Introduction

The recent rapid emergence of antibiotic-resistant bacteria has created a global public health crisis1,2,3 that can be attributed to the misuse and overuse of antibiotic drugs in medical, veterinary, and agricultural practice and food products. During 2019 alone, antibiotic-resistant microorganisms were responsible for 4.95 million human fatalities in 204 countries and territories4. By the year 2050, as many as 10 million deaths per year could be caused by antibiotic-resistant microorganisms, if no effective measures are developed in the interim5. The infectious Diseases Society of America (ISDA) called attention to the six so-called multidrug-resistant ESKAPE pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. They are considered to be the most critical deadly antibiotic-resistant pathogens threatening human health, while acknowledging that other emergent multidrug-resistant bacteria are concerning, if less concerning than the ESKAPE pathogens6. Although the search for novel antibiotic drugs is ongoing, avenues of research that proceed conventionally are expected to produce drugs which will eventually become ineffective due to ongoing evolution of drug-resistant strains. Although we expect to eventually prevent or reduce the socioeconomic burden due to the ongoing spread of antibiotic-resistant microbes, the research on unconventional antibacterial agents or strategies is limited and progressed far too slowly7. Therefore, innovative strategies and effective agents must be rapidly approved and brought to market8, because they are urgently needed to stem the emergent healthcare crisis and prevent massive human suffering caused by antibiotic-resistant bacteria.

Recently, a promising novel strategy on nutrient metabolites has emerged that is based on either improved understanding of bacterial metabolism or the antibiotic-resistant metabolome/metabolic state; they are known as “metabolite-driven” and “metabolic state-driven” approaches, respectively9,10,11,12,13. The metabolite-driven approach relies on empirical data showing that specific exogenous nutrient metabolites potentiate the lethal effects of known antibiotic drugs9. The metabolic state-driven is typically based on metabolome profiling/metabolic state to characterize the antibiotic-resistant metabolome/metabolic state, identify putative metabolic mechanisms underlying antibiotic-resistance, and select critical nutrient metabolites as metabolic reprogramming agents10,11,12. The major difference between the ‘metabolite-driven’ and ‘metabolic state-driven’ approaches is how to determine the metabolic reprogramming agents. This knowledge is then used to develop a protocol for normalizing or reprogramming the antibiotic-resistant metabolome/metabolic state, which potentiates the lethal effects of antibiotic drugs10,12,13. Both of these approaches increase the permeability of the bacterial membrane, stimulate drug uptake, increase the achievable intracellular concentration of antibiotic drugs and thereby increase bactericidal efficacy (See Fig. 1)9,10,12. It is worth noting that this strategy potentiates the efficacy of known antibiotics10,12,14, including β-lactam antibiotics12, but the mechanism is very different from the mechanism by which β-lactamase inhibitors potentiate cell killing (i.e. by inhibiting β-lactamase-mediated cleavage of the β-lactam ring of the drugs)15. This review discusses recent progress in developing metabolic state-driven reprogramming in combination with known antibiotic agents, as an effective approach for restoring susceptibility in multidrug-resistant bacteria, while also providing an overview of metabolite-driven approach.

Fig. 1: Antibiotic-resistant metabolic state is reprogrammed into antibiotic-sensitive metabolic state to be susceptible for antibiotic-mediated killing.
figure 1

Antibiotic-resistant and -sensitive bacteria have respective specific metabolic profiles, designated as antibiotic-resistant and -sensitive metabolic states, which are responsible for low and high antibiotic uptake, respectively. The antibiotic-resistant metabolic state can be reverted into an antibiotic-sensitive metabolic state by metabolic reprogramming mediated by metabolic reprogramming agents to increase membrane permeability. On the other hand, antibiotic resistance is attributed to diverse mechanisms including decreased membrane permeability, expression of chromosomal β-lactamase, high rate of active drug efflux, or mutations that prevent or block antibiotic-mediated inactivation of its intracellular target. The combined impact on drug uptake, degradation, and efflux determines the net effect on the steady-state intracellular drug concentration. High uptake, which rate exceeds the combined rate of drug degradation and efflux, causes high intracellular drug concentration that is lethal to antibiotic-resistant bacteria, whereas low uptake, which rate is a bit higher than the combined rate of drug degradation and efflux, leads to low intracellular concentration that is nonlethal to them. Because of the high uptake, bacterial survival decreases dramatically at or above the MIC of the selected antibiotic. Therefore, antibiotic-mediated killing is greatly elevated. Green, sensitive metabolic state; Pale red, resistant metabolic state.

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Rationale for metabolic state-driven as an approach for restoring susceptibility in multidrug-resistant pathogens

Recent findings demonstrate that the local environment modulates bacterial drug uptake and other aspects of bacterial metabolism10,11,16,17,18. For example, the abundance of cellular indole, adenosine monophosphate, and nutrient metabolites influences transmembrane proton motive force (PMF), drug and nutrient uptake, and bacterial survival in the presence of antibiotic drugs. It is well-recognized that exogenous sugars or other bacterial carbon sources stimulate uptake and potentiate the bactericidal effects of aminoglycosides on both gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) persisters and biofilms9. Therefore, metabolic reprogramming agents from the empirical data can be effective against antibiotic-resistant bacteria19,20,21,22,23,24, which is different from metabolic state-driven approach that is based on the systemic analysis of global metabolic state.

Metabolic state-driven approach involves identifying and quantifying intracellular metabolites and the anabolic and catabolic biochemical pathways by which they are interconverted and interrelated12,25,26,27,28. Because of the degree to which metabolic pathways are inter-connected, overabundance or scarcity of a crucial metabolite can profoundly alter the metabolic state of a unicellular organism29,30. However, this also means that, in some cases, metabolic imbalances can readily be identified and corrected, simply by appropriate forms of complementation. As it turns out, an abundant exogenous source of a single nutrient can in some cases restore metabolic balance, normalize metabolic defects, and revert the antibiotic-resistant phenotype to antibiotic sensitivity10,14,30. In practice, the metabolic state-driven approach is carried out by metabolic reprogramming that involves four consecutive steps. First, metabolome profiles are determined and analytical molecular biological and biochemical analyses are performed to characterize and compare bacterial metabolomes and their correlation with the phenotype(s) of interest, in this case antibiotic-resistance or antibiotic-sensitivity. Second, crucial metabolites linked to the phenotype are identified and their potential as biomarkers and/or metabolic reprogramming agents is tested. Third, the nutrient metabolites are fully characterized, in this case, for their capacity and efficacy in restoring antibiotic susceptibility to a drug-resistant bacterial pathogen. Finally, metabolic reprogramming mechanisms are revealed8,10. Therefore, the metabolic state-driven approach identifies metabolic reprogramming agents by systemic analysis of the global metabolic state and thereby highly effective metabolic reprogramming agents can be determined.

Conceptually, a metabolic reprogramming agent potentiates the efficacy of an antibiotic in the same manner as an adjuvant potentiates the immunogenicity of an antigen. Interestingly, some membrane permeabilizing detergents, surfactants, and chelating agents act as antibiotic adjuvants31,32, demonstrating potent synergistic effects against several bacterial pathogens33. However, these membrane permeabilizing agents are not nutrient metabolites, play no essential metabolic role, and in fact they typically impair membrane function. Therefore, these membrane permeabilizing agents are not discussed further in this review.

Evidence for the efficacy of metabolic state-driven approach to revert antibiotic resistance

In a report published in 2011, James Collins’ and colleagues showed that specific nutrient carbon sources potentiate the lethal effects of aminoglycosides on gram-negative (E. coli) and gram-positive (S. aureus) persisters and biofilms in vitro and in vivo, with a reported 100-10,000-fold increase in killing efficiency in vitro9. Since the report from Collins’ laboratory, evidence has continued to accumulate showing that bacterial metabolic environment can antagonize or synergize with small molecule antibiotic drugs, decreasing or increasing their bactericidal efficacy, respectively34,35,36,37,38,39. This phenomenon, known as metabolite-driven approach, can be effected by carbon-containing nutrient metabolites. Until now, the tested carbon-containing nutrient metabolites include amino acids, sugars, nucleotides, and peptides. Amino acids threonine, glycine, and glutamine potentiate aminoglycosides to kill methicillin-resistant Staphylococcus aureus (MRSA)40,41, while arginine, leucine, and lysine promote gentamicin, sarafloxacin, tetracycline, and β-lactam, respectively, against gram-negative bacteria42,43,44. Sugar D-ribose/nucleotides (uracil adenosine monophosphate) and peptide glutathione are demonstrated to have the similar potentiation through sensitizing aminoglycosides and meropenem, respectively45,46,47,48. Fatty acids also modulate the efficacy of aminoglycosides49. Supplementation of stationary-phase cultures of E. coli, Staphylococcus aureus, and Mycobacterium smegmatis with glucose and a suitable terminal electron acceptor to stimulate respiratory metabolism is sufficient to sensitize cells to quinolone killing50,51. Recent studies also show that NO-releasing polymeric substrates and silver nanomaterials with petal-like structures promote uptake of aminoglycosides and/or norfloxacin22,52. The metabolism of the gut microbiome can also modulate antibiotic efficiency, as noted in high-fat diet-fed mice, such that the tryptophan metabolite indole-3-acetic acid promotes antibiotic efficacy against persisters53. In summary, metabolite-driven approach appears to be a promising approach for improving the efficacy of antibiotics against multidrug-resistant strains of bacteria.

