Total Synthesis and Structural Reassignment of the Antitubercular Natural Product Evybactin.

The escalating threat posed by antibiotic resistance is a global concern and underscores the need for new antibiotics. In this context, the recent discovery of evybactin, a nonribosomal depsipeptide antibiotic that selectively and potently inhibits the growth of M. tuberculosis, is particularly noteworthy. Here, we present the first total synthesis of this natural product, along with a revision of its assigned structure. Our studies revealed a disparity between the structure originally proposed for evybactin and its actual configuration. Specifically, the 3-methylhistidine residue present in the evybactin core macrocycle was found to be of the d-configuration rather than the previously assigned l-His(Me). Having addressed this, we further optimized our solid-phase synthetic route to provide access to evybactin on a multi-hundred-milligram scale. Access to such quantities will allow for more comprehensive studies with this promising antibiotic.

Small molecule produced by Photorhabdus interferes with ubiquinone biosynthesis in Gram-negative bacteria.

We report the identification of 3,6-dihydroxy-1,2-benzisoxazole (DHB) in a screen of Photorhabdus and Xenorhabdus, whose symbiotic relationship with eukaryotic nematodes favors secondary metabolites that meet several requirements matching those for clinically useful antibiotics. DHB is produced by Photorhabdus laumondii and is selective against the Gram-negative species Escherichia coli, Enterobacter cloacae, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii. It is inactive against anaerobic gut bacteria and nontoxic to human cells. Mutants resistant to DHB map to the ubiquinone biosynthesis pathway. DHB binds to 4-hydroxybenzoate octaprenyltransferase (UbiA) and prevents the formation of 4-hydroxy-3-octaprenylbenzoate. Remarkably, DHB itself is prenylated, forming an unusable chimeric product that likely contributes to the toxic effect of this antimicrobial. DHB appears to be both a competitive enzyme inhibitor and a prodrug; this dual mode of action is unusual for an antimicrobial compound. IMPORTANCE: The spread of resistant pathogens has led to the antimicrobial resistance crisis, and the need for new compounds acting against Gram-negative pathogens is especially acute. From a screen of Photorhabdus symbionts of nematodes, we identified 3,6-dihydroxy-1,2-benzisoxazole (DHB) that acts against a range of Gram-negative bacteria, including Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, and Acinetobacter baumannii. DHB had previously been isolated from other bacterial species, but its mechanism of action remained unknown. We show that DHB is unique among antimicrobials, with dual action as an inhibitor of an important enzyme, UbiA, in the biosynthesis pathway of ubiquinone and as a prodrug. DHB is a mimic of the natural substrate, and UbiA modifies it into a toxic product, contributing to the antimicrobial action of this unusual antibiotic. We also uncover the mechanism of DHB selectivity, which depends on a particular fold of the UbiA enzyme.

The Impact of Point-of-Care Ultrasound-Guided Resuscitation on Clinical Outcomes in Patients With Shock: A Systematic Review and Meta-Analysis.

OBJECTIVE: To determine the impact of point-of-care ultrasound (POCUS)-guided resuscitation on clinical outcomes in adult patients with shock. DATA SOURCE: We searched MEDLINE, Embase, and unpublished sources from inception to December 2023. STUDY SELECTION: We included randomized controlled trials (RCTs) that examined the use of POCUS to guide resuscitation in patients with shock. DATA EXTRACTION: We collected data regarding study and patient characteristics, POCUS protocol, control group interventions, and outcomes. DATA SYNTHESIS: We identified 18 eligible RCTs. POCUS slightly influences physicians' plans for IV fluid (IVF) and vasoactive medication prescription (moderate certainty), but results in little to no changes in the administration of IVF (low to high certainty) or inotropes (high certainty). POCUS may result in no change in the number of CT scans performed (low certainty) but probably reduces the number of diagnostic echocardiograms performed (moderate certainty). POCUS-guided resuscitation probably reduces 28-day mortality (relative risk [RR] 0.88; 95% CI, 0.78-0.99), the duration of vasoactive medication (mean difference -0.73 d; 95% CI, -1.16 to -0.30), and the need for renal replacement therapy (RRT) (RR 0.80; 95% CI, 0.63-1.02) (low to moderate certainty evidence), and lactate clearance (high certainty evidence). POCUS-guided resuscitation may results in little to no difference in ICU or hospital admissions, ICU and hospital length of stay, and the need for mechanical ventilation (MV) (low to moderate certainty evidence). There is an uncertain effect on the risk of acute kidney injury and the duration of MV or RRT (very low certainty evidence). CONCLUSIONS: POCUS-guided resuscitation in shock may yield important patient and health system benefits. Due to lack of sufficient evidence, we were unable to explore how the thresholds of operator competency, frequency, and timing of POCUS scans impact patient outcomes.

