Single-bacterial cell insights into mechanisms of ceftriaxone resistance in Neisseria subflava.

The contribution of airway pathobionts to chronic respiratory disease is increasingly recognized, yet the evolutionary processes that shift commensals to pathogens remain poorly understood. Here we investigate how antibiotic pressure drives adaptation in Neisseria subflava, a common airway commensal associated with bronchiectasis. Using serial passage under ceftriaxone exposure, we observe a >300-fold increase in resistance, accompanied by enhanced biofilm formation and genetic reprogramming. Whole-genome sequencing reveals recurrent mutations in the adhesin gene ataA, while single-cell transcriptomics identifies six functionally distinct clusters indicating adaptive programs in growth, metal homeostasis, oxidative stress, and cell-wall remodeling. Notably, biofilm integrity is maintained through compensatory upregulation of comP and bamE, which promotes phagocytic evasion and resistance in experimentally evolved strains and clinical isolates. Iron availability further stabilizes biofilm and modulates antibiotic tolerance, underscoring metal homeostasis as a contributory adaptive axis. Together, these findings reveal a multifaceted strategy by which N. subflava exploits antibiotic selection to transition towards pathogenicity. By integrating experimental evolution with single-cell resolution, we establish a framework for understanding the commensal-to-pathobiont transition, with broad implications for the airway microbiome and antimicrobial resistance in chronic respiratory disease.