Metabolic state-driven approach is an efficient strategy for identifying metabolic imbalances and nutrient metabolites that have potential as metabolic reprogramming agents10. This is because metabolic state determines antibiotic resistance and sensitivity10,12,54. In some cases, a high extracellular concentration of a crucial metabolite is sufficient to restore antibiotic sensitivity to antibiotic-resistant cells and stimulate drug uptake10,11. Peng et al. reported GC-MS data showing that low intracellular glucose and alanine are biomarkers of kanamycin resistance in Edwardsiella tarda10. Furthermore, exposure to exogenous alanine, glucose, fructose, and alanine/glucose restored susceptibility of multidrug-resistant E. tarda to kanamycin by a mechanism involving activation of the pyruvate cycle (the P cycle), increased NADH production and PMF, and increased drug uptake10,55. This pioneering research has laid the foundation for reprogramming metabolomics/metabolic state10,11. Other reports document successful metabolic reprogramming of antibiotic-resistant pathogenic bacteria26,55,56. Zhang et al. demonstrated that intracellular glucose, redox state, membrane potential, ROS, and drug uptake are suppressed in gentamicin-resistant Vibrio alginolyticus relative to control cells14. However, exogenous glucose reprogramed these cells and stimulated/promoted the P cycle, NADH, PMF, ROS, and gentamicin uptake, reverting the phenotype of gentamycin-resistance to gentamycin-sensitivity30. In addition, reduced uptake of gentamicin in a manner of the Na+-NQR system was normalized in a cAMP/CRP complex-dependent manner in the presence of exogenous alanine57. Similarly, Li et al. determined that colistin resistance is associated with reduction of central carbon metabolism and energy metabolism; however, exposure to metabolites in the P cycle promoted the P cycle and the Na+NQR system, increased PMF and biosynthesis of LPS, and promoted binding of colistin to cells58. Furthermore, the metabolic state-driven approach has been successfully adopted in combating antibiotic-resistant pathogens that are seriously harmful to humans. Kuang et al. demonstrated that inactivation of the P cycle, reduced nitric oxide biosynthesis, and reduced nitric oxide, nitrite and NADH are linked to cefoperazone-sulbactam resistance in naturally and artificially evolved strains of Pseudomonas aeruginosa. However, the antibiotic-resistant phenotype reverted to antibiotic-sensitivity in the presence of exogenous fumarate, NADH, nitrate, nitrite, or sodium nitroprusside (a nitric oxide donor)59. Tang et al. also showed that glucose potentiates killing of cefoperazone/sulbactam-resistant P. aeruginosa by activating glucose transport and glucose-mediated downstream metabolism, increasing PMF and stimulating uptake of amikacin60. Similarly, exogenous fumarate reverted aminoglycoside-tolerance in P. aeruginosa PAO161, while ATP/ADP sensitized carbapenem-resistant Acinetobacter baumannii to meropenem62.

