Antibiotic stress triggers α-hemolysin-mediated hemolysis in E. coli. - An overlooked factor for hemolytic driving sepsis.

This study demonstrates that exposure to sub-inhibitory concentrations of antibiotics can activate bacterial virulence in vitro. Under sub-minimum inhibitory concentration (sub-MIC) antibiotic stress, we investigated the regulation of α-hemolysin, a pore-forming exotoxin central to Escherichia coli-mediated cytotoxicity and sepsis. Hemolytic activity was assessed using goat erythrocytes following exposure of the hemolytic strain E. coli MTCC 729 to increasing sub-lethal concentrations of clinically relevant antibiotics, while the non-hemolytic strain E. coli MTCC 723 was included throughout as a biological negative control to validate assay specificity. Extracellular α-hemolysin levels were quantified by the Lowry protein assay, and toxin-induced lytic progression was examined using microscopic imaging and blood agar assays. Among the antibiotics tested, gentamycin (aminoglycoside; 92.15 % hemolysis at 30 min, 260 mg/L extracellular protein) elicited the strongest hemolytic response, significantly exceeding untreated controls (3.14 %), ciprofloxacin (fluoroquinolone; 74.11 %), tetracycline (broad-spectrum; 62.20 %), and ampicillin (β-lactam; 48.21 %) (p < 0.05). The absence of hemolysis in E. coli MTCC 723 cultures, together with Triton X-100 (96 %) and PBS (0 %) controls, confirmed the specificity of α-hemolysin-mediated cytolysis. Morphometric and time-lapse analyses further validated non-osmotic, pore-forming α-hemolysin activity, revealing a progressive cytolytic cascade characterized by erythrocyte crenation, membrane deformation, and terminal rupture. Collectively, these findings highlight the paradoxical nature of antimicrobial therapy, demonstrating that sub-therapeutic antibiotic exposure can potentiate toxin-mediated cytopathogenicity without inhibiting bacterial growth.