Single-entity tracking of intracellular resistance gene degradation by lattice-engineered nFe0 in anoxic groundwater.

Resolving how reactive materials interact with antibiotic-resistant bacteria at the single-entity level is essential for understanding intracellular resistance gene (iARGs) removal in anoxic groundwater, yet such processes remain inaccessible to ensemble-averaged methods. Here, a single-particle-single-cell analytical framework was established using time-of-flight mass spectrometry in combination with exogenous membrane labeling and endogenous elemental fingerprinting. This enabled the identification of single-entity material-membrane association, along with effective assessments of membrane damage and cellular integrity at both the single-particle and single-cell level. A promising groundwater remediation agent (lattice sulfur engineered nanoscale zero-valent iron) was selected as a representative system, where heterogeneous cellular responses within bacterial populations were directly resolved. The single-particle-single-cell analysis revealed that sulfur incorporation enhanced material-membrane association, intensified membrane perturbation, and promoted electron-transfer-associated intracellular DNA damage. These interfacial processes enabled rapid iARGs attenuation, with S-nFe0 achieving over 4-log iARGs removal within 120 min while simultaneously suppressing extracellular resistance gene (eARGs) release and accumulation. Moreover, the framework remained effective in real groundwater matrices and across multiple iron mineral systems, while S-nFe0 maintained stable iARGs and eARGs removal over six consecutive cycles with negligible hydrogen evolution and limited iron leaching. Overall, this work provides a broadly applicable analytical strategy for mechanistically linking interfacial material-cell processes to iARGs removal in groundwater.