeutic agents. In turn, the ability to kill persister cells is believed to significantly improve efforts in eradicating biofilm-related, chronic infections. While much research has focused on elucidating the mechanism(s) by which persister cells form, little is known about the mechanism or factors that enable persister cells to revert to an active and susceptible state. Here, we demonstrate thatcis-2-decenoic acid (cis-DA), a fatty acid signaling molecule, is able to change the status ofPseudomonas aeruginosaandEscherichia colipersister cells from a dormant to a metabolically active state without an increase in cell number. This cell awakening is supported by an increase of the persister cells' respiratory activity together with changes in protein abundance and increases of the transcript expression levels of several metabolic markers, includingacpP, 16S rRNA,atpH, andppx. Given that most antimicrobials target actively growing cells, we also explored the effect ofcis-DA on enhancing antibiotic efficacy in killing persister cells due to their inability to keep a persister cell state. Compared to antimicrobial treatment alone, combinational treatments of persister cell subpopulations with antimicrobials andcis-DA resulted in a significantly greater decrease in cell viability. In addition, the presence ofcis-DA led to a decrease in the number of persister cells isolated. We thus demonstrate the ability of a fatty acid signaling molecule to revert bacterial cells from a tolerant phenotype to a metabolically active, antimicrobial-sensitive state.

scholarly journals Persister cells mediate tolerance to metal oxyanions in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3181-3195 ◽  
Author(s):  
Joe J. Harrison ◽  
Howard Ceri ◽  
Nicole J. Roper ◽  
Erin A. Badry ◽  
Kimberley M. Sproule ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Viable Cell ◽  
Small Population ◽  
Water Soluble ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Term Survival ◽  
E Coli ◽  
Cell Counts ◽  
High Concentrations

Bacterial cultures produce subpopulations of cells termed ‘persisters’, reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (), arsenate (), arsenite (), selenite (), tellurate () and tellurite (). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to or , suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.

scholarly journals Spontaneous Escherichia coli persisters with week-long survival dynamics and lasting memory of a short starvation pulse

2020 ◽  
Author(s):  
Mikkel Skjoldan Svenningsen ◽  
Michael Askvad Sørensen ◽  
Sine Lo Svenningsen ◽  
Namiko Mitarai
Keyword(s):  
Long Memory ◽  
Bacterial Population ◽  
Extended Period ◽  
Bacterial Cells ◽  
High Concentration ◽  
Survival Fraction ◽  
E Coli ◽  
Long Term Survivors ◽  
Small Subpopulation

AbstractThe vast majority of a bacterial population is killed within a time scale comparable to their generation time when treated with a lethal concentration of antibiotics. However, a small subpopulation typically survives for an extended period. To investigate the long-term killing dynamics of bacterial cells we constructed a week-long killing assay and followed the survival fraction of an E. coli K12 strain exposed to a high concentration of ciprofloxacin. We found that long-term survivors were formed during exponential growth in both a wildtype and a relA deletion strain, with some cells surviving at least 7 days. The killing dynamics showed at least three time-scales, in contrast to the commonly assumed biphasic killing. Furthermore, we observed a surprisingly long memory effect of a brief starvation pulse, which was dependent on relA. Specifically, one hour of carbon starvation increased the surviving fraction by nearly 100-fold even after 4 days of antibiotics exposure.

scholarly journals Proteome dynamics in persisting and resuscitating bacterial cells

2020 ◽  
Author(s):  
Maja Semanjski ◽  
Fabio Gratani ◽  
Till Englert ◽  
Viktor Beke ◽  
Nicolas Nalpas ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Treatment Time ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Time Resolved ◽  
E Coli ◽  
Associated Proteins ◽  
Label Incorporation ◽  
Insight Into

AbstractBacterial persister cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of bacterial persistence is hindered by low count of persisting cells and the need for their isolation. Here we used sustained addition of stable isotope-labeled lysine to selectively label the proteome of hipA-induced persisting and hipB-induced resuscitating E. coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-hour measurement of label incorporation allowed detection of over 500 newly synthetized proteins in persister cells, demonstrating low but widespread protein synthesis. Many essential proteins were newly synthesized and several ribosome-associated proteins showed unusually high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized ABC transporters, restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. This dataset provides an unprecedented insight into the processes governing persistence and resuscitation of bacterial cells.

