A metabolomics pipeline highlights microbial metabolism in bloodstream infections.

The growth of antimicrobial resistance (AMR) highlights an urgent need to identify bacterial pathogenic functions that may be targets for clinical intervention. Although severe infections profoundly alter host metabolism, prior studies have largely ignored microbial metabolism in this context. Here, we describe an iterative, comparative metabolomics pipeline to uncover microbial metabolic features in the complex setting of a host and apply it to investigate gram-negative bloodstream infection (BSI) in patients. We find elevated levels of bacterially derived acetylated polyamines during BSI and discover the enzyme responsible for their production (SpeG). Blocking SpeG activity reduces bacterial proliferation and slows pathogenesis. Reduction of SpeG activity also enhances bacterial membrane permeability and increases intracellular antibiotic accumulation, allowing us to overcome AMR in culture and in vivo. This study highlights how tools to study pathogen metabolism in the natural context of infection can reveal and prioritize therapeutic strategies for addressing challenging infections.

Discovery of antimicrobial peptides in the global microbiome with machine learning.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs) within the global microbiome and leverage a vast dataset of 63,410 metagenomes and 87,920 prokaryotic genomes from environmental and host-associated habitats to create the AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, few of which match existing databases. AMPSphere provides insights into the evolutionary origins of peptides, including by duplication or gene truncation of longer sequences, and we observed that AMP production varies by habitat. To validate our predictions, we synthesized and tested 100 AMPs against clinically relevant drug-resistant pathogens and human gut commensals both in vitro and in vivo. A total of 79 peptides were active, with 63 targeting pathogens. These active AMPs exhibited antibacterial activity by disrupting bacterial membranes. In conclusion, our approach identified nearly one million prokaryotic AMP sequences, an open-access resource for antibiotic discovery.

Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens.

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using in vitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy.

Microbial transmission in the social microbiome and host health and disease.

Although social interactions are known to drive pathogen transmission, the contributions of socially transmissible host-associated mutualists and commensals to host health and disease remain poorly explored. We use the concept of the social microbiome-the microbial metacommunity of a social network of hosts-to analyze the implications of social microbial transmission for host health and disease. We investigate the contributions of socially transmissible microbes to both eco-evolutionary microbiome community processes (colonization resistance, the evolution of virulence, and reactions to ecological disturbance) and microbial transmission-based processes (transmission of microbes with metabolic and immune effects, inter-specific transmission, transmission of antibiotic-resistant microbes, and transmission of viruses). We consider the implications of social microbial transmission for communicable and non-communicable diseases and evaluate the importance of a socially transmissible component underlying canonically non-communicable diseases. The social transmission of mutualists and commensals may play a significant, under-appreciated role in the social determinants of health and may act as a hidden force in social evolution.

Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states.

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.

An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.

A Deep Learning Approach to Antibiotic Discovery.

Due to the rapid emergence of antibiotic-resistant bacteria, there is a growing need to discover new antibiotics. To address this challenge, we trained a deep neural network capable of predicting molecules with antibacterial activity. We performed predictions on multiple chemical libraries and discovered a molecule from the Drug Repurposing Hub-halicin-that is structurally divergent from conventional antibiotics and displays bactericidal activity against a wide phylogenetic spectrum of pathogens including Mycobacterium tuberculosis and carbapenem-resistant Enterobacteriaceae. Halicin also effectively treated Clostridioides difficile and pan-resistant Acinetobacter baumannii infections in murine models. Additionally, from a discrete set of 23 empirically tested predictions from >107 million molecules curated from the ZINC15 database, our model identified eight antibacterial compounds that are structurally distant from known antibiotics. This work highlights the utility of deep learning approaches to expand our antibiotic arsenal through the discovery of structurally distinct antibacterial molecules.

Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design.

Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.

Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries.

Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides because the phenotype, death, selects against itself and has caused a scientific bottleneck that confines research to a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial Display (SLAY), a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened ∼800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences, dramatically increasing the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved. VIDEO ABSTRACT.

Lysozyme Counteracts ß-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

Transposase-DNA Complex Structures Reveal Mechanisms for Conjugative Transposition of Antibiotic Resistance.

Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.

Immunomimetic Designer Cells Protect Mice from MRSA Infection.

Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.

Tracing the Enterococci from Paleozoic Origins to the Hospital.

We examined the evolutionary history of leading multidrug resistant hospital pathogens, the enterococci, to their origin hundreds of millions of years ago. Our goal was to understand why, among the vast diversity of gut flora, enterococci are so well adapted to the modern hospital environment. Molecular clock estimation, together with analysis of their environmental distribution, phenotypic diversity, and concordance with host fossil records, place the origins of the enterococci around the time of animal terrestrialization, 425-500 mya. Speciation appears to parallel the diversification of hosts, including the rapid emergence of new enterococcal species following the End Permian Extinction. Major drivers of speciation include changing carbohydrate availability in the host gut. Life on land would have selected for the precise traits that now allow pathogenic enterococci to survive desiccation, starvation, and disinfection in the modern hospital, foreordaining their emergence as leading hospital pathogens.

Lung antimicrobial proteins and peptides: from host defense to therapeutic strategies.

