Persisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to antibiotic therapy. One general theme ...has emerged regarding persisters formed by different bacterial species, namely, a state of relative dormancy characterized by diminished activity of antibiotic targets. Within this framework, a number of studies have linked persister formation to stochastic decreases in energy-generating components, leading to low ATP and target activity. In this study, we screen knockouts in the main global regulators of Escherichia coli for their effect on persisters. A knockout in integration host factor (IHF) had elevated ATP and a diminished level of persisters. This was accompanied by an overexpression of isocitrate dehydrogenase (Icd) and a downregulation of isocitrate lyase (AceA), two genes located at the bifurcation between the tricarboxylic acid (TCA) cycle and the glyoxylate bypass. Using a translational
fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of
produces rare cells with low Icd/high AceA, diverting substrates into the glyoxylate bypass, which decreases ATP, leading to antibiotic-tolerant persisters. We further examine noise in a simple model, the
operon, and show that a knockout of the
repressor increases expression of the operon and decreases persister formation. Our results suggest that noise quenching by overexpression serves as a general approach to determine the nature of persister genes in a variety of bacterial species and conditions.
Persisters are phenotypic variants that survive exposure to antibiotics through temporary dormancy. Mutants with increased levels of persisters have been identified in clinical isolates, and evidence suggests these cells contribute to chronic infections and antibiotic treatment failure. Understanding the underlying mechanism of persister formation and tolerance is important for developing therapeutic approaches to treat chronic infections. In this study, we examine a global regulator, IHF, that plays a role in persister formation. We find that noise in expression of IHF contributes to persister formation, likely by regulating the switch between the TCA cycle that efficiently produces energy and the glyoxylate bypass. We extend this study to a simple model
operon and show that when grown on lactose as the sole carbon source, noise in its expression influences ATP levels and determines persister formation. This noise is quenched by overexpression of the
operon, providing a simple approach to test the involvement of a gene in persister formation.
Abstract
Motivation
Widespread interest in the study of the microbiome has resulted in data proliferation and the development of powerful computational tools. However, many scientific researchers ...lack the time, training, or infrastructure to work with large datasets or to install and use command line tools.
Results
The National Institute of Allergy and Infectious Diseases (NIAID) has created Nephele, a cloud-based microbiome data analysis platform with standardized pipelines and a simple web interface for transforming raw data into biological insights. Nephele integrates common microbiome analysis tools as well as valuable reference datasets like the healthy human subjects cohort of the Human Microbiome Project (HMP). Nephele is built on the Amazon Web Services cloud, which provides centralized and automated storage and compute capacity, thereby reducing the burden on researchers and their institutions.
Availability and implementation
https://nephele.niaid.nih.gov and https://github.com/niaid/Nephele
Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. ...Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed 'eradication' period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways.
The vast majority of microbial species are 'uncultured' and do not grow under laboratory conditions. This has led to the development of a number of methods to culture these organisms in a simulated ...natural environment. Approaches include placing cells in chambers that allow diffusion of compounds from the natural environment, traps enclosed with porous membranes that specifically capture organisms forming hyphae--actinobacteria and microfungi, and growth in the presence of cultivable helper species. Repeated cultivation in situ produces domesticated variants that can grow on regular media in vitro, and can be scaled up for secondary metabolite production. The co-culture approach has led to the identification of the first class of growth factors for uncultured bacteria, iron-chelating siderophores. It appears that many uncultured organisms from diverse taxonomical groups have lost the ability to produce siderophores, and depend on neighboring species for growth. The new cultivation approaches allow for the exploitation of the secondary metabolite potential of the previously inaccessible microorganisms.
The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their ...natural environment. An inoculum is sandwiched between semipermeable (0.03-μm-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and BACTEROIDETES: However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and ACIDOBACTERIA: Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.
Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of ...their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in
,
, and
We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels.
knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.
Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.
Bacterial multidrug tolerance is largely responsible for the inability of antibiotics to eradicate infections and is caused by a small population of dormant bacteria called persisters. HipA is a ...critical Escherichia coli persistence factor that is normally neutralized by HipB, a transcription repressor, which also regulates hipBA expression. Here, we report multiple structures of HipA and a HipA-HipB-DNA complex. HipA has a eukaryotic serine/threonine kinase-like fold and can phosphorylate the translation factor EF-Tu, suggesting a persistence mechanism via cell stasis. The HipA-HipB-DNA structure reveals the HipB-operator binding mechanism, ~70° DNA bending, and unexpected HipA-DNA contacts. Dimeric HipB interacts with two HipA molecules to inhibit its kinase activity through sequestration and conformational inactivation. Combined, these studies suggest mechanisms for HipA-mediated persistence and its neutralization by HipB.
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