The abundant gut commensal Bacteroides thetaiotaomicron is repeatedly challenged by β-lactam exposure in the human intestine, yet its β-lactamase repertoire and dissemination potential remain incompletely characterized. Here, we screened 626 publicly available B. thetaiotaomicron genomes and identified 1059 putative β-lactamase homologs, all belonging to class A or class D families. Four highly prevalent representatives-BTA-1 and CfxA-3 (class A) and OXA-347 and OXA-1327 (class D)-were prioritized for experimental validation. Heterologous expression in Escherichia coli revealed heterogeneous resistance phenotypes, with ones conferring only modest MIC increases (e.g., OXA-347, ~2-fold for amoxicillin) and others producing strong resistance to penicillins (e.g., BTA-1caused a 256-fold increase in amoxicillin MIC). Purified BTA-1 and OXA-347 were active under gut-relevant conditions, with pH optima at 7 and 8 and temperature optima at 40 °C and 30 °C, respectively. Notably, OXA-347 hydrolyzed representatives of penicillins, cephalosporins, carbapenems, and monobactams, and mass spectrometry confirmed β-lactam ring opening. Beyond B. thetaiotaomicron, these β-lactamase alleles were detected across multiple gut commensal genera as well as taxa annotated as opportunistic pathogens. Genomic context analyses showed similar gene-cluster patterns in B. thetaiotaomicron, across gut commensal genera, and in opportunistic-pathogen-associated taxa. Collectively, our findings clarify the diversity, activity, and distribution of B. thetaiotaomicron-associated β-lactamases across the gut microbiome and clinically relevant taxa.

Antibiotic resistance has emerged as a critical global public health challenge. Quorum sensing (QS), a density-dependent regulatory mechanism, plays a pivotal role in bacterial pathogenesis by coordinating virulence factor expression, making it a critical target for antivirulence therapy. Leveraging a drug repositioning strategy, this study investigated the antivirulence potential of drugs in the database of DrugBank on the common opportunistic pathogen Pseudomonas aeruginosa by virtual screening. Molecular docking analysis predicted that the antitumor drug, Tirazone, could bind to the core QS regulatory proteins, LasR, RhlR, and PqsR of P. aeruginosa with abundant active sites, whereas the binding free energies were higher than those of the native QS signals. In vitro experiments demonstrated that Tirazone significantly suppressed virulence factor secretion, cell motilities, and biofilm formation in the model P. aeruginosa strain PAO1, and downregulated the expression of a series of QS-related genes with low effective concentration (≤ 8 μM). A competitive binding model of QS signal molecules further elucidated that Tirazone interfered with QS signaling by competitively inhibiting the function of LasR, RhlR, and PqsR. Additionally, Tirazone treatment significantly protected Caenorhabditis elegans and mouse models from P. aeruginosa infection, and reduced the bacterial loads and pathological lesions in mouse lungs. Moreover, Tirazone demonstrated synergistic effects with polymyxin B, levofloxacin, and amikacin, significantly enhancing their bactericidal efficacy in treating P. aeruginosa. This study reveals the molecular mechanism underlying Tirazone's multi-target intervention in the QS system, and provides an experimental foundation for developing combination therapies based on antivirulence strategies.

Klebsiella pneumoniae causes severe respiratory-associated infections in healthy individuals, and widespread antibiotic resistance in K. pneumoniae poses a global health threat. Although great efforts have been taken to develop effective vaccines against K. pneumoniae, there are no licensed vaccines against K. pneumoniae available up to date. In the current study, we designed a potent subunit vaccine OmpW, which was obtained from an outer membrane protein (OmpW) to maximize host responses in mice. Antibody-mediated responses and protective effects were determined to compare the immunogenicity of the OmpW subunit vaccine in the vaccinated mice. Immunization experiments showed that mice immunized with recombinant OmpW generated high IgG antibody production, which promoted macrophage opsonophagocytic activity and induced strong serum bactericidal activity. The residual bacterial burdens from the different organs were declined in the immunized mice compared with the control group. Notably, immunization with the OmpW subunit vaccine provided better protection against K. pneumoniae challenge in vaccinated mice. The passive transfer of antiserum from OmpW-immunized mice also provided protection against K. pneumoniae infection. This study constitutes an important step toward developing potent antibacterial vaccines against K. pneumoniae infections and prevents further dissemination of K. pneumoniae strains.