Preliminary image evaluation performance of radiographers in one New Zealand District: a 6-month prospective study.

INTRODUCTION: Preliminary image evaluation (PIE) is a system where radiographers alert emergency department referrers to the presence or absence of abnormalities on acute extremity X-ray examinations. PIE and similar systems have been utilised in the United Kingdom (UK) and Australia due to a shortage of radiologists to provide a timely report. As New Zealand (NZ) faces a similar shortage, PIE should be considered to address the negative impact this has on patients. The aim of this study was to determine the effect of regular feedback and education on radiographers' performance when detecting and describing acute abnormalities on extremity X-ray examinations in ED. METHODS: A prospective longitudinal study design was utilised for this study. The sensitivity, specificity, accuracy, and accuracy of PIEs performed by seven radiographers at a public provincial district in NZ were assessed over a 6-month period, with the participants provided monthly results along with regular e-mailed feedback on common errors. RESULTS: The mean for sensitivity, specificity, and accuracy calculated with a 95% confidence interval over the 6-month period were 92.8% (89.9, 95.8), 94.9 (93.1, 96.8), and 94.2 (91.9, 96.5), respectively. When the month-to-month results were analysed, the results demonstrated an improvement in participants' sensitivity, specificity, and accuracy over the 6-month period. CONCLUSION: The results of this study demonstrated that radiographers who participated in the study can perform PIE to a high standard that is comparable with the findings from international studies and demonstrated an improvement over 6 months. Therefore, PIE may be useful in NZ to aid ED clinicians in their clinical decisions when a radiology report is unavailable.

Sophisticated natural products as antibiotics.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

An Approach to Diversifying the Selection of a Guideline Panel-The Process Utilized for the Updated Adult Critical Care Ultrasound Guidelines.

OBJECTIVES: Clinical practice guidelines are essential for promoting evidence-based healthcare. While diversification of panel members can reduce disparities in care, processes for panel selection lack transparency. We aim to share our approach in forming a diverse expert panel for the updated Adult Critical Care Ultrasound Guidelines. DESIGN: This process evaluation aims to understand whether the implementation of a transparent and intentional approach to guideline panel selection would result in the creation of a diverse expert guideline panel. SETTING: This study was conducted in the setting of creating a guideline panel for the updated Adult Critical Care Ultrasound Guidelines. PATIENTS: Understanding that family/patient advocacy in guideline creations can promote the impact of a clinical practice guideline, patient representation on the expert panel was prioritized. INTERVENTIONS: Interventions included creation of a clear definition of expertise, an open invitation to the Society of Critical Care Medicine membership to apply for the panel, additional panel nomination by guideline leadership, voluntary disclosure of pre-identified diversity criteria by potential candidates, and independent review of applications including diversity criteria. This resulted in an overall score per candidate per reviewer and an open forum for discussion and final consensus. MEASUREMENTS AND MAIN RESULTS: The variables of diversity were collected and analyzed after panel selection. These were compared with historical data on panel composition. The final guideline panel comprised of 33 panelists from six countries: 45% women and 79% historically excluded people and groups. The panel has representation from nonphysician professionals and patients advocates. Of the healthcare professionals, there is representation from early, mid, and late career stages. CONCLUSIONS: Our intentional and transparent approach resulted in a panel with improved gender parity and robust diversity along ethnic, racial, and professional lines. We hope it can serve as a starting point as we strive to become a more inclusive and diverse discipline that creates globally representative guidelines.

