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Metabolic reprogramming enhances the susceptibility of multidrug- and carbapenem-resistant bacteria to antibiotics

Nature Microbiology volume 10, pages 2257–2274 (2025)Cite this article

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Abstract

Carbapenem-resistant Enterobacteriaceae and extended-spectrum β-lactamase-resistant bacterial pathogens are a major threat to human and global health. Alternative antibiotics are therefore used to treat infections caused by these pathogens, and approaches to increase their efficacy are needed. Here we used metabolomics, mutant Escherichia coli strains and whole-genome sequencing to examine the metabolic profiles of clinical carbapenem-resistant (CR-ECO), multidrug-resistant (MDR-ECO) and antibiotic-sensitive E. coli (S-ECO) isolates in response to antibiotics in vitro including micronomicin, an aminoglycoside. Downregulation of pyruvate formate-lyase (PFL) alters membrane permeability and reduces the efficacy of micronomicin, the most potent antibiotic, in CR-ECO and MDR-ECO. The metabolism of pyruvate to formate is required to potentiate the effects of micronomicin across multiple bacterial pathogens. Mice infected with CR-ECO and treated with formate plus micronomicin had reduced pathogen growth and spread, and increased survival, compared with mice treated with micronomicin or formate alone. Finally, elevated activity or expression of PFL and increased intracellular CO2, a product of PFL- and formate dehydrogenase-dependent catabolism of formate, are required for antibiotic uptake and pathogen killing. The findings reveal a mechanism of metabolic reprogramming in MDR and CR bacteria for enhanced sensitivity to micronomicin.

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Fig. 1: Metabolic profiles of S-ECO, MDR-ECO and CR-ECO.
Fig. 2: Pyruvate potentiates the lethality of micronomicin with differential drug resistance profiles.
Fig. 3: Formate potentiates the lethality of micronomicin in ECO strains in vitro and in vivo.
Fig. 4: PMF in formate- and micronomicin-treated CR-ECO.
Fig. 5: CO2 downstream of formate potentiates the lethality of micronomicin in CR-ECO.
Fig. 6: Validation of the role of the proposed pflB and proposed model.

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Data availability

Data supporting the findings of this study are available within the article or Supplementary Information. All genomics data were deposited in the National Center for Biotechnology Information with accession number PRJNA1214050. All of the metabolomic raw data were deposited in MetaboLights (http://www.ebi.ac.uk/metabolights/)58 with identifier REQ20250606211036. The reference genome was E. coli K12 MG1655 (https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP010444.1). Source data are provided with this paper.

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Acknowledgements

This study was financially supported by grants from the National Key Research and Development Program of China (grant number 2023YFD1800104), the National Natural Science Foundation of China (32270199, 42276125), the Innovation Group Project of Southern Marine Science and the Engineering Guangdong Laboratory (Zhuhai; number 311020006).

Author information

Authors and Affiliations

  1. State Key Laboratory of Bio-Control, School of Life Sciences, Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Guangdong Province Key Laboratory for Pharmaceutical Functional Genes, Sun Yat-sen University, Guangzhou, China

    Su-fang Kuang, Jiao Xiang, Shao-hua Li, Hui Li, Bo Peng & Xuan-xian Peng

  2. Jiangxi Province Key Laboratory of Natural and Biomimetic Drugs Research, College of Pharmacy, Jiangxi Normal University, Nanchang, China

    Su-fang Kuang

  3. Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, China

    Yu-bin Su

  4. Department of Pediatrics, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

    Zhuang-gui Chen

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  1. Su-fang Kuang
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  3. Shao-hua Li
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  4. Yu-bin Su
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  5. Zhuang-gui Chen
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Contributions

X.P., B.P., H.L. and Z.C. wrote the paper. X.P. and B.P. conceptualized and designed the project. X.P., B.P., H.L., Z.C. and S.K. interpreted the data. S.K., J.X., H.L., S.L. and Y.S. performed data analysis. S.K., J.X., S.L. and Y.S. performed the experiments.

Corresponding authors

Correspondence to Zhuang-gui Chen, Hui Li, Bo Peng or Xuan-xian Peng.

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Competing interests

H.L., B.P., X.P. and S.K. are coinventors in China patent ZL202211021533.9 titled ‘Application of Sodium Formate in the Preparation of Anti-infective Drugs’ and PCT patent PCT/CN2022/139358 with the same title. The other authors declare no competing interests.

