Inactivation kinetics and reaction mechanisms of waterborne pathogens and plasmid DNA reactivity during chlorine dioxide water treatment

This study systematically evaluated the disinfection efficacy and mechanistic behavior of chlorine dioxide (ClO₂) toward representative waterborne pathogens, including Escherichia coli O157:H7, Bacillus subtilis (in vegetative and spore forms), and methicillin-resistant Staphylococcus aureus (MRSA), the latter representing both a clinically relevant pathogen and an antibiotic-resistant bacterium, in aqueous matrices. Microbial inactivation quantified by plate counts followed CT-based second-order kinetics, with rate constants ranging from 1.3(±0.2) to 5.4(±0.4)×10^–1 L·(mg·min) ^–1 for vegetative cells and 3.1(±0.3)×10^–3 L·(mg·min) ^–1 for spores, confirming the markedly higher resistance of spore-forming bacteria. Scanning electron microscopy revealed concentration-dependent morphological damage, including membrane destabilization, vesiculation, cellular deformation, and biomolecule leakage. Complementary biochemical analyses demonstrated increased extracellular protein release and lipid peroxidation (malondialdehyde formation), linking loss of culturability to progressive envelope disruption and oxidative stress rather than instantaneous cell lysis. Beyond cellular inactivation, ClO₂ effectively impaired extracellular genetic material. Quantitative PCR targeting the amp^R gene in plasmid pUC19 showed size-dependent degradation kinetics, with exposure-based rate constants of 1.8(±0.05)×10^–1 to 8.3(±0.01)×10^–1 L·(mg·min)^–1 for 192–851 bp amplicons. Despite substantial loss of qPCR-detectable gene targets, agarose gel electrophoresis indicated preservation of plasmid topology, suggesting that ClO₂ predominantly induces nucleobase damage rather than extensive strand scission. In river water, E. coli inactivation exhibited CT-dependent behavior comparable to that observed in buffered systems, with minimal influence of filtration, demonstrating the robustness of ClO₂ performance in complex matrices. Overall, these results demonstrate that ClO₂ enables consistent inactivation of both microbial cells and antibiotic resistance-associated genetic material across diverse aqueous environments through selective, structure-dependent mechanisms. ClO₂ represents a promising strategy for simultaneous microbial inactivation and gene abatement in water systems, supporting its application in potable reuse, decentralized treatment, and food-processing water management, where consistent disinfection efficacy under non-ideal conditions is required.