Bacterial Cell Wall Synthesis Research Articles

Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.

Comparison of broth microdilution and E-test for susceptibility of MRSA to vancomycin

Introduction: Healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) developed soon after the introduction of methicillin. These bacteria have shown resistance to multiple drugs and therefore vancomycin became the antibiotic of choice for treatment of MRSA infections. Vancomycin is a bactericidal antibiotic that acts by inhibiting bacterial cell wall synthesis. This study aimed to compare broth microdilution (BMD) and E-test in determining vancomycin minimum inhibitory concentration (MIC) against MRSA.Methods: A total of 30 clinical isolates of MRSA were acquired from Colombo South Teaching Hospital, Sri Lanka. These MRSA strains were identified by cefoxitin disk diffusion and the vancomycin MIC was determined through BMD and E-test. The Kruskal-Wallis test was applied to test if the samples originated from the same distribution with post hoc determination of the correlation between methods using the Mann-Whitney U test.Results: All 30 MRSA isolates were 100% vancomycin susceptible (≤2 μg/mL) irrespective of methodology, according to the Clinical and Laboratory Standards Institute (CLSI) established breakpoints. However, the E-test MIC values were 1 to 2 dilutions higher than those of BMD. A statistically significant difference between vancomycin MICs of BMD and E-test (p = <0.00001) was calculated which indicated a difference in accuracy.Conclusion: Due to cost, convenience, and the ability to detect vancomycin intermediate Staphylococcus aureus (VISA), exploring the possibility of using E-test as an alternative to BMD is worthwhile.

Identification of small molecule inhibitors of penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus for the therapeutics of bacterial infection.

The penicillin binding protein 2a (PBP2a) is a key enzyme associated with bacterial cell wall synthesis and bacterial infection. Therefore, targeting PBPa2 offers a promising approach for the therapeutics of bacterial resistance and infection. This study presents a comprehensive analysis of alpha-mangostin as a potential inhibitor of PBPa2. Molecular docking simulations revealed a strong binding affinity between alpha-mangostin and PBP2a, with an affinity score of -6.01 kcal/mol. Notably, alpha-mangostin formed a preferential hydrogen bond with THR216 of PBP2a, alongside several other polar and hydrophobic interactions. ADME and Toxicity predictions indicated that alpha-mangostin possesses favourable pharmacokinetic properties, suggesting its potential as a therapeutic agent. PASS analysis further highlighted its broad range of favourable biological properties. SwissTargetPrediction analysis reinforced these findings, indicating alpha-mangostin's association with various biological processes. Cell toxicity assays demonstrated that alpha-mangostin had no significant impact on the viability of HEK-293 cells, suggesting its potential safety for further development. The IC50 value for alpha-mangostin was found to be 33.43µM. Fluorescence-based binding assays showed that alpha-mangostin effectively inhibited PBP2a activity in a concentration-dependent manner, supporting its role as an inhibitor. In conclusion, the results suggest alpha-mangostin as a promising candidate for inhibiting PBP2a. Further, extensive studies are warranted to explore its clinical applications.

Chitosan‐Stabilized PtAu Nanoparticles with Multienzyme‐Like Activity for Mixed Bacteria Infection Wound Healing and Insights into Its Antibacterial Mechanism

Traditional antibacterial agents are often observed to be ineffective because bacteria evolved to strains with greater antibiotic resistance. Here, vigorous chitosan‐stabilized PtAu nanoparticles (CSPA) with multienzyme‐like activity are successfully fabricated, which serve an effective artificial nanozyme to enhance antibacterial activity for mixed bacterial infection wound treatment. Ultrasmall size CSPA exhibits excellent hydrophilicity and biocompatibility, possesses strong oxidase‐ and peroxidase‐like activity generating a substantial amount of ROS (, 1O2, ·OH) to cause oxidative damage to bacteria, also demonstrates nicotinamide adenine dinucleotide dehydrogenase‐like activity disrupting the bacterial respiratory chains, and subsequently impedes adenosine triphosphate production. CSPA exhibits favorable broad‐spectrum antibacterial activity at very low concentrations, prevents bacterial resistance, and completely inhibits bacterial biofilm formation. Antibacterial Mechanism of CSPA by the transcriptomics is further revealed that CSPA can induce bacterial oxidative stress, hinder bacterial energy metabolism, and disrupt the synthesis and function of bacterial cell walls and cell membranes. In vivo, CSPA inhibits the mixed bacterial population at the wound site and promotes wound healing in rats. This study introduces a novel antibacterial approach, providing important insight into the antibacterial mechanism of CSPA nanozymes and promoting the advancement of nanocatalytic materials in biomedical applications.

Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

Optimizations of lipid II synthesis: an essential glycolipid precursor in bacterial cell wall synthesis and a validated antibiotic target.

Lipid II is an essential glycolipid found in bacteria. Accessing this valuable cell wall precursor is important both for studying cell wall synthesis and for studying/identifying novel antimicrobial compounds. Herein, we describe optimizations to the modular chemical synthesis of lipid II and unnatural analogues. In particular, the glycosylation step, a critical step in the formation of the central disaccharide unit (GlcNAc-MurNAc), was optimized. This was achieved by employing the use of glycosyl donors with diverse leaving groups. The key advantage of this approach lies in its adaptability, allowing for the generation of a wide array of analogues through the incorporation of alternative building blocks at different stages of synthesis.

Antibacterial Potential of Tetrahydrocarbazoles (THCZ): A Review

Abstract: Antibiotic resistance has become a major public threat across the globe associated with human health. Some bacterial and fungal infections produce bacterial resistance, such as methicillin- resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE) and multidrug-resistant (MDR) species Acinetobacter baumannii etc. Tetrahydrocarbazoles (THCz) are a sub-class of indole alkaloids profoundly present in natural products and biologically active compounds and have displayed potential biological activities in literature. THCz exhibit potential antibacterial activities through major bacterial pathways like cell wall synthesis inhibition and DNA gyrase enzyme inhibition with DNA sliding clamp inhibitors and MreB inhibitors. These THCZ also showed significant in vitro antibacterial activities against bacterial-resistant species, such as MRSA, VRE and Acinetobacter baumannii (MDR) in literature. MTDL (Multi Target Direct ligand) approach has been significantly used for the design of THC motif-based antibacterial agents. In this review article, we collected literature on THCz as a potential antibacterial agent from 2014 to date. The review study of THC core-based derivatives found excellent in vitro antibacterial profiles and revealed that they can play a significant role in drug discovery and the development of new antibiotics against various infectious diseases.

Ultrastructural and morphological studies on variables affecting Escherichia coli with selected commercial antibiotics

BackgroundMany studies reported the effects of antibiotic exposure on E. coli bacterial growth and cell modification. However, scarce descriptive information on ultrastructural effects upon exposure of commercial antibiotics. MethodsThis study described the morphological and ultrastructural alterations caused by selected antibiotics (amoxicillin-clavulanate, ceftriaxone, polymyxin B, colistin, gentamicin, and amikacin) that targeted cell wall, plasma membrane, and cytoplasmic density, and also proteins synthesis. We determined extracellular morphological changes of exposure through scanning electron microscopy (FESEM) and intracellular activities through transmission electron microscopy (TEM) investigation. ResultsFESEM and TEM micrograph of E. coli exposed with selected antibiotics shows ultrastructural changes in beta-lactam class (amoxicillin-clavulanate, ceftriaxone) elongated the cells as the cell wall was altered as it inhibits bacterial cell wall synthesis, polymyxin class (polymyxin B, colistin) had plasmid and curli-fimbriae as it breaking down the plasma/cytoplasmic membrane, and aminoglycoside class (gentamicin, and amikacin) reduced ribosome concentration as it inhibits bacterial protein synthesis by binding to 30 s ribosomes. ConclusionMorphological and ultrastructural alterations of E. coli’s mechanism of actions were translated and depicted. This study could be reference for characterization studies for morphological and ultrastructural of E. coli upon exposure to antimicrobial agents.

