Bacterial central carbon metabolism and antibiotic efficacy

The rise of antibiotic treatment failures poses an escalating global crisis, undermining modern medicine and threatening public health. While resistance mechanisms render antibiotics ineffective, tolerance mechanisms such as persistence, enable bacterial populations to withstand transient antibiotic exposure without acquiring genetic resistance. These survival strategies are overlooked by standard susceptibility tests which complicates treatment and increases the likelihood of infection recurrence and development of antibiotic resistance. Since bacterial metabolism is tightly linked to antibiotic tolerance, identifying the pathways that sustain survival is essential for developing effective intervention strategies. However, the role of metabolic mechanisms underlying antibiotic tolerance is not well understood. In-cell nuclear magnetic resonance (NMR) spectroscopy provides a non-invasive approach for real-time metabolic observations in living cells, making it a promising tool for investigating the metabolic shifts that promote antibiotic tolerance.

This thesis utilizes NMR-based methods to investigate the metabolic adaptations driving antibiotic tolerance across three connected studies. The first establishes in-cell hyperpolarized NMR for real-time metabolic tracking in Lactococcus lactis, demonstrating its ability to reveal glycolytic flux alterations and metabolic bottlenecks. These findings highlight the sensitivity of in-cell NMR in detecting shifts in metabolic activity and underscore its potential for investigating bacterial stress responses. The second study examines the evolution of antibiotic tolerance in Salmonella Typhimurium, revealing that tolerance-evolved strains exhibit reduced metabolic activity compared to their ancestral strain while maintaining metabolic function upon antibiotic exposure. This metabolic resilience enables prolonged survival under antibiotic stress, emphasizing the central role of adaptive metabolism in bacterial persistence. The third study explores the impact of uracil supplementation on persistence, showing how it drives metabolic reprogramming, extends the lag phase, and induces morphological adaptations that enhance antibiotic survival. These findings suggest that nucleotide availability serves as a regulatory factor in persistence and may provide novel therapeutic targets.

Addressing antibiotic tolerance is pivotal in the fight against antimicrobial resistance. Integrating real-time metabolic analysis with traditional microbiological assays underscores the potential of NMR-based diagnostics to identify tolerant subpopulations and refine treatment strategies. By uncovering the metabolic foundations of antibiotic tolerance, this research deepens the understanding of bacterial survival strategies and lays the groundwork for more effective therapeutic interventions.