Persistent Gaps in the Ultimate Mechanisms of Antimicrobial-Induced Bacterial Killing.

Antibiotics remain pillars of modern medicine, yet the mechanisms underlying bacterial killing remain incompletely understood. This review addresses unresolved questions in antibiotic lethality, focusing on poorly defined cell-level events. How coinciding stress responses combine to drive killing, and how cells prioritise protective pathways are unclear. Metabolic state strongly modulates lethality, as growth rate, nutrient availability, and respiratory activity determine whether damage reaches a fatal threshold. A small subpopulation of genetically identical cells persists through treatment, but the signals governing entry and maintenance of this state remain elusive. The contribution of reactive oxygen species is context-dependent and debated. Species-specific differences in autolysin activation during cell wall targeting lack unifying principles, while ribosome-targeting antibiotics also induce secondary membrane perturbations whose mechanistic links to translation arrest are unresolved. Biofilms further complicate killing by limiting drug penetration and slowing growth, and host factors such as oxygen tension, pH, and immune pressure reshape bacterial responses in ways that are only beginning to be understood. Addressing these blind spots may reveal new vulnerabilities in bacterial physiology and guide the development of therapeutic strategies that improve killing while limiting tolerance and persistence.