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Abstract Metabolic inhibitors are known to exhibit complex interactions with antibiotics in bacteria, potentially acting as antagonists by inducing cell dormancy and promoting cell survival. However, the specific synergistic or antagonistic effects of these inhibitors depend on factors like their mechanisms of action, concentrations, and treatment timings, which require further investigation. In our study, we systematically explored the synergistic interactions of various metabolic inhibitors—such as chloramphenicol (a translation inhibitor), rifampicin (a transcription inhibitor), arsenate (an ATP production inhibitor), and thioridazine (a PMF inhibitor)—in combination with ofloxacin. We conducted this investigation under pre-, co-, and post-treatment conditions, employing a wide concentration range and utilizing four distinct synergy models. Chloramphenicol, rifampicin, and arsenate consistently showed minimal synergy scores, indicating a notable antagonistic relationship with ofloxacin across all models and conditions. In contrast, thioridazine consistently demonstrated elevated synergy scores, especially in pre- and co-treatment scenarios, albeit its synergy decreased during post-treatment conditions. When multivariable linear regression analyses were used for all drugs and conditions examined, a correlation between the synergy of thioridazine and its ability to suppress cellular energy metabolism became evident, underscoring the potential utility of certain metabolic inhibitors as effective anti-persistence adjuvants. 

Aminoglycoside antibiotics target ribosomes and are effective against a wide range of bacteria. Here, we demonstrated that knockout strains related to energy metabolism inEscherichia colishowed increased tolerance to aminoglycosides during the mid-exponential growth phase. Contrary to expectations, these mutations did not reduce the proton motive force or aminoglycoside uptake, as there were no significant changes in metabolic indicators or intracellular gentamicin levels between wild-type and mutant strains. Our comprehensive proteomics analysis unveiled a noteworthy upregulation of proteins linked to the tricarboxylic acid (TCA) cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research provides valuable insights into the mechanisms of aminoglycoside tolerance, paving the way for novel strategies to combat such cells. 

Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of Escherichia coli cells. Given that aminoglycoside uptake relies on the energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells.