Exogenous adenosine counteracts tigecycline resistance in tet(X3)-harboring Escherichia coli

Abstract

The rapid spread of antibiotic resistance poses a global health crisis. Tigecycline is a last-resort antibiotic, but the recent emergence of the plasmid-borne tet(X3) gene conferring high-level tigecycline resistance is deeply concerning. Here, we report a metabolomics-guided approach to overcome tet(X3)-mediated resistance. Using untargeted metabolomics, we identified adenosine as a key metabolic biomarker associated with tet(X3) expression. Remarkably, supplementation with exogenous adenosine was able to restore tigecycline susceptibility in tet(X3)-positive Escherichia coli both in vitro and in vivo. Our mechanistic investigations reveal that adenosine enhances the bactericidal effects of tigecycline by inducing oxidative stress, DNA/RNA damage, and cell membrane disruption in resistant bacteria. This study establishes a powerful metabolomics-driven strategy to potentiate antibiotic efficacy against drug-resistant pathogens. The adenosine-based adjuvant therapy represents a promising approach to combat the global crisis of antibiotic resistance.IMPORTANCEThe emergence and widespread dissemination of the high-level tigecycline resistance gene tet(X3) have posed a significant challenge to the efficacy of tigecycline, which serves as the "last line of defense" against antimicrobial-resistant bacteria. Although tigecycline has not been approved for veterinary clinical use, constant detection of tet(X3) genes and new subtypes in livestock farming environments poses a substantial threat to public health safety. While developing novel antibiotics is an effective approach to eradicate resistance genes/bacteria, it entails considerable costs and a lengthy timeframe. This study discovered that exogenous adenosine can effectively restore the sensitivity of tet(X3)-positive Escherichia coli to tigecycline through metabolic reprogramming based on a non-targeted metabolomics strategy. The findings are highly significant for exploring comprehensive mechanisms underlying bacterial multidrug resistance, utilizing metabolic reprogramming strategies to curb the spread of novel resistant genes, and treating clinical infections caused by tet(X3)-positive bacteria.

Keywords: adenosine; antimicrobial resistance; metabolomics; tet(X3); tigecycline.

Fig 1

Reprogramming of metabolic profiles in E. coli carrying the tet(X3) gene. (A) Upregulation and downregulation of differential metabolites in tet(X3) gene-positive E. coli. A total of 72 differential metabolites were identified, with 35 upregulated and 37 downregulated in the tet(X3)-carrying strain. The upregulated metabolites primarily consist of pantothenate, ADP, cytosine, glucose, folic acid, and glutamate, while the downregulated metabolites mainly include gallic acid, valine, NAD, and succinic acid. (B) Heatmap analysis of differential metabolite spectrum in tet(X3) gene-positive E. coli. The heatmap showcases the fluctuations of multiple metabolites, with each column representing a specific treatment condition, while each row corresponds to a distinct metabolite. After undergoing normalization, the spectrum of metabolite changes extends from −2 (blue) to 2 (red). The dendrogram positioned at the top showcases the clustering relationship among samples, revealing that specific treatment conditions exhibit a greater resemblance in terms of metabolic patterns.

Fig 2

The tet(X3) gene orchestrates modifications in metabolic pathways. (A) Metabolic pathway enrichment analysis. (B) Metabolite and metabolite interaction network analysis. (C) Metabolic perturbations of key metabolites in pantothenate and CoA biosynthesis. (D) Metabolic perturbations of key metabolites in purine metabolism. (E) Metabolic perturbations of key metabolites in the TCA cycle. (F) Metabolic perturbations of key metabolites in arginine biosynthesis. (G) Metabolic perturbations of key metabolites in histidine metabolism. (H) Metabolic perturbations of key metabolites in alanine, aspartate, and glutamate metabolism. (I) Metabolic perturbations of key metabolites in vitamin B6 metabolism. (J) Metabolic perturbations of key metabolites in one-carbon pool by folate. (K) Metabolic perturbations of key metabolites in nicotinate and nicotinamide metabolism. (L) Metabolic perturbations of key metabolites in pyrimidine metabolism.

