Xylose-induced tricarboxylic acid cycle activation resensitizes gentamicin-resistant Escherichia coli

The escalating antibiotic resistance crisis requires innovative strategies to revitalize existing antibiotics. Here, we report that xylose, a food-grade sugar poorly metabolized by humans but readily utilized by bacteria, acts as a potent metabolic adjuvant to restore gentamicin efficacy against bacteria. Using a laboratory-evolved gentamicin-resistant E. coli strain, we showed that xylose enhances gentamicin-mediated killing in a dose- and time-dependent manner, achieving a 200-fold reduction in viability with a synergy score 25.25. This synergistic bactericidal effect was also confirmed in multidrug-resistant and carbapenem-resistant bacteria and a mouse infection model, boosting survival rates by 30%. Metabolomics and pathway analyses revealed that xylose induced a global metabolic shift, upregulating pyruvate and succinate as biomarkers and activating the tricarboxylic acid (TCA) cycle. Exogenous addition of pyruvate and succinate also contributed to the bactericidal effect of gentamicin. This is accompanied by increased activity of key TCA enzymes (PDH, α-KGDH, SDH, MDH), elevated NADH and ATP levels, and enhanced proton motive force (PMF). However, when TCA cycle enzymes α-ketoglutarate dehydrogenase or succinate dehydrogenase were inhibited via gene knockout or sodium malonate, or when PMF was dissipated by carbonyl cyanide m-chlorophenylhydrazone (CCCP), the synergistic bactericidal effect was abolished. Xylose treatment increased intracellular gentamicin accumulation by 2.34-fold, an effect that was also blocked by TCA cycle or PMF inhibition. Our study established xylose as a bacteria-selective adjuvant that revitalizes gentamicin by reactivating TCA cycle dependent drug uptake, offering a potential strategy to combat antibiotic resistance.IMPORTANCEThis work provided a critical advance in the fight against antibiotic resistance by transforming a common food-grade sugar into a targeted therapeutic tool. Unlike conventional antibiotics or previously reported adjuvants, xylose is uniquely selective: it is efficiently utilized by bacteria but poorly metabolized in humans, thereby minimizing off-target effects. We demonstrated for the first time that xylose reversed resistance by reactivating the silenced TCA cycle in resistant bacteria, going beyond mere substrate provision to restore a critical metabolic function. This mechanism not only revitalized the efficacy of gentamicin but also established a new strategy, exploiting the metabolic differences between host and pathogen to develop "pathogen-focused" adjuvants.