The global dissemination of antibiotic resistance genes (ARGs) across diverse environments has emerged as a critical challenge to public health. As essential primary producers, Cyanobacteria colonize extreme and heterogeneous habitats, coexisting with gut microbiota in wastewater, marine ecosystems, and reservoirs, where they may potentiate the proliferation and transmission of ARGs under antibiotic selective pressures. In this study, three macrolide esterases (NOD-1, OCA-1, and OCB-1) of Cyanobacterial origin were identified through mining of local genomic repositories. These enzymes, classified as serine-dependent alpha/beta -hydrolases, were experimentally validated through antimicrobial susceptibility testing and zone of inhibition assays to inactivate specific 16-membered macrolide antibiotics. Comparative analysis of genomic regions flanking these resistance determinants revealed the presence of mobile genetic elements (MGEs) and co-localized multidrug resistance genes, strongly suggesting the likelihood of horizontal gene transfer (HGT) within Cyanobacterial populations. Such genetic mobility may exacerbate antibiotic resistance dissemination in aquatic ecosystems, underscoring the ecological risks posed by Cyanobacteria as reservoirs and vectors of ARGs.

Atropisomeric architectures with a chiral axis are prevalent in biologically active molecules and natural products, and they are widely put to use in privileged catalysts and chiral ligands. Axially chiral biaryls and heterobiaryls are a fundamental category of atropisomeric compounds with inherent barriers caused by rotational restrictions around carbon–carbon, carbon–nitrogen or nitrogen–nitrogen axes. In this regard, the synthesis of biaryls/hetero-biaryls has witnessed substantial progress, despite the generally complicated traditional synthetic methods. Recent developments in the transition metal-catalyzed substrate activation and subsequent annulation reaction offer a straightforward approach to the preparation of various cyclic (hetero)biaryl atropisomers. In this review, we would like to present recent research advancements in transition metal-catalyzed enantioselective annulation reactions towards (hetero)biaryls featuring atropisomeric optical activity. The focus will be on mechanistic investigations, reaction limitations, and synthetic applications. Additionally, the combination of developing synthetic strategies and representative frameworks is discussed, along with some insights into the developing trend.

Antimicrobial resistance is a critical global public health challenge, with drug-resistant infections contributing to more than one million deaths annually. The widespread dissemination of multidrug-resistant bacteria poses a severe threat to the management of infectious diseases. Bacterial evolution via genetic mutation and horizontal gene transfer diminishes antimicrobial efficacy, often leading to therapeutic failure, increased morbidity and mortality. However, the development of novel antibiotics lags far behind the rapid evolution of drug-resistant bacteria. Therefore, scientists worldwide have committed to exploring alternative therapeutic strategies for bacterial infections. The key question is which strategy holds the greatest promise of addressing the predicament of traditional antibiotics and being recognized as “next-generation anti-infective drugs”. This narrative review summarizes several of the most promising alternative treatment strategies against bacterial infections, emphasizing the core strengths and limitations of each strategy. A critical comparative analysis reveals that no single strategy can simultaneously satisfy the demands of acute therapy, broad patient coverage, and resistance evasion, underscoring the need for context-dependent and sequential deployment. Moreover, among these alternatives, anti-virulence therapeutic strategies, particularly those targeting the bacterial quorum sensing (QS) system, represent a major and extensively studied approach, although their clinical translation remains nascent. This review delineates the molecular mechanisms and therapeutic potential of QS-targeting anti-virulence agents. Furthermore, we candidly assess the extant biological, pharmacological, and clinical barriers impeding their clinical translation, providing perspectives on future research directions to harness these next-generation anti-infective paradigms effectively.