Other data suggest that the efficacy of ROS-dependent antibiotics such as cefoperazone-sulbactam depends on the cellular redox state16,30,63,64,65,66, and that reactive metabolites increase antibiotic efficacy under anaerobic conditions67. Non-targeted metabolomic data from Salmonella show that citrulline, glutamine, and fructose levels are suppressed in apramycin/gentamicin-resistant Salmonella and that exogenous citrulline, glutamine or fructose activates the TCA cycle, increases NADH, raises the PMF, stimulates drug uptake, and restores sensitivity to apramycin/gentamicin55,68. Metabolic reprogramming of the glutathione biosynthesis modulates the resistance of ceftriaxone-resistant Salmonella69. The metabolic state-driven approach is also successfully utilized in gram-positive ampicillin-resistant Streptococcus agalactiae that is reprogrammed by fructose to be sensitive to ampicillin70. A recent report showed that intracellular glutamine is low in clinical multidrug-resistant strains of Escherichia coli and that exogenous glutamine increases cell death in the presence of β-lactam-, aminoglycoside-, quinolone-, and tetracycline-antibiotics12. Exogenous glutamine also increases the efficacy of ampicillin against multidrug-resistant P. aeruginosa, A. baumannii, Klebsiella peneumoniae, E. tarda, V. alginolyticus, and V. parahaemolyticus in vitro and against multidrug-resistant E. coli, P. aeruginosa, A. baumannii, and K. peneumoniae in vivo12. Glucose delays the transition from ampicillin tolerance to resistance in E. coli and E. tarda71. Pyruvate reverses fluoroquinolone tolerance induced by benzaldehyde in company with decreased ROS level in E. coli72. A pyruvate-cysteine-glutathione system/glycine-ROS metabolic pathway is identified as the most key pyruvate-boosting metabolic pathway to increase ROS level that potentiates gentamicin killing in several bacterial pathogens73. The underlying mechanism includes that glycine abundance is elevated to inhibit glutathione reductase and thereby ROS level is increased73. Therefore, metabolic reprogramming is a proven and valuable strategy for stimulating drug uptake and promoting ROS to mitigate the consequences of infection by antibiotic-resistant bacteria, persisters, and biofilms. Importantly, a positive correlation between ROS level and killing efficiency of antibiotics including aminoglycosides and β-lactams is determined30,66,73. Therefore, metabolic reprogramming not only increases intracellular drug concentration but also elevates ROS level that in turn promotes antibiotic killing (Fig. 2)10,30,66,71,73,74. Collectively, metabolic reprogramming agents are among the most promising strategies for stopping the spread of antibiotic-resistant bacterial pathogens.

Fig. 2: Metabolic reprogramming promotes both antibiotic uptake and ROS level that in turn potentiates antibiotic killing in multidrug-resistant E. tarda, carbapenem-resistant P. auroginosa, E. coli, K. pneumonia, and MRSA73.
figure 2

Metabolic reprograming agents modulate metabolism to promote antibiotic uptake and increase intracellular drug concentration by promoting porine abundance and proton motive force (purple). On the other hand, the modulated metabolism elevates ROS production (dark yellow) and inhibits ROS degradation to increase ROS concentration (black), which in turn potentiates antibiotic killing (red). Specifically, activation of the pyruvate cycle (the P cycle), electron transport chain, phosphate pentose pathway (PPP), and riboflavin metabolism increases ROS production, while the pyruvate-flux from the P cycle promotes glycine production that inhibits glutathione reductase (GR). This inhibition affects the reduction of glutathione disulfide (GSSG) to glutathione (GSH), causing GSH decrease and GSSG accumulation. When the decreased GSH is not oxidized to GSSG, the accompanying ROS clearance could not proceed. The increased ROS production and reduced ROS clearance together elevate ROS level to potentiate antibiotic killing.

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It was recently shown that nutrient metabolite-enabled killing of antibiotic-resistant pathogens is a successful strategy in mice, an important advance in the field. More specifically, mannitol improved outcomes following treatment of chronic E. coli biofilm infections with gentamycin in a mouse model for urinary tract infections9 and indole-3-acetic acid and ciprofloxacin synergistically promoted survival of high-fat diet-fed mice infected with persistent methicillin-resistant Staphylococcus aureus (MRSA)53. Similarly, when pathogenic E. tarda EIB202 and ATCC15947 biofilms were seeded via catheter into the urinary tracts of mice, kanamycin plus alanine and/or glucose or fructose more effectively inhibited infection of mouse urinary tract and kidneys than kanamycin alone10,55. Pyruvate, citrulline, and glutamine plus aminoglycosides more effectively inhibited infection of mouse blood, liver, and spleen by gentamicin-resistant V. alginolyticus or apramycin-resistant Salmonella than antibiotic alone56,75. Extending these in vivo observations to another species, glucose, pyruvate, or fructose plus gentamicin synergistically inhibited bacterial infections in zebrafish or chicken and were more effective than gentamicin alone14,68,75. More importantly, a combined antibacterial agent with an antibiotic and glutamine12, which is identified by metabolic state-driven approach, is being developed by Guangdong Litai Pharmaceutical Co., LTD., China. These data support future clinical applications using synergistic combinations of metabolites and antibiotics to prevent or mitigate infections with antibiotic-resistant bacterial pathogens. Notably, nutrient metabolites exceeding bacterial physiological doses need to be used to achieve high-efficiency antibacterial.

