Structural, proteomic, and metabolomic insights into the antibacterial efficacy of nalidixic acid against Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a facultative intracellular pathogen that causes caseous lymphadenitis, a disease responsible for substantial economic losses in the livestock industry. Although nalidixic acid (NA) has been shown to inhibit C. pseudotuberculosis biofilm formation, the molecular mechanisms underlying its efficacy remain poorly characterized. This study aimed to characterize the structural, proteomic, and metabolomic alterations induced by NA in C. pseudotuberculosis at effective concentrations. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of NA against planktonic cells were determined. Its antibiofilm efficacy was assessed through pellicle formation, crystal violet (biomass), and resazurin (metabolic viability) assays. Biofilm structure was visualized using field emission scanning electron microscopy (FESEM), and biochemical composition was analyzed via Fourier-transform infrared (FTIR) spectroscopy. Proteomic profiling was conducted using SDS-PAGE combined with HPLC-MS/MS, while metabolomic profiling was performed using UPLC/Q-TOF-MS. NA exhibited an MIC of 12.5 μg/mL and was bactericidal at 100 μg/mL (MBC). It inhibited pellicle formation and significantly (p < 0.05) decreased biofilm biomass and viability. At 100 μg/mL, FESEM revealed a near-complete loss of structural integrity and extracellular matrix, a finding corroborated by FTIR spectra indicating altered biochemical composition. Proteomic analysis revealed the differential expression of key hub proteins involved in energy production (AtpA, AtpD), translation (ribosomal proteins), and amino acid metabolism (GlyA). Metabolomic analysis identified 118 significantly dysregulated metabolites, pointing to a collapse of central carbon metabolism, most notably in the tricarboxylic acid (TCA) cycle. This study demonstrates that NA induces a systems-level disruption in C. pseudotuberculosis biofilm as inferred from integrative proteomic and metabolomic network analysis.