Tigecycline, the first member of glycylcycline class antibiotic, is often considered one of the effective antibiotics against multidrug-resistant (MDR) infections. However, the emergence and wide distribution of two novel plasmid-mediated tigecycline resistance genes, tet (X3) and tet (X4), pose a great threat to the clinical use of tigecycline.

The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against Escherichia coli was 32–64 µg/mL, including strains carrying bla_NDM-5, which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against E. coli by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene upp increased the MIC of 5-FU against E. coli by 32-fold, indicating the role of the upp gene in 5-FU resistance. Additionally, transcriptomic analysis of E. coli treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD⁺/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on E. coli through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.

It is crucial to discover novel antimicrobial drugs to combat resistance. This study investigated the antibacterial properties of halicin (SU3327), an AI-identified anti-diabetic drug, against 13 kinds of common clinical pathogens of animal origin, including multidrug-resistant strains. Employing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assessments, halicin demonstrated a broad-spectrum antibacterial effect. Time-killing assays revealed its concentration-dependent bactericidal activity against Escherichia coli ATCC 25922 (E. coli ATCC 25922), Staphylococcus aureus ATCC 29213 (S. aureus ATCC 29213), and Actinobacillus pleuropneumoniae S6 (APP S6) after 4 h of treatment at concentrations above the MIC. Halicin exhibited longer post-antibiotic effects (PAEs) and sub-MIC effects (PA-SMEs) for E. coli 25922, S. aureus 29213, and APP S6 compared to ceftiofur and ciprofloxacin, the commonly used veterinary antimicrobial agents, indicating sustained antibacterial action. Additionally, the results of consecutive passaging experiments over 40 d at sub-inhibitory concentrations showed that bacteria exhibited difficulty in developing resistance to halicin. Toxicology studies confirmed that halicin exhibited low acute toxicity, being non-mutagenic, non-reproductive-toxic, and non-genotoxic. Blood biochemical results suggested that halicin has no significant impact on hematological parameters, liver function, and kidney function. Furthermore, halicin effectively treated respiratory A. pleuropneumoniae infections in murine models. These results underscore the potential of halicin as a new antibacterial agent with applications against clinically relevant pathogens in veterinary medicine.

The emergence and worldwide dissemination of mobile tigecycline resistance genes tet(X3)/tet(X4) posed an enormous threat to the public health. Urgently, feasible strategies must be implemented to restore the clinical efficacy of tetracyclines and prolong the lifespan of existing drugs to address the emerging global antimicrobial resistance threat. Herein, versatile structural scaffolds of quinones for antibiotic adjuvants discovery enlightened a promising and underappreciated reservoir to circumvent the antibiotic resistance. 2-methoxy-1,4-naphthoquinone (MNQ) exhibited the potent potentiation (4 to 32-fold) with tetracyclines, along with effective inhibition on biofilm formation. Mechanistic studies revealed that MNQ synergistically operates with tetracyclines by inhibiting the enzymatic activity of Tet(X3)/Tet(X4) proteins through interaction with their active residues. Furthermore, exposure to MNQ significantly dissipate the proton motive force, leading to a cascade of membrane structural damage and metabolic homeostasis imbalance. Encouragingly, the MNQ-tetracyclines combination showcased substantial therapeutic benefits in two in vivo infection models, as evidenced by the reduced bacterial burden and mitigated pathological injury. Our findings propose a potential therapeutic option and a novel tetracyclines' adjuvant against drug-resistant pathogens carrying Tet(X3)/Tet(X4).

The global emergence and spread of carbapenem-resistant Enterobacteriaceae in clinical settings have driven the search for inhibitors that can counteract carbapenemases. These enzymes include several serine β-lactamases (SBLs, such as KPC) and metallo-β-lactamases (MBLs, such as NDM) that hydrolyse almost all β-lactams including carbapenems. This endeavour has successful developed some SBL inhibitors, including the boron-containing compound vaborbactam. However, the challenge posed by MBLs remains unresolved. A high-throughput screening was conducted on 1718 FDA-approved drugs as potential adjuvants to meropenem. The synergistic effect was determined by checkerboard assay. The underlying mechanisms were elucidated using enzyme inhibition assays, molecular docking and dynamics simulations. The safety and efficacy were evaluated using a murine model. We have identified another boron-containing broad-spectrum serine and metallo-β-lactamase inhibitor, the benzoxaborole antifungal agent tavaborole. In vitro, tavaborole enhances the antibacterial activity of multiple β-lactam antibiotics against bacteria producing either SBLs or MBLs. In vivo, injectable administration of tavaborole has demonstrated good safety in mice and has restored the efficacy of meropenem against bla_NDM-5 and bla_KPC-2-positive bacterial infection in a mouse intraperitoneal model. Tavaborole may effectively inhibit the activity of SBLs and MBLs by covalently bonding with the active serine residue of SBLs and chelating the Zn²⁺ at the active center of MBLs. Tavaborole shows good potential as an agent for use in combination with β-lactam antibiotics for treating multidrug-resistant Gram-negative bacterial infections. National Key Research and Development Program of China, National Natural Science Foundation of China, Pinduoduo-China Agricultural University Research Fund.

• Scientific questions: The widespread dissemination of the mobile tigecycline resistance gene tet(X4) poses a significant threat to public health, as it has been detected in multiple species of Enterobacterales across various sources, including animals, the environment, animal-derived food, and humans. However, the prevalence and spread of tet(X4) in the clinically important pathogen Klebsiella pneumonia (K. pneumonia) have not been well understood. • Evidence before this study: Previous research on tet(X4) primarily focused on its presence in Escherichia coli, with only sporadic reports of its detection in K. pneumoniae from different sources. However, in our previous study, we have already identified the presence of tet(X4) in clinical K. pneumoniae obtained from the feces of hospitalized patients. • New findings: In this study, we have made new findings regarding tet(X4)-positive K. pneumoniae isolates. We discovered a relatively high prevalence of multidrug-resistant tet(X4)-positive K. pneumoniae isolates in schools and markets. In addition, our investigation revealed that both clonal spread, indicating transmission within specific K. pneumoniae, and horizontal transfer, referring to the transfer of the tet(X4) gene between different strains, contribute to the spread of tigecycline resistance. These findings highlight the urgent need for appropriate surveillance and control measures to prevent further dissemination. • Significance of the study: This study underscores the importance of continuous surveillance of tet(X4)-positive K. pneumoniae, particularly in raw meat, as it can serve as a potential source of transmission. These findings emphasize the necessity of implementing effective measures to prevent the spread of tet(X4)-positive K. pneumoniae and to address the associated multidrug resistance.