Loss of Gre factors leads to phenotypic heterogeneity and cheating in populations under nitric oxide stress.

Nitric oxide (·NO) is one of the toxic metabolites that bacteria can be exposed to within phagosomes. Gre factors, which are also known as transcript cleavage factors or transcription elongation factors, relieve back-tracked transcription elongation complexes by cleaving nascent RNAs, which allows transcription to resume after stalling. Here we discovered that loss of both Gre factors in , GreA and GreB, significantly compromised ·NO detoxification due to ·NO-induced phenotypic heterogeneity in ΔΔ populations, which did not occur in wild-type cultures.

Differential impacts of DNA repair machinery on fluoroquinolone persisters with different chromosome abundances.

Unlabelled: DNA repair machinery has been found to be indispensable for fluoroquinolone (FQ) persistence of . Previously, we found that cells harboring two copies of the chromosome (2Chr) in stationary-phase cultures were more likely to yield FQ persisters than those with one copy of the chromosome (1Chr). Furthermore, we found that RecA and RecB were required to observe that difference, and that loss of either more significantly impacted 2Chr persisters than 1Chr persisters.

Cephalosporin resistance, tolerance, and approaches to improve their activities.

Cephalosporins comprise a β-lactam antibiotic class whose first members were discovered in 1945 from the fungus Cephalosporium acremonium. Their clinical use for Gram-negative bacterial infections is widespread due to their ability to traverse outer membranes through porins to gain access to the periplasm and disrupt peptidoglycan synthesis. More recent members of the cephalosporin class are administered as last resort treatments for complicated urinary tract infections, MRSA, and other multi-drug resistant pathogens, such as Neisseria gonorrhoeae.

Amino acids can deplete ATP and impair nitric oxide detoxification by Escherichia coli.

Nitric oxide (·NO) is a prevalent antimicrobial that is known to damage iron-containing enzymes in amino acid (AA) biosynthesis pathways. With Escherichia coli, ·NO is detoxified in aerobic environments by Hmp, which is an enzyme that is synthesized de novo in response to ·NO. With this knowledgebase, it is expected that the availability of AAs in the extracellular environment would enhance ·NO detoxification, because AAs would foster translation of Hmp.

High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin.

The complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria.

Genome-wide mapping of fluoroquinolone-stabilized DNA gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence.

Bacterial persisters are rare phenotypic variants that are suspected to be culprits of recurrent infections. Fluoroquinolones (FQs) are a class of antibiotics that facilitate bacterial killing by stabilizing bacterial type II topoisomerases when they are in a complex with cleaved DNA. In Escherichia coli, DNA gyrase is the primary FQ target, and previous work has demonstrated that persisters are not spared from FQ-induced DNA damage.

Causes of polymyxin treatment failure and new derivatives to fill the gap.

Polymyxins are a class of antibiotics that were discovered in 1947 from programs searching for compounds effective in the treatment of Gram-negative infections. Produced by the Gram-positive bacterium Paenibacillus polymyxa and composed of a cyclic peptide chain with a peptide-fatty acyl tail, polymyxins exert bactericidal effects through membrane disruption. Currently, polymyxin B and colistin (polymyxin E) have been developed for clinical use, where they are reserved as "last-line" therapies for multidrug-resistant (MDR) infections.

Translational Fusion to Hmp Improves Heterologous Protein Expression.

Flavohemoglobins, which are widely distributed in prokaryotes and eukaryotes, play key roles in oxygen (O) transport and nitric oxide (·NO) defense. Hmp is the flavohemoglobin of , and here we report that the translational fusion of Hmp to the N-terminus of heterologous proteins increases their expression in . The effect required the fusion of the proteins, and was independent of both the O-binding and catalytic activity of Hmp.

Fluoroquinolone Persistence in Requires DNA Repair despite Differing between Starving Populations.

When faced with nutritional deprivation, bacteria undergo a range of metabolic, regulatory, and biosynthetic changes. Those adjustments, which can be specific or independent of the missing nutrient, often alter bacterial tolerance to antibiotics. Here, using fluoroquinolones, we quantified persister levels in cultures experiencing starvation from a lack of carbon (C), nitrogen (N), phosphorous (P), or magnesium (Mg).

Robustness of nitric oxide detoxification to nitrogen starvation in Escherichia coli requires RelA.

Reactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes.

Counting Chromosomes in Individual Bacteria to Quantify Their Impacts on Persistence.

Persisters are phenotypic variants within bacterial populations that tolerate antibiotic treatments considerably better than the majority of cells. A phenotypic quality that varies within bacterial populations is the chromosome number of individual cells. One, two, four, or more chromosomes per cell have been observed previously, and the impact of genome copy number can range from gene dosage effects to an inability to perform specific DNA repair functions, such as homologous recombination.

Rifamycin antibiotics and the mechanisms of their failure.