A Review on the Use of Third-Generation Cephalosporins on Gram-Positive and Gram-Negative Bacteria Based on its Spectrum of Activity

This review explores the clinical usage of third-generation cephalosporins, a class of broad-spectrum antibiotics that are essential for treating gram-positive and gram-negative bacterial infections in a variety of infectious conditions. These antibiotics have bactericidal action by inhibiting the synthesis of bacterial cell walls. Among the well-known members of this class are cefoperazone, cefpodoxime proxetil, ceftibuten, ceftriaxone, cefotaxime, and ceftazidime. Inhibiting transpeptidase is the mechanism of action, which, in the case of gram-negative bacteria, results in cell lysis and death. Third-generation cephalosporins are used to treat a wide range of conditions, including meningitis, genitourinary tract infections, pneumonia, infections of the skin and soft tissues, and bone and joint infections. Each cephalosporin within this generation exhibits a distinct spectrum of activity. For instance, cefpodoxime proxetil acts against Staphylococcus aureus and Klebsiella pneumoniae, whereas cefixime acts against Streptococcus pneumoniae and Escherichia coli. Neisseria gonorrhoea and Pseudomonas aeruginosa are only two examples of the many gram-positive and gram-negative bacteria that ceftriaxone is active against. This review also highlights the synergistic effects of combining third-generation cephalosporins with other antibiotics, demonstrating their versatility in managing complex infections. Despite their effectiveness, third-generation cephalosporins may not be effective against Chlamydia trachomatis. Furthermore, careful consideration of each antibiotic's spectrum guides their specific use in treating conditions such as urinary tract infections, otitis media, bronchitis, and gynecologic infections. In conclusion, the comprehensive spectrum of action of third-generation cephalosporins makes them valuable in addressing a diverse array of bacterial infections. However, their use should be judicious, considering the evolving landscape of antibiotic resistance and the specific bacterial targets for optimal therapeutic outcomes.

Propene-bridged cyanurate tetramers decorated on carbon nanosheets with antibacterial activity: insights from molecular modeling and in vitro studies

Propene-bridged cyanurate tetramers (PBCTs) nested onto 2D nanosheets (CNHs) exhibit antibacterial activity by disrupting bacterial cell wall synthesis.

Metabolomics reveals the mechanism of action of meropenem and amikacin combined in the treatment of Pseudomonas aeruginosa.

The treatment of Pseudomonas aeruginosa infection often involves the combined use of β-lactam and aminoglycoside antibiotics. In this study, we employed metabolomic analysis to investigate the mechanism responsible for the synergistic activities of meropenem/amikacin combination therapy against multidrug-resistant P. aeruginosa strains harboring OXA-50 and PAO genes. Antibiotic concentrations for meropenem (2 mg/L) monotherapy, amikacin (16 mg/L) monotherapy, and meropenem/amikacin (2/16 mg/L) combination therapy were selected based on clinical breakpoint considerations. Metabolomic analysis revealed significant alterations in relevant metabolites involved in bacterial cell membrane and cell wall synthesis within 15 min of combined drug administration. These alterations encompassed various metabolic pathways, including fatty acid metabolism, peptidoglycan synthesis, and lipopolysaccharide metabolism. Furthermore, at 1 h and 4 h, the combination therapy exhibited significant interference with amino acid metabolism, nucleotide metabolism, and central carbon metabolism pathways, including the tricarboxylic acid cycle and pentose phosphate pathway. In contrast, the substances affected by single drug administration at 1 h and 4 h demonstrated a noticeable reduction. Meropenem/amikacin combination resulted in notable perturbations of metabolic pathways essential for survival of P. aeruginosa, whereas monotherapies had comparatively diminished impacts.

Antibacterial and biofilm prevention metabolites from Acanthophora spicifera

Abstract Acanthophora spicifera harbors a diverse array of secondary metabolites with therapeutic potential. The aim of this study is to isolate and characterize secondary metabolites from A. spicifera and then evaluate the antiproliferation, antibacterial, and biofilm prevention properties, followed by an analysis of molecular docking experiments. By employing chromatographic analysis and NMR spectroscopy, the isolated compounds were, the known flavonol, 8-hydroxyquercetagetin (1), three recognized steroids cholest-4-ene-3,6-dione (2), cholest-5-en-3β-ol (3), and 5α-cholestane-3,6-dione (4), and 2-bromohexadecanoic acid (5). These compounds exhibited antimicrobial effects against various Gram-negative and Gram-positive bacteria with inhibition zones ranging from 6.5 ± 0.2 to 17.2 ± 0.12 mm and 7.0 ± 0.4 to 15.3 ± 0.60 mm, respectively. Compounds 1 and 2 inhibited biofilm formation in P. aeruginosa and S. aureus. Compounds 1–4 demonstrated binding affinity values between −7.5 and −9.4 kcal/mol to protein 1A0G. These binding affinity values were akin to that of amoxicillin, implying that one potential antibacterial mechanism of action of these compounds may involve the inhibition of bacterial cell wall synthesis. All compounds showed no toxicity against Artemia salina and weak activity against Lymphoma and Lewis lung carcinoma cell lines with LD50 > 100 μg/mL.

