Metabolomic Changes in Biofilm of Pseudomonas aeruginosa Under Early-Stage Tobramycin Treatment Identified by 2D NMR-Based COLMARq

Abstract Biofilms are estimated to account for a large proportion of persistent microbial infections and are associated with substantial mortality in hospitals. Pseudomonas aeruginosa biofilms exhibit higher resistance to antibiotics compared to their planktonic growth form, making them difficult to eradicate and contributing to the mounting global challenge posed by infectious diseases. To gain deeper insights into the metabolic response of biofilms to antibiotic pressure, we conducted a quantitative, untargeted metabolomics analysis using heteronuclear 2D NMR spectroscopy. Biofilms were grown in the presence of sub-inhibitory concentrations of tobramycin, and metabolic alterations were identified and quantified using the COLMARq analysis platform. Our analysis revealed significant metabolic shifts even at low doses of tobramycin with key metabolites such as UDP-glucose increasing and putrescine decreasing. Pathway enrichment analysis highlighted alterations in amino acid biosynthesis, energy metabolism, and stress response pathways. Notably, the accumulation of glutathione disulfide indicates enhanced oxidative stress, while the depletion of β-alanine, succinate, and GABA suggested metabolic shifts linked to biofilm adaptation. These findings provide new insights into the metabolic reprogramming of biofilms under antibiotic treatment and offer perspectives into biofilm behavior in the case of Pseudomonas aeruginosa when exposed to antibiotics from an early stage of infection.