Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Evaluation of a Clinical Decision Support System and an Automated Electronic Health Record Alert on Outpatient Prescribing of Cefdinir.

Acute bacterial upper respiratory infections are common indications for antibiotics in pediatrics, and many prescriptions may be inappropriate. Novel approaches to outpatient antimicrobial stewardship interventions are needed. This quasi-experimental study of an order set and best practice advisory alert targeting cefdinir prescriptions demonstrated an 8.4% decrease in cefdinir prescribing (P ≤ .001).

Synthesis, Structure-Activity Relationship Study, Bioactivity, and Nephrotoxicity Evaluation of the Proposed Structure of the Cyclic Lipodepsipeptide Brevicidine B.

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.

Catalyst-free late-stage functionalization to assemble α-acyloxyenamide electrophiles for selectively profiling conserved lysine residues.

Covalent probes coupled with chemical proteomics represent a powerful method for investigating small molecule and protein interactions. However, the creation of a reactive warhead within various ligands to form covalent probes has been a major obstacle. Herein, we report a convenient and robust process to assemble a unique electrophile, an α-acyloxyenamide, through a one-step late-stage coupling reaction. This procedure demonstrates remarkable tolerance towards other functional groups and facilitates ligand-directed labeling in proteins of interest. The reactive group has been successfully incorporated into a clinical drug targeting the EGFR L858R mutant, erlotinib, and a pan-kinase inhibitor. The resulting probes have been shown to be able to covalently engage a lysine residue proximal to the ATP-binding pocket of the EGFR L858R mutant. A series of active sites, and Mg2+, ATP-binding sites of kinases, such as K33 of CDK1, CDK2, CDK5 were detected. This is the first report of engaging these conserved catalytic lysine residues in kinases with covalent inhibition. Further application of this methodology to natural products has demonstrated its success in profiling ligandable conserved lysine residues in whole proteome. These findings offer insights for the development of new targeted covalent inhibitors (TCIs).

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

Amino acid 17 in QRDR of Gyrase A plays a key role in fluoroquinolones susceptibility in mycobacteria.

IMPORTANCE: Fluoroquinolones (FQs) play a key role in the treatment regimens against tuberculosis and non-tuberculous mycobacterial infections. However, there are significant differences in the sensitivities of different mycobacteria to FQs. In this study, we proved that this is associated with the polymorphism at amino acid 17 of quinolone resistance-determining region of Gyrase A by gene editing. This is the first study using CRISPR-associated recombination for gene editing in Mycobacterium abscessus to underscore the contribution of the amino acid substitutions in GyrA to FQ susceptibilities in mycobacteria.

The two-component system CroRS acts as a master regulator of cell envelope homeostasis to confer antimicrobial tolerance in the bacterial pathogen Enterococcus faecalis.

Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we have carried out RNA-seq analyses in the E. faecalis wild-type and isogenic ∆ croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and demonstrate that CroRS upregulates biosynthesis of all major components of the enterococcal cell envelope in response to teixobactin. This suggests a coordinating role of this regulatory system in maintaining integrity of the multiple layers of the enterococcal envelope during antimicrobial stress, likely contributing to bacterial survival. Using experimental evolution, we observed that truncation of HppS, a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone, was sufficient to rescue tolerance in the croRS deletion strain. This highlights a key role for isoprenoid biosynthesis in antimicrobial tolerance in E. faecalis. Here, we propose a model of CroRS acting as a master regulator of cell envelope biogenesis and a gate-keeper between isoprenoid biosynthesis and respiration to ensure tolerance against antimicrobial challenge.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments.

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas.

German brass for Benin Bronzes: Geochemical analysis insights into the early Atlantic trade.

Utilizing geochemical analysis, this study identifies the sources of European brass used in the casting of the renowned Benin Bronzes, produced by the Edo people of Nigeria. It is commonly believed that distinctive brass rings known as "manillas", used as currency in the European trade in West Africa, also served as a metal source for the making of the Bronzes. However, prior to the current study, no research had conclusively connected the Benin artworks and the European manillas. For this research, manillas from shipwrecks in African, American and European waters dating between the 16th and 19th Century were analysed using ICP-MS analysis. Comparing trace elements and lead isotope ratios of manillas and Benin Bronzes identifies Germany as the principal source of the manillas used in the West African trade between the 15th and 18th centuries before British industries took over the brass trade in the late 18th century.

Structural basis for bacterial energy extraction from atmospheric hydrogen.

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.

The evolution of antibiotic resistance is associated with collateral drug phenotypes in Mycobacterium tuberculosis.

The increasing incidence of drug resistance in Mycobacterium tuberculosis has diminished the efficacy of almost all available antibiotics, complicating efforts to combat the spread of this global health burden. Alongside the development of new drugs, optimised drug combinations are needed to improve treatment success and prevent the further spread of antibiotic resistance. Typically, antibiotic resistance leads to reduced sensitivity, yet in some cases the evolution of drug resistance can lead to enhanced sensitivity to unrelated drugs. This phenomenon of collateral sensitivity is largely unexplored in M. tuberculosis but has the potential to identify alternative therapeutic strategies to combat drug-resistant strains that are unresponsive to current treatments. Here, by using drug susceptibility profiling, genomics and evolutionary studies we provide evidence for the existence of collateral drug sensitivities in an isogenic collection M. tuberculosis drug-resistant strains. Furthermore, in proof-of-concept studies, we demonstrate how collateral drug phenotypes can be exploited to select against and prevent the emergence of drug-resistant strains. This study highlights that the evolution of drug resistance in M. tuberculosis leads to collateral drug responses that can be exploited to design improved drug regimens.

Discovery of 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as new cytochrome bd oxidase inhibitors for tuberculosis therapy.