Bacitracin enhances ceftriaxone susceptibility of the high-level ceftriaxone-resistant gonococcal FC428 clone

ABSTRACT Neisseria gonorrhoeae is a multidrug-resistant bacterial pathogen for which ceftriaxone mono-antimicrobial therapy and ceftriaxone plus azithromycin dual-antimicrobial therapy are the only remaining effective therapies. However, ceftriaxone efficacy is threatened by the global dissemination of the high-level ceftriaxone-resistant FC428 clone, while azithromycin has in recent years been removed from the recommended dual therapy in some countries as a result of increasing (high-level) azithromycin resistance. Inclusion of an alternative second antimicrobial could be an effective strategy to protect ceftriaxone from (further) resistance development. In this study, we investigated gonococcal susceptibility to bacitracin and determined its synergistic anti-gonococcal activity with ceftriaxone. Gonococcal susceptibility to bacitracin was investigated for 449 contemporary clinical isolates using the agar dilution method. Bacitracin displayed consistent activity against the N. gonorrhoeae strain collection, with a minimum inhibitory concentration (MIC) range of 2–32 mg/L and a MIC 90 of 32 mg/L. Furthermore, bacitracin was bactericidal in time-kill assays, particularly against the tested FC428-associated isolate, which showed 10 5 -fold inactivation after 8 hours of exposure to a 4× MIC dose. Importantly, bacitracin and ceftriaxone displayed synergistic activity, with a fractional inhibitory concentration index ≤0.5 for the majority of tested strains. Finally, spot assays showed strong synergistic activity against the FC428-associated isolates, with up to 200-fold reduced plating efficacy for bacitracin combined with ceftriaxone compared with ceftriaxone only. In conclusion, bacitracin shows synergistic anti-gonococcal activity with ceftriaxone and might therefore be an interesting candidate for inclusion in a dual-antimicrobial therapy with ceftriaxone. IMPORTANCE Ceftriaxone-based antimicrobial therapies for gonorrhea are threatened by waning ceftriaxone susceptibility levels and the global dissemination of the high-level ceftriaxone-resistant gonococcal FC428 clone. Combination therapy can be an effective strategy to restrain the development of ceftriaxone resistance, and for that purpose, it is important to find an alternative antimicrobial to replace azithromycin, which has recently been removed in some countries from the recommended ceftriaxone plus azithromycin dual-antimicrobial therapy. Ideally, the second antimicrobial should display synergistic activity with ceftriaxone. We hypothesized that bacitracin might display synergistic activity with ceftriaxone because of their distinct mechanisms targeting bacterial cell wall synthesis. In this study, we showed that bacitracin indeed displays synergistic activity with ceftriaxone against Neisseria gonorrhoeae . Importantly, strains associated with the FC428 clone appeared to be particularly susceptible to the bacitracin plus ceftriaxone combination, which might therefore be an interesting dual therapy for further in vivo testing.

Super-Resolution Microscopy of the Bacterial Cell Wall Labeled by Fluorescent D-Amino Acids.

Fluorescent D-amino acids (FDAAs) enable in situ visualization of bacterial cell wall synthesis via their incorporation into peptidoglycan (PG) crosslinks. When combined with super-resolution microscopy, FDAAs allow the details of cell wall synthesis to be resolved beyond the diffraction limit of visible light. Here, we describe using the super-resolution method of single-molecule localization microscopy (SMLM) in conjunction with two newly synthesized FDAAs (sCy5DA and sCy5DL_amide) to resolve bacterial PG at the nanoscale in a variety of species, including Gram-negative, Gram-positive, and mycobacteria.