Acinetobacter baumannii is a notorious clinical pathogen that predominantly causes nosocomial infections. The multidrug resistant Acinetobacter baumannii clinical isolates are becoming more prevalent on a global scale. In this study, we aimed to determine the potential synergistic antibacterial activity of contezolid in combination with polymyxin B nonapeptide (PBNP) against various A. baumannii strains. Contezolid, a commonly-used agents targeting Gram-positive bacteria, in combination with PBNP (CP) was used to explore the potential synergistic antibacterial activity against A. baumannii in vitro and Caenorhabditis elegans models. We found that CP treatment exhibited a strong synergistic effect on A. baumannii or multidrug resistant A. baumannii. CP also inhibited the biofilm formation and alter morphology of A. baumannii compared to the control group. Moreover, the residual bacteria were significantly decreased in the CP-treated murine alveolar macrophages (MH-S). Importantly, CP treatment improved the survival of C. elegans compared to the control or monotherapy group. This strategy improved antibacterial activity spectrum of agents targeting Gram-positive bacteria like contezolid. Nevertheless, CP combination therapy requires further validation in mammalian infection models.

Asymmetric transition metal catalysis is of great significance for preparing chiral pharmaceuticals and natural products through inert chemical bond cleavage and new carbon-carbon and carbon-heteroatom bond construction, generally exhibiting good compatibility with various functional groups. Electrochemical synthesis utilizing electric current as the energy source has emerged as an eco-friendly and safe methodology to promote redox reactions. The combination of asymmetric transition metal catalysis and electrochemistry has provided a mild alternative to access enantio‑enriched compounds through a straightforward pathway that is often hard to realize by traditional methodologies. There remain, however, some challenging problems in the field of such integration owing to the formation of highly reactive intermediates, leading to undesired byproducts and low stereoselectivity. Therefore, in this review, we focus on the mechanistic insights involving the activation pathways and bond generation procedures of these methodologies. Moreover, these approaches are illustrated by the representative examples and analyzed by detailed cyclovoltametric investigations. We hope this review could contribute to a better understanding and appreciation of asymmetric electrochemical synthesis catalyzed by transition metals.

Macrolides are widely used in both human and veterinary medicine, and enzymatic inactivation represents an important yet underexplored resistance mechanism beyond the well-studied Ere family. Here, we functionally characterized ECO-1, an Est-type macrolide esterase identified from Escherichia coli. Phylogenetic analysis placed ECO-1 as a distinct branch within the α/β-hydrolase superfamily, showing low sequence identity (37.8–46.7%) to previously characterized macrolide esterases. Heterologous expression of eco-1 in E. coli selectively increased resistance to 16-membered macrolides, with marked MIC elevation for tylosin (32-fold), tildipirosin (8-fold) and tilmicosin (4-fold), while showing minimal effects on 14- and 15-membered macrolides. Consistently, IC₅₀ values for five 16-membered macrolides (tylosin, tilmicosin, tildipirosin, spiramycin and kitasamycin) increased in ECO-1-expressing strains. Using purified recombinant ECO-1, agar diffusion assays demonstrated substantial loss of antibacterial activity after enzyme treatment. ESI-MS further confirmed hydrolysis products with a characteristic + 18 Da mass shift for all five substrates, supporting ester bond cleavage as the inactivation mechanism. Genomic context analysis revealed eco-1 embedded in a conserved multidrug resistance region associated with Tn3-family transposition and co-localized with clinically relevant resistance determinants including blaTEM-1 and tetracycline resistance genes (tet(B)/tetR(B)/tet(C)). A large-scale database search identified 136 ECO-1—positive genomes across 14 countries, predominantly in E. coli and Salmonella enterica, spanning animal, food, human, and environmental sources. Collectively, ECO-1 expands the repertoire of Est-type macrolide esterases with selective activity toward 16-membered macrolides and highlights the potential dissemination risk associated with mobile resistance gene clusters.