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Nature Microbiology thanks Evgeny Nudler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Antibiotic selection and effect of the combination of micronomicin and pyruvate on S-ECO, MDR-ECO, and CR-ECO.

a, Percent survival of MDR-ECO44 and CR-ECO28 in the absence or presence of 10 mM sodium pyruvate (Pyr-Na) with the indicated antibiotics. GEN (Gentamicin, 60 μg/mL), AMI (amikacin, 60 μg/mL), MCR (micronomicin, 60 μg/mL), MEM (meropenem, 1 μg/mL), IPM (imipenem, 1 μg/mL), CAZ (ceftazidime, 20 μg/mL), SCF (cefoperazone-sulbactam, 20 μg/mL), BLFX (balofloxacin, 10 μg/mL), and CIP (ciprofloxacin, 10 μg/mL) for 10 h. n = 3. ****p < 0.0001 at GEN, AMI, MCR, CIP; p = 0.0002 at BLFX in MDR-ECO44. b, Percent survival of S-ECO, MDR-ECO, and CR-ECO in the presence or absence of 10 mM sodium pyruvate and plus 20 μg/mL, 60 μg/mL, and 240 μg/mL micronomicin, respectively, for 10 h. b, n = 3. ****p < 0.0001 for all but ***p = 0.0002 (51). All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

Source data

Extended Data Fig. 2 Schematic diagram of pyruvate metabolism and percent survival of mutants with genes encoding pyruvate metabolism.

a, Schematic diagram. b, percent survival of K12 ΔpoxB, ΔaceE, ΔaceF, ΔpflB, ΔtdcE, ΔidhA, Δdld in the presence or absence of 1 μg/mL micronomicin, 10 mM sodium pyruvate or both. n = 3. **p = 0.0050 at ΔaceE, ***p = 0.0003 at ΔaceF, **p = 0.0064 at ΔpflB. c, RT-PCR for expression of dld. n = 8. d, Western blot for abundance of PflB in S-ECO, MDR-ECO, and CR-ECO. n = 10. *p = 0.0169 at MDR-ECO, ****p < 0.0001 at CR-ECO. All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data (b) were performed by two-way ANOVA followed by Sidak’s multiple comparison test. Data (c, d) were performed by one-way ANOVA followed by Tukey’s post hoc test.

Source data

Extended Data Fig. 3 Level of metabolites and expression of acs.

a-c, Levels of formate (a), acetate (b), and acetyl-CoA (c) of S-ECO 61, MDR-ECO44, and CR-ECO28 in absence and presence of 10 mM sodium pyruvate and or 20 mM sodium acetate (only for acetyl-CoA). n = 3. a, ****p = 0.0001 (1 vs 2 or 3, 4 vs 6), *p = 0.0197 (2 vs 5). b, **p = 0.0067 (1 vs 4), **p = 0.0013 (2 vs 5), *p = 0.0175 (3 vs 6). c, *p = 0.0131 (1 vs 2), ****p <0.0001 (1 vs 3), ***p = 0.0004 (2 vs 3), *p = 0.019 (1 vs 4), **p = 0.0012 (1 vs 7). d, qRT-PCR for expression of acs in the absence or presence of 20 mM acetate cross S-ECO, MDR-ECO, and CR-ECO. n = 8. **p = 0.0015 (1 vs 2), **p = 0.0035 (1 vs 3), ****p = 0.0001 (1,2,3 vs 4,5,6, respectively). All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by one-way ANOVA followed by Tukey’s post hoc test.

Source data

Extended Data Fig. 4 Percent survival of S-ECO61, MDR-ECO44, and CR-ECO28 in the absence or presence of the indicated metabolites and micronomicin, respectively.

Percent survival of S-ECO61 (a), MDR-ECO44 (b), and CR-ECO28 (c) in the absence or presence of the indicated metabolites plus 20 μg/mL, 60 μg/mL, and 60 μg/mL micronomicin, respectively. n = 3. a,****p = 0.0001 at formate, glucose, glucose + mannitol, **p = 0.0011 at mannitol, ***p = 0.0010 at alanine, **p = 0.0012 at glutamate. b, ****<0.0001 for all comparisions. c, ***p = 0.0006 at formate. All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

Source data

Extended Data Fig. 5 Percent survival of clinically isolated in absence or presence of formate plus micronomicin.

