Pyrimidine biosynthesis repression fuels cost and survival of trimethoprim/sulfamethoxazole - Resistant E. coli

Decades of extensive application have led to the rapid emergence and global dissemination of trimethoprim/sulfamethoxazole (TMP/SMZ)-resistant strains, yet the associated fitness costs and underlying adaptive mechanisms remain poorly defined. This study systematically investigated the physiological and molecular adaptations of resistant E. coli under 0-0.125/2.375 μg/mL TMP/SMZ stress. In the absence of antibiotics, resistant bacteria (R) incurred significant fitness costs, evidenced by impaired growth, a 30% reduction in biofilm formation, a 47% thicker cell wall, a diminished capacity for carbon source utilization, and altered nitrogen metabolism - characterized by reduced GS yet elevated GDH and Ca²⁺/Mg²⁺-ATPase activities - compared to the susceptible strain (S; P < 0.05). In competitive co-culture, the R was outcompeted (W < 1). Multi-omics analyses revealed the R strain downregulated energy-intensive processes while maintaining primed oxidative stress defenses. Conversely, under 0.125/2.375 μg/mL TMP/SMZ pressure, R strain demonstrated a pronounced survival advantage. While approximately half of the susceptible population lysed, the R strain exhibited enhanced growth, improved capacity for carbon source utilization and superior competitive fitness (W > 1). This adaptive advantage was underpinned by a fundamental metabolic reconfiguration, characterized by: 1) repression of pyrimidine biosynthesis and resource reallocation to the shikimate pathway and polyamine biosynthesis; 2) a shift in central carbon flux from oxidative phosphorylation toward fermentative pathways; and 3) robust activation of glutathione metabolism and NAD(P)H pools to mitigate oxidative stress. This study reveals the fitness cost and stress-response mechanisms of TMP/SMZ-resistant E. coli, providing novel insights into resistance persistence and a theoretical basis for curbing its environmental spread.