Increasing intracellular antibiotic by increasing uptake to overcome the degradation and efflux

Conventional dogma is that there are four distinct mechanisms by which bacteria acquire resistance to small molecule antibiotics: i) non-specific decrease in bacterial membrane permeability; ii) increase in capacity to degrade, destabilize or inactivate an antibiotic or a class of similar antibiotics (e.g., induction of chromosomal beta-lactamase); iii) increase in efflux pump capacity; and iv) genetic mutations that render the bacteria resistant to the antibiotic (e.g., acquired capacity to express an isoform of the target molecule with low or no affinity for an antibiotic or a class of antibiotics)76,77,78,79. The combined impact via mechanisms i, ii and/or iii on drug uptake, degradation, and efflux must be considered together, to determine the net effect on the steady-state intracellular drug concentration (see Fig. 1). Unfortunately, little progress has been made in understanding and manipulating bacterial membrane permeability, and this impasse is largely responsible for limited progress in the field80,81.

The metabolic state-driven approach is a promising therapeutic approach that could potentially overcome limited uptake of antibiotics by drug-resistant bacteria8,82. The elevated intracellular drug concentrations are quantified using commercial kits or LC-MS analysis. For example, Peng et al. and Su et al. demonstrated that exogenous alanine, glucose, or fructose plus kanamycin stimulated drug uptake several-fold more than needed to compensate for 9.5 ng/mL lower intracellular steady-state kanamycin in kanamycin-resistant E. coli than in kanamycin-sensitive E. coli10. Furthermore, fumarate stimulated respiratory activity and uptake of tobramycin, which lowered viability of drug-tolerant Pseudomonas aeruginosa61. In the presence of glucose, uptake of amikacin by cefoperazone/sulbactam-resistant P. aeruginosa increased 1.5-fold and uptake of gentamicin by gentamicin-resistant V. alginolyticus increased 2-fold14,58. D-ribose also increased drug uptake approximately 2-fold and increased the lethality of gentamicin in drug-resistant Salmonella spp46. Endogenous succinate and glutamate from lung epithelial cells also stimulated uptake of aminoglycosides by drug-resistant clinical pathogens83 and triclosan inhibited biosynthesis of fatty acids and stimulated uptake of ciprofloxacin by P. aeruginosa84. In E. coli, glutamine and other metabolites such as inosine promoted uptake of several classes of drugs including penicillins (ampicillin), cephalosporins (ceftriaxone), aminoglycosides (gentamicin), quinolones (balofloxacin), and tetracyclines (tetracycline)12; Succinate and inosine promoted intracellular ciprofloxacin or/and tigecycline concentration85,86. These reports suggest that clinically common classes of antibacterials are included in the nutrient metabolites-potentiated drug uptake. Therefore, the nutrient metabolites-potentiated drug uptake is effective approach to combat antibiotic resistance.