a and b, Percent survival of MDR-ECO (a) and CR-ECO (b) in the presence or absence of 20 mM sodium formate and 60 μg/mL of micronomicin, except for 30 μg/mL for MDR-ECO31, MDR-ECO37, MDR-ECO42, 20 μg/mL for MDR-ECO38, 5 μg/mL for MDR-ECO33, MDR-ECO40, 2 μg/mL for MDR-ECO34, 1 μg/mL for MDR-ECO47, MDR-ECO48, MDR-ECO50, 0.5 μg/mL for MDR-ECO32, 30 μg/mL for CR-ECO1, 20 μg/mL for CR-ECO10, CR-ECO13, 1 μg/mL for CR-ECO5, CR-ECO19, CR-ECO22, and 0.5 μg/mL for CR-ECO12. n = 3. a, ****p < 0.0001 for all comparisons between MCR+for-Na and the other three groups. b, ****p < 0.0001 for all comparisons between MCR+for-Na and the other three groups. c, Percent survival of MDR-K. pneumonia in the presence or absence of 20 mM sodium formate and micronomicin 60 μg/mL for MDR-KPN23 and ATCC700603, 1.5 μg/mL for MDR-KPN4, 1 μg/mL for MDR-KPN42. n = 3. ****p < 0.0001 for comparisons between MCR+for-Na and the other three groups. d, Percent survival of CR- and MDR-P. aeruginosa in the presence or absence of 20 mM sodium formate and micronomicin 40 μg/mL for CR-PAE-A2, 5 μg/mL for CR-PAE-C3, 2 μg/mL for MDR-PAE3 and PAE-S1, and 0.5 μg/mL for CR-PAE-D2. n = 3. ****p < 0.0001 for all comparisons between MCR+for-Na and the other three groups. e, Percent survival of methicillin-resistant and -sensitive Staphylococcus aureus (MRSA and MSSA, respectively) in the presence or absence of 20 mM sodium formate and micronomicin 10 μg/mL for MRSA and 1 μg/mL for MSSA. n = 3. ****p < 0.0001 for all comparisons between MCR+for-Na and the other three groups. f, Percent survival of the indicated antibiotic-resistant pathogens in the presence or absence of 20 mM sodium formate and 5 μg/mL micronomicin for Vibrio paraheamolyticus ZNV4 and ZNV10, and Edwardsiella tarda EIB202. n = 3. ****p < 0.0001 for all comparisons between MCR+for-Na and the other three groups. All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

Source data

Extended Data Fig. 6 Percent survival of CR-ECO28 in absence or presence of sodium formate, micronomicin or both.

Percent survival of CR-ECO28 in absence or presence of 20 mM sodium formate, 60 μg/mL micronomicin or both. n = 3. ****p < 0.0001 for all comparisons between MCR+for-Na and the other three groups. All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

Source data

Extended Data Fig. 7 Formate potentiates effect of micronomicin on CR/MDR biofilm and load in mouse organs.

a, Bacterial load of spleen, kidney, and liver of mice infected with CR-ECO18 in the absence or presence of formate, micronomicin or/and both. n = 6. ****p < 0.0001 (MCR vs MCR+for-Na at spleen, liver). *p = 0.0026 (MCR vs MCR+for-Na at kidney). b, Bacterial load of catheters in mouse urinary tracts. Mice were catheterized in urinary tracts and infected with CR-ECO18. These mice were treated with saline (control), 50 mg/kg sodium formate, 4 mg/kg micronomicin, or both once daily for 3 days. n = 6. *p = 0.0153 (MCR vs MCR+for-Na). c, Kidney biopsies were obtained from (b), and CFU/g kidney tissue was measured. *p = 0.0116 (MCR vs MCR+for-Na). d and e, Bacterial load of spleen, kidney, and liver of mice infected with A. baumannii CR-ABA54 (e) or P. aeruginosa CR-PAEC3 (f) in the absence or presence of sodium formate, micronomicin or/and both. n = 6(d), n = 4(e). ****p < 0.0001 for all comparisons but e, ***p = 0.0004 at kidney (MCR vs MCR+for-Na). f and g, Bacterial load of liver, kidney, and spleen of mice infected with MDR-ECO44 (f) and CR-ECO18 (g) in the absence or presence of sodium pyruvate, micronomicin or/and both. n = 6. g, ****p < 0.0001 for all comparisons (MCR vs MCR+for-Na). All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

Source data

Extended Data Fig. 8 LC-MS and Oxford cup for intracellular micronomicin.