The cytochrome bcc-aa3 oxidase (Cyt-bcc) of Mycobacterium tuberculosis (Mtb) is a promising anti-tuberculosis target. However, when Cyt-bcc is inhibited, cytochrome bd terminal oxidase (Cyt-bd) can still maintain the activity of the respiratory chain and drive ATP synthesis. Through virtual screening and biological validation, we discovered two FDA-approved drugs, ivacaftor and roquinimex, exhibited moderate binding affinity to Cyt-bd. Structural modifications of them led to 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as potent new Cyt-bd inhibitors. Compound 8d binds to Cyt-bd with a Kd value of 4.17 µM and inhibits the growth of the Cyt-bcc knock-out strain (ΔqcrCAB, Cyt-bd+) with a MIC value of 6.25 µM. The combination of 8d with the Cyt-bcc inhibitor Q203 completely inhibited oxygen consumption of the wild-type strain and the inverted-membrane vesicles expressing M. tuberculosis Cyt-bd (ΔcydAB::MtbCydAB+). Our study provides a promising starting point for the development of novel dual chemotherapies for tuberculosis.

Alkyltriphenylphosphonium turns naphthoquinoneimidazoles into potent membrane depolarizers against mycobacteria.

Due to its central role in energy generation and bacterial viability, mycobacterial bioenergetics is an attractive therapeutic target for anti-tuberculosis drug discovery. Building upon our work on antimycobacterial dioxonaphthoimidazoliums that were activated by a proximal positive charge and generated reactive oxygen species upon reduction by Type II NADH dehydrogenase, we herein studied the effect of a distal positive charge on the antimycobacterial activity of naphthoquinoneimidazoles by incorporating a trialkylphosphonium cation. The potency-enhancing properties of the linker length were affirmed by structure-activity relationship studies. The most active compound against M. tb H37Rv displayed good selectivity index (SI = 34) and strong bactericidal activity in the low micromolar range, which occurred through rapid bacterial membrane depolarization that resulted in depletion of intracellular ATP. Through this work, we demonstrated a switch of the scaffold's mode-of-action via relocation of positive charge while retaining its excellent antibacterial activity and selectivity.

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d-Phe-6, and the N-Terminus Enabled by S-Lipidation.

With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol-ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics.

Characterization of Genetic Variants Associated with Rifampicin Resistance Level in Mycobacterium tuberculosis Clinical Isolates Collected in Guangzhou Chest Hospital, China.

Objective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF. Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay. Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to >64 µg/mL) of which 57 were MDR and 81 pre-XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426-452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284-1289 CCAGCT), (1295-1303 AATTCATGG), and insertion at (1300-1302 TTC) were detected within RRDR of three RIFR strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIFR strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively. Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIFR clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings.

Synthesis and Biological Evaluation of Aurachin D Analogues as Inhibitors of Mycobacterium tuberculosis Cytochrome bd Oxidase.

A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 µM).

Complete Genome Sequence of a New Zealand Mycobacterium tuberculosis Strain Responsible for Ongoing Transmission over the Past 30 Years.

We report here the complete genome sequence of Mycobacterium tuberculosis strain Colonial S-type 1 (CS1), which has been responsible for ongoing outbreaks of tuberculosis in New Zealand over the past 30 years. CS1 appears to be highly transmissible, with greater rates of progression to active disease, compared to other circulating M. tuberculosis strains; therefore, comparison of its genomic content is of interest.

RNase HI Depletion Strongly Potentiates Cell Killing by Rifampicin in Mycobacteria.

Multidrug-resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and they can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin, and rifampicin was increased, the latter by a striking near 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and rifampicin. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action and providing the first step in developing a new class of antimycobacterial drug.

Uncovering interactions between mycobacterial respiratory complexes to target drug-resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Mycobacterial bioenergetics has emerged as a promising space for the development of novel therapeutics. Further to this, unique combinations of respiratory inhibitors have been shown to have synergistic or synthetic lethal interactions, suggesting that combinations of bioenergetic inhibitors could drastically shorten treatment times. Realizing the full potential of this unique target space requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest interactions and potential in a clinical setting. In this review, we discuss (i) chemical-interaction, (ii) genetic-interaction and (iii) chemical-genetic interaction studies to explore the consequences of inhibiting multiple mycobacterial respiratory components. We provide potential mechanisms to describe the basis for the strongest interactions. Finally, whilst we place an emphasis on interactions that occur with existing bioenergetic inhibitors, by highlighting interactions that occur with alternative respiratory components we envision that this information will provide a rational to further explore alternative proteins as potential drug targets and as part of unique drug combinations.

Antimicrobial tolerance and its role in the development of resistance: Lessons from enterococci.

Bacteria have developed resistance against every antimicrobial in clinical use at an alarming rate. There is a critical need for more effective use of antimicrobials to both extend their shelf life and prevent resistance from arising. Significantly, antimicrobial tolerance, i.e., the ability to survive but not proliferate during antimicrobial exposure, has been shown to precede the development of bona fide antimicrobial resistance (AMR), sparking a renewed and rapidly increasing interest in this field. As a consequence, problematic infections for the first time are now being investigated for antimicrobial tolerance, with increasing reports demonstrating in-host evolution of antimicrobial tolerance. Tolerance has been identified in a wide array of bacterial species to all bactericidal antimicrobials. Of particular interest are enterococci, which contain the opportunistic bacterial pathogens Enterococcus faecalis and Enterococcus faecium. Enterococci are one of the leading causes of hospital-acquired infection and possess intrinsic tolerance to a number of antimicrobial classes. Persistence of these infections in the clinic is of growing concern, particularly for the immunocompromised. Here, we review current known mechanisms of antimicrobial tolerance, and include an in-depth analysis of those identified in enterococci with implications for both the development and prevention of AMR.