Representing severe morbidity and mortality globally, respiratory infections associated with chronic respiratory diseases, including complicated pneumonia, asthma, interstitial lung disease, and chronic obstructive pulmonary disease, are a major public health concern. Lung health and the prevention of pulmonary disease rely on the mechanisms of airway surface fluid secretion, mucociliary clearance, and adequate immune response to eradicate inhaled pathogens and particulate matter from the environment. The antimicrobial proteins and peptides contribute to maintaining an antimicrobial milieu in human lungs to eliminate pathogens and prevent them from causing pulmonary diseases. The predominant antimicrobial molecules of the lung environment include human α- and ß-defensins and cathelicidins, among numerous other host defense molecules with antimicrobial and antibiofilm activity such as PLUNC (palate, lung, and nasal epithelium clone) family proteins, elafin, collectins, lactoferrin, lysozymes, mucins, secretory leukocyte proteinase inhibitor, surfactant proteins SP-A and SP-D, and RNases. It has been demonstrated that changes in antimicrobial molecule expression levels are associated with regulating inflammation, potentiating exacerbations, pathological changes, and modifications in chronic lung disease severity. Antimicrobial molecules also display roles in both anticancer and tumorigenic effects. Lung antimicrobial proteins and peptides are promising alternative therapeutics for treating and preventing multidrug-resistant bacterial infections and anticancer therapies.

ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells.

Antibiotic resistance is a global health threat and often results from new mutations. Antibiotics can induce mutations via mechanisms activated by stress responses, which both reveal environmental cues of mutagenesis and are weak links in mutagenesis networks. Network inhibition could slow the evolution of resistance during antibiotic therapies. Despite its pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the Escherichia coli stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin-induced mutagenesis. Binding of response-activator ppGpp to RNA polymerase (RNAP) at two sites leads to an antibiotic-induced mutable gambler-cell subpopulation. Each activates a stress response required for mutagenic DNA-break repair: surprisingly, ppGpp-site-1-RNAP triggers the DNA-damage response, and ppGpp-site-2-RNAP induces σS-response activity. We propose that RNAP regulates DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNAP-regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.

Mechanisms of Methicillin Resistance in Staphylococcus aureus.

Staphylococcus aureus is a major human and veterinary pathogen worldwide. Methicillin-resistant S. aureus (MRSA) poses a significant and enduring problem to the treatment of infection by such strains. Resistance is usually conferred by the acquisition of a nonnative gene encoding a penicillin-binding protein (PBP2a), with significantly lower affinity for ß-lactams. This resistance allows cell-wall biosynthesis, the target of ß-lactams, to continue even in the presence of typically inhibitory concentrations of antibiotic. PBP2a is encoded by the mecA gene, which is carried on a distinct mobile genetic element (SCCmec), the expression of which is controlled through a proteolytic signal transduction pathway comprising a sensor protein (MecR1) and a repressor (MecI). Many of the molecular and biochemical mechanisms underlying methicillin resistance in S. aureus have been elucidated, including regulatory events and the structure of key proteins. Here we review recent advances in this area.

Efflux-linked accelerated evolution of antibiotic resistance at a population edge.

Efflux is a common mechanism of resistance to antibiotics. We show that efflux itself promotes accumulation of antibiotic-resistance mutations (ARMs). This phenomenon was initially discovered in a bacterial swarm where the linked phenotypes of high efflux and high mutation frequencies spatially segregated to the edge, driven there by motility. We have uncovered and validated a global regulatory network connecting high efflux to downregulation of specific DNA-repair pathways even in non-swarming states. The efflux-DNA repair link was corroborated in a clinical "resistome" database: genomes with mutations that increase efflux exhibit a significant increase in ARMs. Accordingly, efflux inhibitors decreased evolvability to antibiotic resistance. Swarms also revealed how bacterial populations serve as a reservoir of ARMs even in the absence of antibiotic selection pressure. High efflux at the edge births mutants that, despite compromised fitness, survive there because of reduced competition. This finding is relevant to biofilms where efflux activity is high.

HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are potent inhibitors of prokaryotic RNA polymerase (RNAP) used to treat tuberculosis and other bacterial infections. Although resistance arises in the clinic principally through mutations in RNAP, many bacteria possess highly specific enzyme-mediated resistance mechanisms that modify and inactivate rifamycins. The expression of these enzymes is controlled by a 19-bp cis-acting rifamycin-associated element (RAE). Guided by the presence of RAE sequences, we identify a helicase-like protein, HelR, in Streptomyces venezuelae that confers broad-spectrum rifamycin resistance. We show that HelR also promotes tolerance to rifamycins, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP and rescues transcription inhibition by displacing rifamycins from RNAP, thereby providing resistance by target protection . Furthermore, HelRs are broadly distributed in Actinobacteria, including several opportunistic Mycobacterial pathogens, offering yet another challenge for developing new rifamycin antibiotics.

Reactive metabolic byproducts contribute to antibiotic lethality under anaerobic conditions.

Understanding how bactericidal antibiotics kill bacteria remains an open question. Previous work has proposed that primary drug-target corruption leads to increased energetic demands, resulting in the generation of reactive metabolic byproducts (RMBs), particularly reactive oxygen species, that contribute to antibiotic-induced cell death. Studies have challenged this hypothesis by pointing to antibiotic lethality under anaerobic conditions. Here, we show that treatment of Escherichia coli with bactericidal antibiotics under anaerobic conditions leads to changes in the intracellular concentrations of central carbon metabolites, as well as the production of RMBs, particularly reactive electrophilic species (RES). We show that antibiotic treatment results in DNA double-strand breaks and membrane damage and demonstrate that antibiotic lethality under anaerobic conditions can be decreased by RMB scavengers, which reduce RES accumulation and mitigate associated macromolecular damage. This work indicates that RMBs, generated in response to antibiotic-induced energetic demands, contribute in part to antibiotic lethality under anaerobic conditions.