a. n = 3. LC-MS for intracellular micronomicin of mutants ∆ompC, ∆fhuE, ∆fepA, and ∆tsx in the presence of 20 mM sodium acetate, 20 mM sodium formate, or both plus 20 μg/mL micronomicin. ****p < 0.0001 for all comparisons but 1 (*p = 0.0115), 8 (***p = 0.0046), 9 (**p = 0.0012), 10 (*p = 0.0172), 15 (***p = 0.0008), 16 (***p = 0.0003), 18 (**p = 0.0031), 24 (**p = 0.0044), 25 (*p = 0139), 26 (**p = 0.0017), 28 (**p = 0.0026), 29 (**p = 0.0056), 30(**p = 0.0098), 35 (***p = 0.0009), 36 (***p = 0.0004), 37 (***p = 0.0004), 38 (***p = 0.0019), 44 (***p = 0.0008), 45 (*p = 0.0212), 49 (**p = 0.0070), 51 (***p = 0.0004), 55 (*p = 0.0147), 60 (***p = 0.0003). b. n = 3. Oxford cup for intracellular micronomicin in MDR-ECO and CR-ECO in the presence of 60 μg/mL micronomicin with or without 20 mM sodium formate. ****p < 0.0001 for all comparisons but 29 (*p = 0.0322), 30 (***p = 0.0002), 52 (**p = 0.0015). All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

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Extended Data Fig. 9 Percent survival of mutants of genes encoding E. coli outer membrane proteins in the presence of sodium acetate, sodium formate, or both plus micronomicin.

n = 3. For group with the combination of MCR and for-Na: ****p < 0.0001 for all comparisons but *p = 0.0292 (1 vs 6), **p = 0.005 (1 vs 9), ***p = 0.0004 (1 vs 11), *p = 0.017 (1 vs 14), ***p = 0.0003 (1 vs 17), ***p = 0.0004 (1 vs 20). For group with the combination of MCR and ace-Na: ****p < 0.0001 for all comparisons but ***p = 0.0002 (1 vs 9), *p = 0.049 (1 vs 14), ***p = 0.0003 (1 vs 17), ***p = 0.0006 (1 vs 20), *p = 0.038 (1 vs 22), p = 0.378 (1 vs 23), *p = 0.023 (1 vs 24), *p = 0.023 (1 vs 27), ***p = 0.0003 (1 vs 31). All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by two-way ANOVA followed by Sidak’s multiple comparison test.

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Extended Data Fig. 10 Intracellular meropenem concentration of pflB-deleted mutants in the indicated concentrations of meropenem.

(a) and percent survival and intracellular meropenem concentration of CR-ECO28 (b). n = 3. a, for S2, ****p < 0.0001 for all comparisons; for S13, ****p < 0.0001 for all comparisons; for S-ECO61, ****p < 0.0001 for all comparisons. b, n = 3. **p = 00.0098 at 240 for percent survival; ****p < 0.0001 for intracellular meropenem. All error bars represent standard deviation. All statistical tests were two-sided, created in Graphpad Prism 10. Data were performed by one-way ANOVA followed by Tukey’s post hoc test.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–5.

Reporting Summary

Supplementary Table 1

MIC of S-ECO (sensitive to all drugs tested except for partial strains resistant to PIP or/and SAM of penicillins or/and CIP of quinolones), MDR-ECO (resistant to at least three classes of drugs but sensitive to MEM and/or IEM) and CR-ECO (resistant to at least classes of drugs plus MEM and/or IEM) strains.

Supplementary Table 2

β-lactamase of sensitive, MDR and CR E. coli strains.

Supplementary Table 3

Whole-genome sequencing of 13 strains (4 S-ECO, 5 MDR-ECO, 4 CR-ECO) revealed 86 resistance genes identified.

Supplementary Table 4

Among 86 resistance genes identified, 24 were shared across groups, with 19 exclusive to CR-ECO.

Supplementary Table 5

SNP analysis across MDR-ECO and CR-ECO strains. A total of 188,343 mutation sites were identified, including missense, nonsense, insertion, deletion and duplication mutations. Among these, 33 conserved mutations were exclusively missense. Comparative analysis with S-ECO revealed 30 genes with mutations common to all CR-ECO strains and 3 genes shared among all MDR-ECO strains, with CR-ECO harbouring significantly more mutations. These mutated genes map to 9 metabolic pathways, with ABC transporters being the only pathway overlapping between MDR-ECO and CR-ECO; the remaining 8 pathways were unique to CR-ECO.

Supplementary Table 6

Primers used for qPCR or sequencing of E. coli.

Supplementary Table 7

Comparative analysis of bactericidal activity in micronomicin when combined with sodium acetate versus sodium formate. After deletion of the outer membrane protein-coding gene, the degree of reduction of the bactericidal efficiency of sodium acetate plus micronomicin was compared with that of sodium formate plus micronomicin.

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Kuang, Sf., Xiang, J., Li, Sh. et al. Metabolic reprogramming enhances the susceptibility of multidrug- and carbapenem-resistant bacteria to antibiotics. Nat Microbiol 10, 2257–2274 (2025). https://doi.org/10.1038/s41564-025-02083-8

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