Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells ...represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.
The current antibiotic resistance crisis has led to increased pressure to prioritize strategies to tackle the issue, with a strong focus being placed on the development of novel antimicrobials. ...However, one major obstacle that is often overlooked is persister cells, which are refractory to antibiotic treatment. Tackling persistence is a challenge because these cell types are extremely difficult to study and, consequently, little is known about their physiology and the factors that lead to their emergence. Here, four experts contemplate the main physiological features that define persistence and the implications of persistence for antibiotic treatment regimens, and consider what the study of bacterial persistence has taught us about the heterogeneity of bacterial populations.
The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens
. These microorganisms have a highly restrictive permeability barrier, which limits the ...penetration of most compounds
. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s
. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.
The gut microbiota affects many important host functions, including the immune response and the nervous system
. However, while substantial progress has been made in growing diverse microorganisms of ...the microbiota
, 23-65% of species residing in the human gut remain uncultured
, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ
. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression.
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.
We present a scalable and facile technique for noncovalent functionalization of graphene with 1-pyrenecarboxylic acid that exfoliates single-, few-, and multilayered graphene flakes into stable ...aqueous dispersions. The exfoliation mechanism is established using stringent control experiments and detailed characterization steps. Using the exfoliated graphene, we demonstrate highly sensitive and selective conductometric sensors (whose resistance rapidly changes >10,000% in saturated ethanol vapor), and ultracapacitors with extremely high specific capacitance (∼ 120 F/g), power density (∼ 105 kW/kg), and energy density (∼ 9.2 Wh/kg).
Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil ...microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.
Persisters represent a small subpopulation of non- or slow-growing bacterial cells that are tolerant to killing by antibiotics. Despite their prominent role in the recalcitrance of chronic infections ...to antibiotic therapy, the mechanism of their formation has remained elusive. We show that sorted cells of Escherichia coli with low levels of energy-generating enzymes are better able to survive antibiotic killing. Using microfluidics time-lapse microscopy and a fluorescent reporter for in vivo ATP measurements, we find that a subpopulation of cells with a low level of ATP survives killing by ampicillin. We propose that these low ATP cells are formed stochastically as a result of fluctuations in the abundance of energy-generating components. These findings point to a general "low energy" mechanism of persister formation.
Tuberculosis is difficult to treat due to dormant cells formed in response to immune stress and stochastically formed persisters, both of which are tolerant of antibiotics. Bactericidal antibiotics ...kill by corrupting their energy-dependent targets. We reasoned that stochastic variation, or noise, in the expression of an energy-generating component will produce rare persister cells. In sorted M. tuberculosis cells grown on acetate, there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing
sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters. Entrance into a low-energy state driven by noise in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters.
M. tuberculosis infection requires the administration of multiple antibiotics for a prolonged period of time. Treatment difficulty is generally attributed to M. tuberculosis entrance into a nonreplicative, antibiotic-tolerant state. M. tuberculosis enters this nonreplicative state in response to immune stress. However, a small population of cells enter a nonreplicative, multidrug-tolerant state under normal growth conditions, absent any stress. These cells are termed persisters. The mechanisms by which persisters enter a nonreplicative state are largely unknown. Here, we show that, as with other bacteria, M. tuberculosis persisters are low-energy cells formed stochastically during normal growth. Additionally, we identify the natural variation in the expression of energy producing genes as a source of the stochastic entrance of M. tuberculosis into the low-energy persister state. These findings have important implications for understanding the heterogeneous nature of M. tuberculosis infection and will aid in designing better treatment regimens against this important human pathogen.
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