Evybactin is a DNA gyrase inhibitor that selectively kills Mycobacterium tuberculosis.

The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity.

A Silent Operon of Photorhabdus luminescens Encodes a Prodrug Mimic of GTP.

With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra, a betaproteobacterium; odilorhabdins, from Xenorhabdus, are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk, kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. IMPORTANCE Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus, gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express in vitro. To gain access to silent operons, we first fractionated supernatant from Photorhabdus and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.

Computational identification of a systemic antibiotic for gram-negative bacteria.

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 ß-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.

Pulse Dosing of Antibiotic Enhances Killing of a Staphylococcus aureus Biofilm.

Biofilms are highly tolerant to antibiotics and underlie the recalcitrance of many chronic infections. We demonstrate that mature Staphylococcus aureus biofilms can be substantially sensitized to the treatment by pulse dosing of an antibiotic - in this case, oxacillin. Pulse (periodic) dosing was compared to continuous application of antibiotic and was studied in a novel in vitro flow system which allowed for robust biofilm growth and tractable pharmacokinetics of dosing regimens. Our results highlight that a subpopulation of the biofilm survives antibiotic without becoming resistant, a population we refer to as persister bacteria. When oxacillin was continuously present the persister level did not decline, but, importantly, providing correctly timed periodic breaks decreased the surviving population. We found that the length of the periodic break impacted efficacy, and there was an optimal length that sensitized the biofilm to repeat treatment without allowing resistance expansion. Periodic dosing provides a potential simple solution to a complicated problem.