The Minisci-type reaction is arguably one of the most straightforward methods for constructing C–C bonds through functionalization of heteroarenes. This radical-based strategy has been particularly valuable for preparing polysubstituted N-heterocycles and has garnered significant attention due to the predictability of the reaction outcomes based on the properties of the carbon-centered radicals and heteroarenes involved. In recent years, a novel approach has emerged: light-induced Minisci-type transformations, where C-H functionalization reactions can be promoted under mild conditions. Notably, direct decarboxylative reactions via photocatalysis have been developed without the need for pretreatment of the reaction partners, resulting in highly economical and efficient procedures. This review summarizes recent advancements in photoinduced decarboxylative Minisci-type reactions, including mechanistic explorations of the formation of diverse radicals. Moreover, these transformations are categorized by the types of radical precursors used. The potential applications and existing limitations of each protocol are also discussed in detail, aiming to provide a comprehensive toolbox and guidance for drug discovery.

Reversible alterations at DNA sequence or epigenetic levels can result in phenotypes that are unstably inherited. The reversibility of these inheritable changes might be uniquely beneficial for adaption to possible fluctuations in environment. However, unstable changes are always ignored for the genetic instability in traditional studies, especially in the cause of drug resistance. In this study, we conduct a specific genetic screen in fission yeast using rapamycin (+caffeine) and obtain 173 resistant isolates. In contrast to the common strategy of isolating stable genetic mutants, we passage the cell culture with rapamycin resistance on drug free condition and test the resistance of offspring every five days, and obtain 14 strains that exhibit unstable resistance to rapamycin (the drug resistance is lost randomly among the cell progenies without drug selection pressure). Further studies show that the unstable genetic resistance of some strains is regulated by reversible DNA sequence alterationat the ssp1 gene locus. This study provides new insights and relevant scientific basis for the regulatory mechanism of unstable drug resistance in the process of rapamycin as a clinical anti-tumor drug, and a new possible target for solving the problem of drug resistance.

Akkermansia muciniphila, a member of the Verrucomicrobiota phylum, is recognized as a key gut microbe and has emerged as a potential next-generation probiotic. Assessment of antibiotic resistance in probiotics is a prerequisite for their application, while very few is studied in Akkermansia species. To address this, eight representative class A (3-lactamases (36.90 %-41.30 % identity with known (3-lactamases) from the Akkermansia species were screened and found to increase the minimum inhibitory concentration (MIC) of Escherichia coli to (3-lactams (2-1024 fold). Secondly, four (3-lactamases were successfully purified and identified as extended-spectrum (3-lactamase because they exhibited hydrolase activity against (3-lactams from penicillin, cephalosporins, and monobactam classes. Based on predicted three-dimensional structure, we hypothesized and validated that serine at 51 position was catalytic amino acid. Thirdly, the genomic context analysis revealed the absence of mobile genetic elements or other antibiotic resistance genes surrounding (3-lactamase genes, suggesting they may not be transferable. This study provides a foundational basis for the safety evaluation of Akkermansia species as probiotics.

Polymicrobial infections are recognized as a direct cause of exacerbation in chronic respiratory infections. In patients with reduced lung function, the transfer of oral microbiota to the lungs is often higher than in healthy individuals. However, the mechanisms underlying the interaction between dominant pathogens and oral microbiota, as well as their relationship with antibiotics, remain poorly understood. Using Pseudomonas aeruginosa and Streptococcus salivarius, co-isolated from the bronchial fluid of chronic obstructive pulmonary disease (COPD) patients, as a model, we investigated their interspecies invasion dynamics under antibiotic intervention. This study employed a combination of phenotypic and transcriptomic analyses to elucidate interaction dynamics under antibiotic intervention, providing insight into the gene expression and virulence impacts of antibiotics during co-infection. Our findings revealed that P. aeruginosa significantly inhibited the growth of S. salivarius on blank agar plates. However, in the presence of the β-lactam antibiotic aztreonam, at a sub-inhibitory concentration, the quorum sensing (QS) system of P. aeruginosa was suppressed, and genes related to growth and metabolism were downregulated. Conversely, the intervention of aztreonam activated S. salivarius in co-culture, enabling its successful invasion of P. aeruginosa. Although S. salivarius exhibited inhibitory activity against P. aeruginosa, antibiotic intervention played a critical role in reversing their inherent competitive relationship. This study suggests that the repeated use of antibiotics may contribute to the persistent coexistence of polymicrobial communities within the host, leading to complex microbial dynamics that impact disease progression. It underscores the critical need to explore bacterial interactions in detail, as understanding these relationships could revolutionize our approach to chronic diseases caused by polymicrobial infections, offering new therapeutic strategies that go beyond traditional pathogen-targeted treatments.