Rifamycins are a class of antibiotics that were first discovered in 1957 and are known for their use in treating tuberculosis (TB). Rifamycins exhibit bactericidal activity against many Gram-positive and Gram-negative bacteria by inhibiting RNA polymerase (RNAP); however, resistance is prevalent and the mechanisms range from primary target modification and antibiotic inactivation to cytoplasmic exclusion. Further, phenotypic resistance, in which only a subpopulation of bacteria grow in concentrations exceeding their minimum inhibitory concentration, and tolerance, which is characterized by reduced rates of bacterial cell death, have been identified as additional causes of rifamycin failure.

Toxin Induction or Inhibition of Transcription or Translation Posttreatment Increases Persistence to Fluoroquinolones.

Toxin-antitoxin modules are widespread in prokaryotes, and the capacity of toxin accumulation to increase the tolerances of bacteria to antibiotics has been well documented. The conventional model for this functionality implies that an overabundance of toxin arrests bacterial growth, which inhibits processes targeted by antibiotics and thereby limits their corruption and the lethal damage that would ensue. Implicit in this model is that toxins exert their influence on antibiotic lethality before and/or during treatment, even though they are also present and functional after treatment concludes.

Phagosome-Bacteria Interactions from the Bottom Up.

When attempting to propagate infections, bacterial pathogens encounter phagocytes that encase them in vacuoles called phagosomes. Within phagosomes, bacteria are bombarded with a plethora of stresses that often lead to their demise. However, pathogens have evolved numerous strategies to counter those host defenses and facilitate survival.

Pseudomonas aeruginosa prioritizes detoxification of hydrogen peroxide over nitric oxide.

Objective: Bacteria are exposed to multiple concurrent antimicrobial stressors within phagosomes. Among the antimicrobials produced, hydrogen peroxide and nitric oxide are two of the most deleterious products. In a previous study, we discovered that when faced with both stressors simultaneously, Escherichia coli prioritized detoxification of hydrogen peroxide over nitric oxide.

Ploidy is an important determinant of fluoroquinolone persister survival.

Genetic mutants have demonstrated the importance of homologous recombination (HR) to fluoroquinolone (FQ) persistence, which suggests that single-cell chromosome (Chr) abundance might be a phenotypic variable of importance to persisters. Here, we sorted stationary-phase E. coli based on ploidy and subjected the subpopulations to tolerance assays.

Metabolites Potentiate Nitrofurans in Nongrowing Escherichia coli.

Nitrofurantoin (NIT) is a broad-spectrum bactericidal antibiotic used in the treatment of urinary tract infections. It is a prodrug that once activated by nitroreductases goes on to inhibit bacterial DNA, RNA, cell wall, and protein synthesis. Previous work has suggested that NIT retains considerable activity against nongrowing bacteria.

Synergy Screening Identifies a Compound That Selectively Enhances the Antibacterial Activity of Nitric Oxide.

Antibiotic resistance poses a serious threat to global health. To reinforce the anti-infective arsenal, many novel therapeutic strategies to fight bacterial infections are being explored. Among them, anti-virulence therapies, which target pathways important for virulence, have attracted much attention.

Quantitative Modeling Extends the Antibacterial Activity of Nitric Oxide.

Numerous materials have been developed to try and harness the antimicrobial properties of nitric oxide (NO). However, the short half-life and reactivity of NO have made precise, tunable delivery difficult. As such, conventional methodologies have generally relied on donors that spontaneously release NO at different rates, and delivery profiles have largely been constrained to decaying dynamics.

Quantifying Nitric Oxide Flux Distributions.

Nitric oxide (NO) is a radical that is used as an attack molecule by immune cells. NO can interact and damage a range of biomolecules, and the biological outcome for bacteria assaulted with NO will be governed by how the radical distributes within their biochemical reaction networks. Measurement of those NO fluxes is complicated by the low abundance and transience of many of its reaction products.

Stationary phase persister formation in Escherichia coli can be suppressed by piperacillin and PBP3 inhibition.

Background: Persisters are rare phenotypic variants within a bacterial population that are capable of tolerating lethal antibiotic concentrations. Passage through stationary phase is associated with the formation of persisters (type I), and a major physiological response of Escherichia coli during stationary phase is cell wall restructuring. Given the concurrence of these processes, we sought to assess whether perturbation to cell wall synthesis during stationary phase impacts type I persister formation.

Transcriptional Regulation Contributes to Prioritized Detoxification of Hydrogen Peroxide over Nitric Oxide.

Hydrogen peroxide (HO) and nitric oxide (NO·) are toxic metabolites that immune cells use to attack pathogens. These antimicrobials can be present at the same time in phagosomes, and it remains unclear how bacteria deal with these insults when simultaneously present. Here, using , we observed that simultaneous exposure to HO and NO· leads to prioritized detoxification, where enzymatic removal of NO· is impeded until HO has been eliminated.

Publisher Correction: Definitions and guidelines for research on antibiotic persistence.

In Figure 2b, the minimal duration for killing (MDK) 99% of tolerant cells was erroneously labelled as MDK99.99 instead of MDK99. This has now been corrected in all versions of the Review.

Definitions and guidelines for research on antibiotic persistence.

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.