Rational Engineering of Escherichia coli Nissle 1917 as Live Biotherapeutic to Degrade Uremic Toxin Precursors.

Uremic toxins (UTs) are microbiota-derived metabolites that accelerate the progression of kidney damage in patients with chronic kidney disease (CKD). One of the major UTs involved in CKD progression is p-cresol-sulfate (PCS), derived from dietary l-tyrosine (l-Tyr). Here, we engineered a probiotic strain of Escherichia coli Nissle 1917, to convert l-Tyr to the nontoxic compound p-coumaric acid via tyrosine ammonia lyase (TAL). First, a small metagenomic library was assessed to identify the TAL with the greatest whole-cell activity. Second, accessory genes implicated in the import of l-Tyr and export of PCA were overexpressed to enhance l-Tyr degradation by 106% and 56%, respectively. Last, random mutagenesis coupled to a novel selection and screening strategy was developed that identified a TAL variant with a 25% increase in whole-cell activity. Taken together, the final strain exhibits a 183% improvement over initial whole-cell activity and provides a promising candidate to degrade l-Tyr mediated PCS accumulation.

A Resistance-Evading Antibiotic for Treating Anthrax.

The antimicrobial resistance crisis (AMR) is associated with millions of deaths and undermines the franchise of medicine. Of particular concern is the threat of bioweapons, exemplified by anthrax. Introduction of novel antibiotics helps mitigate AMR, but does not address the threat of bioweapons with engineered resistance. We reasoned that teixobactin, an antibiotic with no detectable resistance, is uniquely suited to address the challenge of weaponized anthrax. Teixobactinbinds to immutable targets, precursors of cell wall polymers. Here we show that teixobactinis highly efficacious in a rabbit model of inhalation anthrax. Inhaling spores of Bacillus anthracis causes overwhelming morbidity and mortality. Treating rabbits with teixobactinafter the onset of disease rapidly eliminates the pathogen from blood and tissues, normalizes body temperature, and prevents tissue damage. Teixobactinassembles into an irreversible supramolecular structure of the surface of B. anthracis membrane, likely contributing to its unusually high potency against anthrax. Antibiotics evading resistance provide a rational solution to both AMR and engineered bioweapons.

Crystal structure of the N-terminal domain of MtClpC1 in complex with the anti-mycobacterial natural peptide Lassomycin.

Antibiotics form our frontline therapy against disease-causing bacteria. Unfortunately, antibiotic resistance is becoming more common, threatening a future where these medications can no longer cure infections. Furthermore, the emergence of multidrug-resistant (MDR), totally drug-resistant (TDR), and extensively drug-resistant (XDR) tuberculosis has increased the urgency of discovering new therapeutic leads with unique modes of action. Some natural peptides derived from actinomycetes, such as Cyclomarin A, Lassomycin, Rufomycin I, and Ecumicin, have potent and specific bactericidal activity against Mycobacterium tuberculosis, with the specificity owing to the fact that these peptides target the ClpC1 ATPase, an essential enzyme in mycobacteria, and inhibit/activate the proteolytic activity of the ClpC1/P1/P2 complex that participates in protein homeostasis. Here, we report the high-resolution crystal structure of the N-terminal domain of ClpC1 (ClpC1 NTD) in complex with Lassomycin, showing the specific binding mode of Lassomycin. In addition, the work also compares the Lassomycin complex structure with the previously known structures of ClpC1 NTD in complex with other natural peptides such as Cyclomarin A, Rufomycin I, and Ecumicin.