Polypharmacological assessment of Amoxicillin and its analogues against the bacterial DNA gyrase B using molecular docking, DFT and molecular dynamics simulation

There has been an increase in the emergence and spread of drug-resistant pathogens, leading to a steep incline in the cases of antimicrobial resistance. Due to this, there is an imperative need for the development and identification of new antimicrobials to combat this menace of antimicrobial resistance. But this need is not being completely fulfilled by conventional drug discovery focused on a one molecule-one target approach. Polypharmacology, i.e., designing a drug in a way that acts on multiple cellular or molecular targets, a new approach for the identification of antimicrobial compounds, has been gaining attention. DNA gyrase B is one of the critical proteins involved in DNA replication and cell division in E. coli. In this study, the polypharmacological effect of amoxicillin and its analogues was studied on the DNA gyrase B and various other proteins of E. coli, using multiple in silico approaches like molecular docking, structural similarity, DFT, and molecular dynamics simulation. Both amoxicillin and its analogue, Cefaclor, tend to disrupt bacterial cell wall synthesis, but this study, based on in silico analysis, suggests a probable additional mode of action involving DNA gyrase B of E. coli which can be further explored to design novel dual-target inhibitors.

Penicillin-binding protein (PBP) inhibitor development: A 10-year chemical perspective.

Bacterial cell wall formation is essential for cellular survival and morphogenesis. The peptidoglycan (PG), a heteropolymer that surrounds the bacterial membrane, is a key component of the cell wall, and its multistep biosynthetic process is an attractive antibacterial development target. Penicillin-binding proteins (PBPs) are responsible for cross-linking PG stem peptides, and their central role in bacterial cell wall synthesis has made them the target of successful antibiotics, including β-lactams, that have been used worldwide for decades. Following the discovery of penicillin, several other compounds with antibiotic activity have been discovered and, since then, have saved millions of lives. However, since pathogens inevitably become resistant to antibiotics, the search for new active compounds is continuous. The present review highlights the ongoing development of inhibitors acting mainly in the transpeptidase domain of PBPs with potential therapeutic applications for the development of new antibiotic agents. Both the critical aspects of the strategy, design, and structure-activity relationships (SAR) are discussed, covering the main published articles over the last 10 years. Some of the molecules described display activities against main bacterial pathogens and could open avenues toward the development of new, efficient antibacterial drugs.

Synthesis of Peptidyl-tRNA Mimics for Structural Biology Applications.

ConspectusProtein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry.Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule-NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis.Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs.In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3'-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging.Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.

Unraveling the unexpected aggregation behavior of Pyrazole-Based compounds Targeting Mycobacterium tuberculosis UDP-Galactopyranose mutase

A pyrazole-based compound, MS208, was previously identified as an inhibitor of UDP-Galactopyranose Mutase from Mycobacterium tuberculosis (MtUGM). Targeting this enzyme is a novel therapeutic strategy for the development of new antituberculosis agents because MtUGM is an essential enzyme for the bacterial cell wall synthesis and it is not present in human. It was proposed that MS208 targets an allosteric site in MtUGM as MS208 followed a mixed inhibition model. DA10, an MS208 analogue, showed competitive inhibition rather than mixed inhibition. In this paper, we have used an integrated biophysical approach, including thermal shift assays, dynamic light scattering and nuclear magnetic resonance experiments, to show that MS208 and many analogues displayed unexpected aggregation behavior against MtUGM.

N4-Substituted Piperazinyl Norfloxacin Derivatives with Broad-Spectrum Activity and Multiple Mechanisms on Gyrase, Topoisomerase IV, and Bacterial Cell Wall Synthesis.

Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gram-negative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.

Cattle-compost-soil: The transfer of antibiotic resistance in livestock agriculture.

Antibiotic resistance is a major global health threat. Agricultural use of antibiotics is considered to be a main contributor to the issue, influencing both animals and humans as defined by the One Health approach. The purpose of the present study was to determine the abundance of antibiotic-resistant bacterial populations and the overall bacterial diversity of cattle farm soils that have been treated with animal manure compost. Soil and manure samples were collected from different sites at Tullimba farm, NSW. Cultures were grown from these samples in the presence of 11 commonly used antibiotics and antibiotic-resistant bacteria (ARB) colonies were identified. Soil and manure bacterial diversity was also determined using 16S ribosomal RNA next-generation sequencing. Results showed that ARB abundance was greatest in fresh manure and significantly lower in composted manure. However, the application of composted manure on paddock soil led to a significant increase in soil ARB abundance. Of the antibiotics tested, the number of ARB in each sample was greatest for antibiotics that inhibited the bacterial cell wall and protein synthesis. Collectively, these results suggest that the transfer of antibiotic resistance from composted animal manure to soil may not be solely mediated through the application of live bacteria and highlight the need for further research into the mechanism of antibiotic resistance transfer.