As antibiotic resistance escalates into a global health crisis, novel therapeutic approaches against infectious diseases are in urgent need. Pseudomonas aeruginosa, an adaptable opportunistic pathogen, poses substantial challenges in treating a range of infections. The quorum-sensing (QS) system plays a pivotal role in orchestrating the production of a large set of virulence factors in a cell density-dependent manner, and the anti-virulence strategy targeting QS may show huge potential. Here, we present a comprehensive investigation into the potential of the synthesized compound 3-(benzo[d][1,3]dioxol-4-yl)oxazolidin-2-one (OZDO, C10H9NO4) as a QS inhibitor to curb the virulence of P. aeruginosa. By employing an integrated approach encompassing in silico screening, in vitro and in vivo functional identification, we elucidated the multifaceted effects of OZDO. Molecular docking predicted that OZDO interfered with three core regulatory proteins of P. aeruginosa QS system. Notably, OZDO exhibited significant inhibition on the production of pyocyanin, rhamnolipid and extracellular proteases, biofilm formation, and cell motilities of P. aeruginosa. Transcriptomic analysis and quantitative real-time PCR displayed the down-regulation of QS-controlled genes in OZDO-treated PAO1, reaffirming the QS-inhibition activity of OZDO. In vivo assessments using a Caenorhabditis elegans-infection model demonstrated OZDO mitigated P. aeruginosa pathogenicity, particularly against the hypervirulent strain PA14. Moreover, OZDO in combination with polymyxin B and aztreonam presented a promising avenue for innovative anti-infective therapy. Our study sheds light on the multifaceted potential of OZDO as an anti-virulence agent and its significance in combating P. aeruginosa-associated infections.

On the basis of the co-culture strategy, five previously undescribed S-bridged pyranonaphthoquinones, crepidamycins A-E (1-5) and five known analogues (6-10) were isolated from a medicinal plant endophytic Streptomyces sp. MG-F-1 in Dendrobium crepidatum with Bacillus cereus MG-1. The structures and absolute configurations of 1-5 were elucidated by the interpretation of data from detailed spectroscopic analysis and electronic circular dichroism spectra, together with consideration of the biogenetic origins. Interestingly, these previously undescribed compounds were only found in the co-cultures and absent from the pure culture controls. Compounds 1-10 were assayed for their anti-inflammatory potential using LPS-stimulated RAW264.7 cells, of which compound 4 showed strong nitric oxide inhibitory effect with an IC50 of 0.22 ± 0.16 μM. The results of extracellular acidification rate and molecular docking suggest that it may play a role by regulating PKM2-mediated glycolysis.

Macrolide antibiotics are vital for controlling infections in humans, animals, and agriculture, yet their effectiveness is increasingly compromised by antimicrobial resistance. Macrolide esterases (MLEs) are key mediators of macrolide resistance but have only been detected in Gram-negative bacteria, with no evidence in Gram-positive species. Here, we mined over 500 000 Gram-positive genomes and identified 8707 candidate proteins. Six representative MLEs were functionally validated, conferring resistance to 16-membered macrolides and increasing minimum inhibitory concentrations (MICs) up to 16-fold in Escherichia coli and 128-fold in Bacillus subtilis. Moreover, two exhibited broad-spectrum activity against all clinically and veterinary relevant 16-membered macrolides. Temporal analysis revealed that Gram-positive MLEs originated at least 2.7 million years ago, contrasting with their emergence in Gram-negative bacteria after the introduction of antibiotics. Genomic surveys further demonstrated the global distribution of MLE-carrying Gram-positive bacteria across 97 countries and diverse ecosystems, including clinical, food, agricultural, and natural environments. These findings highlight Gram-positive MLEs as an underrecognized risk and underscore the need for a One Health-oriented strategy to monitor, assess, and mitigate the spread of macrolide resistance across interconnected ecosystems.