scholarly journals Persister Cells Resuscitate Using Membrane Sensors that Activate Chemotaxis, Lower cAMP Levels, and Revive Ribosomes

2018 ◽  
Author(s):  
Ryota Yamasaki ◽  
Sooyeon Song ◽  
Michael J. Benedik ◽  
Thomas K. Wood
Keyword(s):  
Escherichia Coli ◽  
Single Cell ◽  
Nutrient Transport ◽  
Carbon Sources ◽  
Membrane Receptors ◽  
Persister Cells ◽  
Secondary Messenger ◽  
Persister Cell ◽  
Camp Levels ◽  
Tolerant State

ABSTRACTPersistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined thatEscherichia colipersister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors which relay the signal to ribosomes via the secondary messenger cAMP, and persisters wake and utilize chemotaxis to acquire nutrients.

scholarly journals Interkingdom Signal Indole Inhibits Pseudomonas aeruginosa Persister Cell Waking

2019 ◽  
Author(s):  
Weiwei Zhang ◽  
Ryota Yamasaki ◽  
Thomas K. Wood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Large Population ◽  
Opportunistic Pathogen ◽  
Commensal Bacteria ◽  
Persister Cells ◽  
E Coli ◽  
Carbonyl Cyanide ◽  
Persister Cell ◽  
Stressed Cells ◽  
Insight Into

ABSTRACTAimsPersister cells are stressed cells that have transient tolerance to antibiotics; these cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how persisters resuscitate, so we explored the waking of a cells in the presence of the interkingdom signal indole.Methods and ResultsTo generate a large population of persister cells, we induced the persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pre-treating cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single cell observations, that proline is sufficient to wake the persister cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa persister cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa persister cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary phase cultures, the indole produced by E. coli completely inhibits persister cell resuscitation of P. aeruginosa.ConclusionsTherefore, E. col has devised a method to outcompete its competitors by preventing their resuscitation with indole.SignificanceThis work provides insight into why indole is produced by commensal bacteria.

scholarly journals Variations in the Morphology, Mechanics and Adhesion of Persister and Resister E. coli Cells in Response to Ampicillin: AFM Study

Antibiotics ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 235 ◽  
Author(s):  
Samuel C. Uzoechi ◽  
Nehal I. Abu-Lail
Keyword(s):  
Surface Area ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Bacterial Surface ◽  
E Coli ◽  
Surface Areas ◽  
Atomic Force ◽  
Grafting Density ◽  
New Antibiotics ◽  
Surface Biopolymers

Persister bacterial cells are great at surviving antibiotics. The phenotypic means by which they do that are underexplored. As such, atomic force microscope (AFM) was used to quantify the contributions of the surface properties of the outer membrane of multidrug resistance (MDR)-Escherichia coli Strains (A5 and A9) in the presence of ampicillin at minimum inhibitory concentration (MIC) (resistant cells) and at 20× MIC (persistent cells). The properties quantified were morphology, root mean square (RMS) roughness, adhesion, elasticity, and bacterial surface biopolymers’ thickness and grafting density. Compared to untreated cells, persister cells of E. coli A5 increased their RMS, adhesion, apparent grafting density, and elasticity by 1.2, 3.4, 2.0, and 3.3 folds, respectively, and decreased their surface area and brush thickness by 1.3 and 1.2 folds, respectively. Similarly, compared to untreated cells, persister cells of E. coli A9 increased their RMS, adhesion and elasticity by 1.6, 4.4, and 4.5 folds, respectively; decreased their surface area and brush thickness by 1.4 and 1.6 folds, respectively; and did not change their grafting densities. Our results indicate that resistant and persistent E. coli A5 cells battled ampicillin by decreasing their size and going through dormancy. The resistant E. coli A9 cells resisted ampicillin through elongation, increased surface area, and adhesion. In contrast, the persistent E. coli A9 cells resisted ampicillin through increased roughness, increased surface biopolymers’ grafting densities, increased cellular elasticities, and decreased surface areas. Mechanistic insights into how the resistant and persistent E. coli cells respond to ampicillin’s treatment are instrumental to guide design efforts exploring the development of new antibiotics or renovating the existing antibiotics that may kill persistent bacteria by combining more than one mechanism of action.