The above findings demonstrate that exogenous nutrient metabolites can be used to stimulate drug uptake; however, not all studies compare the steady-state intracellular concentration of drug in the presence or absence of exogenous metabolites. This question was addressed in a recent study on metabolic state-driven nutrient based approach using ATCC35218, a β-lactamase–producing strain of E. coli, to demonstrate that exogenous glutamine clearly increases the intracellular concentration of ampicillin12. This study used a commercial kit to quantify intracellular ampicillin. It demonstrated that 47.2, 96.7, and 141.5 nM intracellular ampicillin conferred 50, 90, and 99% loss in cell viability, and these endpoints were achieved when E. coli culture media contained 5.2, 15.1, and 24.3 mM ampicillin in the absence of glutamine, while the same drug efficacy was observed when cells were cultured in 0.5, 1.2, and 2.8 mM ampicillin in the presence of glutamine, respectively. At the same time, the velocity of β-lactamase hydrolysis (Vh, nmol/mg per second) of ampicillin increased approximately 2-fold and the rate of drug efflux (Ve, nmol/mg per second) increased 30%. At the minimum inhibitory concentration (MIC), the velocity of influx (Vin, nmol/mg per second) was approximately equal to 4 (Ve + Vh). Therefore, exogenous glutamine can stimulate drug uptake to the point where uptake outpaces the combined rates of β-lactamase–dependent drug degradation and drug efflux, such that the intracellular concentration of antibiotic exceeds the threshold required to kill the cell. This is likely mediated by activation of the glutamine-inosine-CpxA/R-OmpF pathway12 (see Fig. 3), suggesting that nutrient metabolite-manipulated two-component system-porine plays a key role in promoting drug uptake. Very recently, the relationship between gentamicin influx (Vin) and efflux (Ve) is also established in gentamicin-resistant V. alginolyticus through comparison in medium with or without pyruvate. Pyruvate leads to a 7.21-fold increase in intracellular gentamicin75. Therefore, pyruvate promotes gentamicin influx and thereby reverses gentamicin resistance. These progresses support the call that the next generation of antimicrobials should promote antimicrobial uptake based on nutrient metabolites8.

Fig. 3: Glutamine reprograms the metabolic state and reverts the antibiotic-resistant phenotype to antibiotic-sensitivity in E. coli12.
figure 3

Antibiotic-resistant bacteria have antibiotic-resistant metabolic state, whereas antibiotic-sensitive bacteria have antibiotic-sensitive metabolic states. They, respectively, refuse and accept uptake of extracellular antibiotics. Exogenous glutamine reprograms the antibiotic-resistant metabolic state to an antibiotic-sensitive metabolic state, elevates membrane permeability, and increases drug uptake. When extracellular concentration of antibiotics is higher than MIC, more drugs are pumped out and hydrolyzed in the presence than absence of glutamine. However, the resulting high rate of drug influx exceeds the combined rate of drug degradation by β-lactamase and drug efflux, thereby elevating intracellular drug concentration to kill the antibiotic-resistant bacteria. Mechanistically, glutamine is converted to inosine via promoting purine biosynthesis and in turn promotes phosphorylation of CpxA and dephosphorylation of CpxR, which upregulates OmpF, an outer membrane channel for antibiotic uptake. Blue, inactivation; Red, activation.

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Besides porin channels, antibiotic transport is also regulated by metabolites87. For example, UhpT is an inducible transporter for fosfomycin that potentiates fosfomycin-mediated killing of Escherichia, Citrobacter, Enterobacter, or Klebsiella species in the presence of glucose-6-phosphate88. On the other hand, fructose, phosphoenolpyruvate, and glyceraldehyde-3-phosphate increase fosfomycin efficacy in Stenotrophomonas maltophilia strains that lack UhpT89, suggesting that membrane permeability can be modulated by a specific metabolite. In the latter case, the presence of the metabolite leads to a rate of drug influx that exceeds the combined rates of drug degradation and efflux.

Metabolic reprogramming agents slow down the development of antibiotic resistance

β-lactam antibiotics and β-lactamase inhibitors are increasingly used simultaneously (i.e., as combination therapy) to treat infections caused by pathogens that express β-lactamase90. However, cross-resistance to β-lactam antibiotics and β-lactamase inhibitors has been documented91,92, which makes such combination therapy ineffective93,94. Furthermore, β-lactamase inhibitors do not inhibit all known β-lactamase enzymes94,95. Because metabolic reprogramming agents are typically essential nutrients, it is difficult for bacteria to restrict their uptake/transport across the bacterial membrane, and this is not expected to change over time. For example, three antibiotic-sensitive and three antibiotic-resistant E. coli strains were propagated in M9 medium containing increasing concentrations of ampicillin with or without 20 mM glutamine for 30 days at 1 cycle/day, during which time the MIC increased due to gradual emergence of acquired resistance to ampicillin. However, the increase in MIC was approximately four-fold lower in the presence than in the absence of glutamine. These bacteria remained sensitive to ampicillin plus glutamine after being injected into mice and the efficacy of glutamine plus ampicillin was retained over five treatment cycles. These results suggest that the bacteria acquired resistance to ampicillin plus glutamine more slowly than to ampicillin alone12. This argues strongly in favor metabolic state-driven approach as a strategy to fight antibiotic-resistant bacteria and to increase and prolong the efficacy of antibiotic drugs.