AIM:Pseudomonas aeruginosa employs the quorum sensing (QS) system, a sophisticated cell-to-cell communication mechanism, to modulate the synthesis and secretion of a range of virulence factors, which contribute to the establishment of acute or chronic infections in hosts. This study seeks to attenuate the virulence of P. aeruginosa by inhibiting the QS system, thereby reducing its pathogenicity as a promising alternative to traditional antibiotics. METHODS AND RESULTS:Two compounds with an amino-substituted diphenyl fumaramide core, N1-(4-bromophenyl)-N4-(4'-oxo-3',4'-dihydro-1'H-spiro [cyclopentane-1,2'-quinazolin]-6'-yl) fumaramide (10D) and N1-(3-chloro-4-fluorophenyl)-N4-(4-oxo-3,4,4',5'-tetrahydro-1H,2'H-spiro [quinazoline-2,3'-thiophen]-6-yl) fumaramide (12A), were identified through in-silico screening. The QS inhibitory potential of both compounds was explored in vitro and in vivo. In in vitro experiments, neither compound exhibited bactericidal effects but significantly inhibited the production of QS-regulated extracellular protease and pyocyanin. Quantitative PCR analysis revealed that QS-activated genes and downstream virulence genes were transcriptionally suppressed by 10D or 12A. Molecular docking and molecular dynamics simulations predicted stable interactions between these compounds and the key QS regulators LasR and PqsR. When combined with polymyxin B, kanamycin, and levofloxacin, 10D and 12A exhibited synergistic antibacterial activity. Furthermore, compounds 10D and 12A significantly improved the survival of mice challenged with P. aeruginosa and effectively reduced the bacterial load in the lungs. CONCLUSION:This study indicates that 10D and 12A possess considerable QS inhibitory potential, effectively attenuating the pathogenicity of P. aeruginosa. Moreover, the study offers structural insights and methodological guidance for the advancement of anti-virulence drug development.

Antibiotics, particularly β-lactams, are emerging environmental contaminants that induce antibiotic-resistant bacteria or genes, which threaten public health. Enzymatic degradation is an environmentally friendly approach to reducing residual antibiotics; however, its application is limited by enzyme's narrow substrate spectrum, which cannot deal with the co-occurrence of multiple β-lactam classes in the environment. To address this issue, four representative β-lactamases were biochemically characterized and two of them (CTX-33 to VIM-1) were selected because of their broader substrate spectrum and better stability. Compared to a single enzyme that can only degrade the ring structure of three classes of β-lactam antibiotics at most, the enzyme cocktail (with a ratio of CTX-33 to VIM-1 of 4:1) was able to hydrolyze nineteen antibiotics from four different β-lactam families. In laboratory experiments, the enzyme cocktail demonstrated degradation efficiencies of over 99% for the four classes of β-lactams (penicillin, cephalosporin, carbapenem, and monobactam) within the concentration range of 1 to 100 mg/L, both individually and simultaneously. Moreover, when applied to pharmaceutical industry wastewater, pig farm wastewater, and river water, over 99% simultaneous degradation of β-lactams was observed at 1 mg/L within 60 min and at 100 mg/L within 5 h. This is the first report of an enzymatic method capable of simultaneously removing antibiotics from four β-lactam classes. This study offers a novel and eco-friendly approach to β-lactam degradation.

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P seud omon as aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa . Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P . aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P . aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P . aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P . aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. Key points • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.