Viability of Edwardsiella tarda and Esherichia coli preserved with glycerol-tryptone soy broth (TSB) kept at freezing temperature

2013 ◽  
Vol 1 (2) ◽  
pp. 154
Author(s):  
Abdur Rohman ◽  
Frans Ijong ◽  
I K Suwetja
Keyword(s):  
Research And Development ◽  
Germ Plasm ◽  
Freezing Temperature ◽  
Experimental Methods ◽  
Edwardsiella Tarda ◽  
Diagnostic Tools ◽  
Glycerol Concentration ◽  
E Coli ◽  
Esherichia Coli ◽  
Escherchia Coli

Preservation of bacteria carried out in relation to the collection and preservation of germ plasm microbe is useful for research and development or for the establishment of diagnostic tools. Glycerol is a good preservation media but it is not known what doses should be used for effective preservation.  This research used two experimental  methods consisting of 2 factors and 3 treatments. This study aimed to find the best glycerol concentration that can be used to preserve Edwarsiella tarda and Escherchia coli in the -20ºC environment, to understand the viability of bacteria after being preserved and to describe the characteristics of the preserved bacteria. Treatments applied were 10%, 15% and 20%  glycerol in TSB. Viability of the bacteria was analyzed after 7, 14, 28, 35, and 42 days of preservation. Results showed that E.coli bacteria preserved in 15%  glycerol had the highest viability, i.e. 84% and preserved in 10% glycerol had the lowest viability, i.e. 80%. But for E. tarda bacteria preserved in 10% glycerol had the highest viability, i.e. 1.83% and preseved in 15% glycerol had the lowest viability, i.e. 0,55%. Preservasi bakteri dilakukan dalam kaitannya dengan koleksi dan konservasi plasma nutfah mikroba yang berguna untuk penelitian dan pengembangan atau untuk pembentukan alat diagnosa. Gliserol merupakan bahan preservasi yang baik, tetapi belum diketahui dosis yang baik dan efektif untuk preservasi bakteri Edwarsiella tarda dan Escherchia coli pada suhu -20ºC. Penelitian ini dilakukan dengan metode eksperimen yang terdiri dari 2 faktor dan 3 taraf perlakuan, masing-masing perlakuan dengan 3 kali ulangan, media preservasi yang digunakan adalah TSB dan gliserol dengan konsentrasi 10%, 15% dan 20%. Parameter yang diukur adalah viabilitas dan kecocokan/penyimpangan karakteristik biokimia. Penelitian ini dilaksanakan di Laboratorium Balai Karantina Ikan Pengendalian Mutu dan Keamanan Hasil Perikanan Manado, dari bulan September sampai dengan November 2013. Tujuan Penelitian ini adalah untuk menentukan konsentrasi gliserol dalam TSB sebagai media preservasi yang efektif dan efisien pada bakteri  Edwarsiella tarda dan Escherchia coli yang dipreservasi dengan suhu -20ºC dan disimpan selama 42  hari. Hasil penelitian menunjukkan adanya penurunan laju pertumbuhan bakteri selama preservasi. Persentase viabilitas  bakteri E. coli yang tertinggi selama preservasi diperoleh dengan penggunaan gliserol konsentrasi 15% dengan jumlah 84% dan yang terendah adalah dengan penggunaan konsentrasi 10% yakni sebesar 80%, sedangkan untuk E. tarda persentase viabilitas  bakteri yang tertinggi selama preservasi diperoleh dengan penggunaan gliserol konsentrasi 10% dengan jumlah 1,83% dan yang terendah adalah dengan penggunaan konsentrasi 15% yakni sebesar 0,55%. Berdasarkan uji statistik analisis variasi (ANAVA) didapat hasil F hitung E. tarda dan E. coli yang lebih besar  dari FTabel dengan tingkat kepercayaan 95 %.

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