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.

Structural insights into the mechanism of overcoming Erm-mediated resistance by macrolides acting together with hygromycin-A.

The ever-growing rise of antibiotic resistance among bacterial pathogens is one of the top healthcare threats today. Although combination antibiotic therapies represent a potential approach to more efficiently combat infections caused by susceptible and drug-resistant bacteria, only a few known drug pairs exhibit synergy/cooperativity in killing bacteria. Here, we discover that well-known ribosomal antibiotics, hygromycin A (HygA) and macrolides, which target peptidyl transferase center and peptide exit tunnel, respectively, can act cooperatively against susceptible and drug-resistant bacteria. Remarkably, HygA slows down macrolide dissociation from the ribosome by 60-fold and enhances the otherwise weak antimicrobial activity of the newest-generation macrolide drugs known as ketolides against macrolide-resistant bacteria. By determining a set of high-resolution X-ray crystal structures of drug-sensitive wild-type and macrolide-resistant Erm-methylated 70S ribosomes in complex with three HygA-macrolide pairs, we provide a structural rationale for the binding cooperativity of these drugs and also uncover the molecular mechanism of overcoming Erm-type resistance by macrolides acting together with hygromycin A. Altogether our structural, biochemical, and microbiological findings lay the foundation for the subsequent development of synergistic antibiotic tandems with improved bactericidal properties against drug-resistant pathogens, including those expressing erm genes.

A new 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, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). 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 irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

An investigation into oral surgery referrals in Wales.

Introduction With waiting list time increasing in all specialties in the aftermath of the COVID-19 pandemic, it is vital to make sure that patients are receiving treatment in an appropriate setting. Most oral surgery undertaken in secondary care could be successfully carried out in a primary care setting by specialist oral surgeons or general dental practitioners (GDPs) with a special interest in oral surgery.Aim To investigate reasons for oral surgery referrals to secondary care.Method A pilot study looking at oral surgery referrals to secondary care was completed to identify key themes for referrals. From this, a questionnaire was designed. An electronic copy of the questionnaire was distributed to all GDPs registered with Health Education and Improvement Wales (HEIW) throughout Wales.Results Five main themes for referrals, which corresponded with the pilot study findings were: contract limitations; the perception that recently trained dentists do not have the practical skills to undertake oral surgery; limited communication between the oral and maxillofacial surgery departments and GDPs; limited practice resources; and GDPs being less risk averse in undertaking oral surgery in primary care.Outcome Following the results from this research, an All-Wales oral surgery referral handbook for general dental practitioners was published, hosted by HEIW, describing oral surgery patient care pathways. Formation of the Oral Surgery Managed Clinical Networks in Wales and the All-Wales Oral Surgery Strategic Advisory Forum will help further develop robust, sustainable patient care pathways, in collaboration with the health boards.

Deletions of DNA in cancer and their possible uses for therapy.

Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which a pair of sequence-specific roadblocks is placed solely at two cancer-confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed "replication blocks specific for deletions" (RBSD), and another deletions-based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer-specific toxicity of RBSD.

Systematic comparisons between Lyme disease and post-treatment Lyme disease syndrome in the U.S. with administrative claims data.

