The discovery of penicillin revolutionized medicine, marking the beginning of the antibiotic era and driving advancements in medical research and pharmaceutical development [1]. However, the widespread and indiscriminate use of antibiotics has accelerated the emergence of multidrug-resistant (MDR) bacteria [2,3]. These MDR strains have led to increased mortality rates and soaring healthcare costs, posing a formidable challenge to global public health [4]. Klebsiella pneumoniae, a common Gram-negative bacterium of the Enterobacteriaceae family, is a major cause of nosocomial infections and often results in the failure of empirical antibiotic therapy [5,6]. Tigecycline, a glycylcycline antibiotic, is now considered a last-resort option for treating complex infections caused by MDR Gram-negative pathogens [7,8]. However, the emergence of the tmexCD1-toprJ1 gene cluster, which confers resistance to multiple antibiotic classes, including tetracyclines, cephalosporins, aminoglycosides, phenicols, and quinolones, poses a new challenge [9,10]. Epidemiologic investigations identified plasmid-encoded variants of transmissible RND efflux pump genes, such as tmexCD1-toprJ1 from chicken farm in China [9], tmexCD2-toprJ2 from diseased patients in China [11], and tmexCD3-toprJ3 in Klebsiella spp. from municipal sludge in Vietnam [12], suggesting that plasmid-mediated mechanisms for RND efflux are emerging and spreading globally on multiple scales. Indeed, the dissemination of the tmexCD-toprJ gene cluster and its variants represents a critical threat to global public health. Hence, there is an urgent need to monitor and control the spread of the tmexCD-toprJ gene cluster and explore effective strategies for combating tmexCD-toprJ-positive bacterial infections. A major challenge in combating antibiotic-resistant bacteria is the limited intracellular concentration of antibiotics, particularly in Gram-negative species, due to enhanced efflux or reduced influx [13]. Under antibiotic stress, bacteria can reallocate metabolic resources and alter metabolic pathway activities, directly affecting their susceptibility to antibiotics [14,15]. For instance, activating or inhibiting certain metabolic pathways can change bacterial growth rates and physiological states, thereby influencing antibiotic efficacy. Several studies have successfully reprogrammed bacterial metabolism using nucleotides and amino acids, focusing on enhancing pyruvate cycle metabolism to increase the uptake of antibiotics [16,17]. Metabolic reprogramming via the supplementation of exogenous metabolites offers a novel approach to enhance intracellular accumulation of antibiotics, thereby effectively reversing antibiotic resistance [[17], [18], [19], [20], [21], [22]]. In our previous study, we compared the metabolic profiles of tmexCD1-toprJ1-positive and -negative bacteria via metabolomics and identified 168 differentially abundant metabolites [23]. Specifically, we identified low abundance of cytidine, taurine, cytosine, glutathione, thymine, indole-3-acetamide, kynurenic acid, L-5-hydroxytryptophan, inosine, and lysine as a critical metabolic feature of bacteria carrying tmexCD1-toprJ1. While we previously demonstrated that inosine could potentiate tigecycline efficacy, the potential roles of other metabolites in reversing tmexCD1-toprJ1-mediated multidrug resistance remain to be explored.

An increasing body of evidence suggests that antibiotic adjuvant strategies have the potential to prolong the effectiveness of current antibiotics [24]. The function and underlying mechanisms of lysine as a co-adjuvant for tigecycline against K. pneumoniae harboring the tmexCD1-toprJ1 gene cluster remain to be fully elucidated. Thus, this study focused on tigecycline resistance driven by the efflux pump gene cluster tmexCD1-toprJ1, aiming to tackle the clinically significant issue of drug resistance in K. pneumoniae. This investigation highlights the potential of lysine to serve as a synergistic adjuvant for tigecycline in combating infections caused by tmexCD-toprJ-positive pathogens.