Fig 3

Synergistic antibacterial effect of adenosine in combination with tigecycline or tetracycline. (A) The checkerboard method was employed to assess the synergistic antibacterial effect of adenosine in combination with tetracycline on engineered strains. (B) The checkerboard method was utilized to evaluate the synergistic antibacterial effect of adenosine in combination with tetracycline on natural tet(X3)-carrying strain. (C) The checkerboard method was applied to determine the synergistic antibacterial effect of adenosine in combination with doxycycline on engineered strains. (D) The checkerboard method was employed to ascertain the synergistic antibacterial effect of adenosine in combination with doxycycline on natural tet(X3)-carrying strain. (E) Adenosine and tetracycline were combined for generating a synergy killing curve using engineered strains. (F) Adenosine and tetracycline were combined for generating a synergy killing curve using natural tet(X3)-carrying strain. (G) Adenosine and doxycycline were combined for generating a synergy killing curve using engineered strains. (H) Adenosine and doxycycline were combined for generating a synergy killing curve using natural tet(X3)-carrying strain. (N = 3 independent experiments, each performed in duplicate), bars indicate means, and error bars indicate standard deviation.

Fig 4

Co-administration of Ado and TGC leads to intracellular accumulation of ROS. (A) Alterations in intracellular ROS accumulation in engineered strains. (B) Modifications in intracellular ROS accumulation in natural tet(X3)-carrying strain. N = 3 independent experiments, each performed in duplicate, bars indicate means, and error bars indicate standard deviation, ** represents P＜0.01.

Fig 5

The effect of ROS scavengers on the reversal of tigecycline resistance in tet(X3)-positive E. coli. (A) Impact of ROS inhibitors on the survival rate of engineered strains. (B) Influence of ROS inhibitors on the survival rate of natural tet(X3)-carrying strain. AA indicates ascorbic acid, ALA indicates α-lipoic acid, and NAC indicates N-acetylcysteine. N = 3 independent experiments, each performed in duplicate, bars indicate means, and error bars indicate standard deviation, ** represents P＜0.01.

Fig 6

Intracellular oxidative stress damage in tet(X3)-positive E. coli. (A) Effects of adenosine and tigecycline combination on intracellular 8-OHdG levels. (B) Effects of adenosine and tigecycline combination on intracellular 8-OHG levels. N = 3 independent experiments, each performed in duplicate, bars indicate means, and error bars indicate standard deviation, * represents P＜0.05, ** represents P＜0.01.

Fig 7

Scanning electron microscopic characterization of morphological changes in tet(X3)-harboring E. coli DH5α (pUC19/tet(X3)). (A) Morphological changes in the control group bacteria (10,000×). (B) Morphological changes in the control group bacteria (20,000×). (C) Morphological changes in the adenosine-treated group bacteria (10,000×). (D) Morphological changes in the adenosine-treated group bacteria (20,000×). (E) Morphological changes in the TGC-treated group bacteria (10,000×). (F) Morphological changes in the TGC-treated group bacteria (20,000×). (G) Bacterial morphological changes in the adenosine and TGC combination treatment group (10,000×). (H) Bacterial morphological changes in the adenosine and TGC combination treatment group (20,000×). The red boxes delineate areas demonstrating bacterial cytoplasmic content leakage.

Fig 8

In vivo therapeutic efficacy of Ado demonstrates remarkable synergistic effects. (A) The bacterial load of engineered strain in the liver of mice (N = 6). (B) The bacterial load of engineered strain in the spleen of mice (N = 6). (C) The bacterial load of natural tet(X3)-carrying strain in the liver of mice (N = 6). (D) The bacterial load of natural tet(X3)-carrying strain in the spleen of mice (N = 6). (E) Seven-day survival rate of mice infected with engineered strains (N = 15). (F) Seven-day survival rate of mice infected with natural tet(X3)-carrying strain (N = 6). (G) Histopathological observation of mouse liver and spleen. ** represents P＜0.01. Bars indicate means, and error bars indicate standard deviation. The “normal control” group consisted of mice injected with physiological saline, while the “model” group comprised mice with induced bacterial resistance that received no therapeutic intervention. The red arrows indicate areas of necrosis, green arrows denote lipid degeneration, blue arrows highlight extramedullary hematopoiesis in the spleen, orange arrows indicate fibroblasts, yellow arrows represent neutrophil proliferation in the splenic red pulp, purple arrows indicate areas of congestion, black arrows point to lymphocytes, and gray arrows indicate loss of splenic marginal zone.

Fig 9

Metabolic reprogramming strategy for metabolite screening in tigecycline-resistant tet(X3)-positive bacteria and elucidation of their synergistic bactericidal mechanism. The process begins with the cultivation of strains, followed by sample preparation for metabolomic analysis. The prepared samples are analyzed using UPLC-QE-Orbitrap-MS to profile metabolites. Visual data comparisons reveal significant metabolic reprogramming in tet(X3)-positive strains, particularly in response to adenosine and tigecycline, suggesting potential antibiotic resistance mechanisms. Pathway mapping indicates alterations in the purine metabolic pathway, illustrating the adaptive responses of these strains under antibiotic stress.