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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. 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 ihfA-mVenus fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of ihf 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 lac operon, and show that a knockout of the lacI 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. IMPORTANCE 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 lac 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 lac operon, providing a simple approach to test the involvement of a gene in persister formation.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus , a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its β-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development. Computational search identifies dynobactin A which is a systemically active, natural-product peptide antibiotic that kills Gram-negative bacteria.

The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm. Environmental strains of the soil bacterium Bacillus subtilis have valuable applications in agriculture, industry, and biotechnology; however, environmental strains are genetically less accessible. This reduced accessibility is in sharp contrast to laboratory strains, which are well known for their natural competence, and a limitation in their applications. In this study, we observed that robust biofilm formation by environmental strains of B. subtilis greatly reduced the frequency of competent cells in the biofilm. By using model strain 3610, we revealed a cross-pathway regulation that allows biofilm matrix producers and competence-developing cells to undergo mutually exclusive cell differentiation. We further demonstrated that the competence activator ComK represses the key biofilm regulatory gene sinI by directly binding to the sinI promoter, thus blocking competent cells from simultaneously becoming matrix producers. In parallel, the biofilm activator SlrR represses competence through three distinct mechanisms involving both genetic regulation and cell morphological changes. Finally, we discuss the potential implications of limiting competence in a bacterial biofilm. IMPORTANCE The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm.

Antibiotic resistance and recalcitrant infections are challenges faced in the clinic every day. One perplexing aspect of this daily battle is the fact that many of these infections are caused by antibiotic susceptible bacteria, and yet cannot be cleared. The presence of persister cells in a bacterial population may explain these puzzling observations. Persisters are phenotypic variants that survive exposure to antibiotics, due to temporary dormancy. Therefore, understanding how persisters are formed can improve patient treatment. This research investigates persisters of Escherichia coli from several avenues. First we screened for a global regulator of persister formation. It has been demonstrated that fluctuations in expression of metabolic genes can affect persister formation, so we screened for a global regulator that creates global changes in the cell by controlling multiple genes in metabolism. We identified a deletion in ihfA which resulted in decreased persister levels when grown on various carbon sources. Further investigation revealed that ihfA was modulating persister formation through the glyoxylate bypass. Second, we investigated noise quenching of gene expression and its impact on persister formation. Persisters can form stochastically and noise in expression of metabolic genes has been shown to affect persister formation. We aimed to quench noise in a simple and defined system and examine the impact on persister formation. In a study of the inherently noisy lac operon, we determined that a deletion of lacI, the noise generating gene in the lac operon, had less persisters than the wild type. Finally, we interrogated the role of high persister (hip) mutations in vivo. Hip mutants have been identified in clinical isolates collected from patients, which suggests a link between persisters and clinical infection. We used an antibiotic-treated murine thigh infection model to assess the advantage, if any, hip mutants had in vivo. The data show that a hipA7 high persister mutant survived better in the murine model when competed directly with the wild type. These three avenues of research serve to broaden our understanding of persisters and their mechanism of formation. Understanding the underlying mechanism of persister formation, as well as the clinical implication of persisters can help us better eradicate these cells. Ultimately, determining how persisters form will guide patient treatment in the future. In knowing this relationship, researchers may be able to find target genes for future anti-persister antibiotics, thus abolishing their role in chronic infection.

Environmental strains of the soil bacterium Bacillus subtilis have valuable applications in agriculture, industry, and biotechnology. They are capable of forming robust biofilms and demonstrate excellent biological control activities in plant protection. However, environmental strains are genetically less accessible, a sharp contrast to the laboratory strains well known for their natural competence and a limitation toward their application. In this study, we observed that robust biofilm formation of the environmental strains greatly reduces the rate of competent cells within the biofilm. By using the model strain 3610, we reveal a cross-pathway regulation that allows biofilm matrix producers and competence-developing cells to undergo mutually exclusive cell differentiation. We show that the competence activator ComK represses the key biofilm regulatory gene sinI by directly binding to the sinI promoter, thus blocking competent cells from simultaneously becoming matrix producers. In parallel, the biofilm activator SlrR represses competence through three distinct mechanisms, involving both genetic regulation and cell morphological changes. We discuss potential implications of limiting competence in a bacterial biofilm.

Pharmacologic management of infections in elderly patients presents multiple challenges to health care professionals due to variable pharmacokinetics, pharmacodynamics, and immune function. Age is a well-established risk factor for infection, but furthermore is a risk factor for prolonged length of hospital stay, increased incidence of complications, and significant and sustained decline in baseline functional status. In 2014, 46.2 million Americans were aged ≥65 years, accounting for 14.5% of the total population. By 2033, for the first time, the population of persons aged ≥65 years is projected to outnumber the people <18 years of age. According to the National Ambulatory Medical Care Survey and the National Hospital Ambulatory Medical Care Survey, 154 million prescriptions for antimicrobials were estimated to have been written in doctors' offices and emergency departments during a 1-year time period. In 2014, 266.1 million courses of antimicrobials were dispensed to outpatients by US community pharmacies. A study that evaluated 2007-2009 Medicare Part D data found that patients aged ≥65 years used more antimicrobials, at 1.10 per person per year, compared to 0.88 antimicrobials used per person per year in patients aged 0-64 years. With the abundance of antimicrobial prescriptions and the current growth in the number and proportion of older adults in the US, it is essential that health care providers understand appropriate antimicrobial pharmacotherapy in the elderly patient. This review focuses on the use and implications of antimicrobial agents in the elderly population.