BACKGROUND: Post-treatment Lyme disease syndrome (PTLDS) is used to describe Lyme disease patients who have the infection cleared by antibiotic but then experienced persisting symptoms of pain, fatigue, or cognitive impairment. Currently, little is known about the cause or epidemiology of PTLDS. METHODS: We conducted a data-driven study with a large nationwide administrative dataset, which consists of more than 98 billion billing and 1.4 billion prescription records between 2008 and 2016, to identify unique aspects of PTLDS that could have diagnostic and etiologic values. We defined PTLDS based on its symptomatology and compared the demographic, longitudinal changes of comorbidity, and antibiotic prescriptions between patients who have Lyme with absence of prolonged symptoms (APS) and PTLDS. FINDINGS: The age and temporal distributions were similar between Lyme APS and PTLDS. The PTLDS-to-Lyme APS case ratio was 3.42%. The co-occurrence of 3 out of 19 chronic conditions were significantly higher in PTLDS versus Lyme APS-odds ratio and 95% CI for anemia, hyperlipidemia, and osteoarthrosis were 1.46 (1.11-1.92), 1.39 (1.15-1.68), and 1.62 (1.23-2.12) respectively. We did not find significant differences between PTLDS and Lyme APS for the number of types of antibiotics prescribed (incidence rate ratio = 1.009, p = 0.90) and for the prescription of each of the five antibiotics (FDR adjusted p values 0.72-0.95). INTERPRETATION: PTLDS cases have more codes corresponding to anemia, hyperlipidemia, and osteoarthrosis compared to Lyme APS. Our finding of hyperlipidemia is consistent with a dysregulation of fat metabolism reported by other researchers, and further investigation should be conducted to understand the potential biological relationship between the two. FUNDING: Steven & Alexandra Cohen Foundation, Global Lyme Alliance, and the Pazala Foundation; National Institutes of Health R01ES032470.

Disulfiram: Mechanisms, Applications, and Challenges.

Background: Since disulfiram's discovery in the 1940s and its FDA approval for alcohol use disorder, other indications have been investigated. This review describes potential clinical applications, associated risks, and challenges. Methods: For this narrative review, a PubMed search was conducted for articles addressing in vivo studies of disulfiram with an emphasis on drug repurposing for the treatment of human diseases. The key search terms were "disulfiram" and "Antabuse". Animal studies and in vitro studies highlighting important mechanisms and safety issues were also included. Results: In total, 196 sources addressing our research focus spanning 1948-2022 were selected for inclusion. In addition to alcohol use disorder, emerging data support a potential role for disulfiram in the treatment of other addictions (e.g., cocaine), infections (e.g., bacteria such as Staphylococcus aureus and Borrelia burgdorferi, viruses, parasites), inflammatory conditions, neurological diseases, and cancers. The side effects range from minor to life-threatening, with lower doses conveying less risk. Caution in human use is needed due to the considerable inter-subject variability in disulfiram pharmacokinetics. Conclusions: While disulfiram has promise as a "repurposed" agent in human disease, its risk profile is of concern. Animal studies and well-controlled clinical trials are needed to assess its safety and efficacy for non-alcohol-related indications.

Identification of a growth factor required for culturing specific fastidious oral bacteria.

Aims: The aim of this research was to isolate oral bacteria that are dependent for growth on adjacent bacteria producing a required growth factor and to identify the chemical structure of the growth factor. Methods: Porphyromonas pasteri strain KLE1280, could be cultivated with Staphylococcus hominis and Escherichia coli as helper strains. A deletion mutant library of E. coli was screened to determine genes involved in production of the growth factor. Compounds produced by the growth factor's pathway were screened to see if they would stimulate growth of strain P. pasteri KLE1280. The genomes of species related to P. pasteri KLE1280 were screened for presence of the factor's synthetic pathway. Results: Analysis of the E. coli deletion mutant library and growth studies identified 1,2-dihydroxy-2-naphthoic acid (DHNA) and menaquinone-4 (MK4) as the growth factors. Strain P. pasteri KLE1280 was shown to lack five genes in the menaquinone synthesis pathway but to possess the two genes necessary to convert DHNA to menaquinone. Genome analysis found that 8 species in genera Porphyromonas and Tannerella lack five genes in the menaquinone synthesis pathway. Conclusions: Addition of DHNA to culture media allows isolation of strains of several oral species that are not recovered using standard media.

Noise in a Metabolic Pathway Leads to Persister Formation in Mycobacterium tuberculosis.

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 ackA 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. IMPORTANCE 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.