Conclusions and perspectives

Successful use of the metabolic state-driven nutrient-based strategy to combat antibiotic resistance depends on in-depth understanding of bacterial metabolism and the ability to assess normal vs altered bacterial metabolic states. When metabolome profiling reveals specific downregulation of a metabolite (or several metabolites from the same pathway) in antibiotic-resistant vs. antibiotic-sensitive bacteria, those metabolites are likely candidates for use as metabolic reprogramming agents. In many cases, by providing an exogenous source of the downregulated metabolite(s) and complementing the apparent deficit, the metabolic profile can be remodeled and normalized, while at the same time, the antibiotic-resistant phenotype reverts to a phenotype of antibiotic sensitivity. This is because antibiotic-sensitive and -resistant bacteria have antibiotic-sensitive and -resistant metabolic states, respectively, which is highly related to antibiotic killing efficiency. On the hand, the metabolic reprogramming agents overcomes membrane permeability barrier to promote drug uptake. The increased uptake exceeds the combined rates of enzyme–dependent drug degradation and drug efflux, leading high intracellular drug and thereby causing a high killing. A typical example is the activation of the glutamine-inosine-CpxA/R-OmpF pathway12. On the other hand, antibiotic killing efficiency is related to metabolites’ transformation products such as ROS. The higher ROS concentration, the greater the bactericidal efficiency of aminoglycosides and β-lactams66,73. Therefore, metabolic reprogramming agents can effectively promote antibiotic killing.

Metabolism-based intervention is a simple, straightforward way to effectively increase membrane permeability and stimulate uptake of an antibacterial drug. The phenotype of antibiotic resistance often correlates with downregulation of membrane porin channels that facilitate influx of nutrients and small molecule drugs. This is consistent with the fact that exogenous metabolites often upregulate transcription factors and two-component systems that stimulate porin biosynthesis and mobilization. However, questions remain about how metabolites exert their downstream effects. Furthermore, the expression of antibiotic class-specific influx channels and their functional overlap, and the fact that many metabolites impact several metabolic pathways, complicate the design of effective interventions targeting membrane permeability. A thorough understanding of how different metabolite fluxes intersect and influence each other’s impact on drug uptake and antibiotic resistance is needed in order to exploit the full potential of metabolic reprogramming and facilitate development of successful metabolism-based strategies to combat antibiotic resistance.

Because many bacterial metabolites are safe and non-toxic human nutrients, it is unlikely that pilot or cohort studies in human subjects will be delayed due to safety concerns. As this research moves forward, we suggest that metabolites could be delivered for clinical use via at least three distinct routes: i) clinical grade nutrients could be prepared by an independent process and then used with antibiotic drugs; ii) nutrients and antibiotics could be prepared as a compound drug for clinical use, iii) nutrients and antibiotics could be chemically crosslinked and treated essentially as distinct pharmaceuticals for clinical use.

Finally, translating the concept into novel therapies appears easy and straightforward, but massive hurdles exist. This is because clinical applications must be carried out based on drugs that have already been approved. Overcoming these hurdles is essential for implementing this strategy. In any case, developing new drugs is a prerequisite for achieving the translating. However, developing new drugs based on the new theory involves various stakeholders such as the government, venture capital, pharmaceutical companies, hospitals, and patients. This requires them to truly understand that metabolic state determines the bactericidal efficiency of antibiotics, nutritional metabolites are more beneficial than inhibitors for human beings, and increasing the uptake of antibiotics is preferable to the inhibition of drug enzymatic hydrolysis. Additionally, economic considerations are necessary. Nutritional metabolites are much cheaper than inhibitors, thus offering profit potential. While addressing these understanding issues, it is essential in clinical trials to determine the optimal concentration of reprogrammed metabolites, the best antibiotic ratios, the most effective administration times and routes, the most suitable types of antibiotic-resistant bacteria, and the ideal patient conditions. Notably, in some countries such as China, the use of previously approved drugs for a new implication allows to explore. Thus, an alternative approach can speed up the clinical applications when nutrient metabolites are drugs themselves, such as glucose and inosine. This can be requested by interested doctors and implemented after approval from the hospital’s ethics committee.