Effects of Hydrogen Peroxide on Biofilm Formation and Antibiotic Susceptibility of Clinical Bacterial Isolates

Abstract

Background: Reactive oxygen species (ROS) are products of aerobic metabolism that play dual roles as signaling molecules and as inducers of oxidative damage. Their impact on bacterial physiology, particularly biofilm formation and antibiotic resistance, is of growing clinical significance. Objective: This study aimed to investigate the effects of hydrogen peroxide (H₂O₂)-induced oxidative stress on biofilm formation and antibiotic susceptibility among clinical bacterial isolates. Methods: Seventy bacterial isolates were collected from clinical cases at Tikrit Teaching Hospital, Iraq, and identified as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus viridans. Minimum inhibitory concentrations (MIC) and sub-MIC values of H₂O₂ were determined, followed by assessments of biofilm production (tube method) and antibiotic susceptibility (modified Bauer–Kirby disk diffusion) before and after H₂O₂ exposure. Results: Sub-MIC values of H₂O₂ varied across species (30 mM for E. coli, 2 mM for K. pneumoniae, 70 mM for S. aureus, and 40 mM for S. viridans). Biofilm assays revealed significant modulation, with S. aureus losing its weak biofilm phenotype post-exposure, while S. viridans shifted from weak to strong biofilm production. Antibiotic susceptibility profiles demonstrated species-dependent alterations, including increased resistance to vancomycin and rifampin in S. viridans, linked to enhanced biofilm formation, and loss of resistance in other species. These changes are attributed to oxidative stress-induced genetic mutations, plasmid rearrangements, and quorum sensing regulation. Conclusion: Sub-lethal oxidative stress induced by H₂O₂ profoundly modulates bacterial adaptive mechanisms, particularly biofilm formation and antibiotic resistance. These findings highlight the complex interplay between oxidative stress, microbial survival strategies, and antimicrobial efficacy, providing insights into the evolutionary dynamics of bacterial pathogenesis and potential therapeutic challenges.