Characterizing ceftriaxone tolerance in <i>Neisseria gonorrhoeae</i> across <i>in vitro</i> and <i>in vivo</i> models

This study aims to characterize the phenotypic behavior and <i>in vivo</i> persistence of a ceftriaxone-tolerant <i>Neisseria gonorrhoeae</i> clinical isolate from a single patient and evaluate the potential role of tolerance in treatment failure. A previously identified ceftriaxone-tolerant vaginal isolate was compared with isogenic and clinical non-tolerant strains. Bacterial growth was assessed <i>in vitro</i>, and tolerance was quantified using the minimum duration required to kill 99% of the population (MDK99), and persistence was evaluated in an <i>in vivo Galleria mellonella</i> infection model. Whole-genome sequencing (WGS) and transcriptomic (RNA-sequencing [RNA-seq]) profiling were performed to identify tolerance-associated genetic and transcriptional signatures. The tolerant strain exhibited prolonged MDK99 values across ceftriaxone concentrations, persisting for up to 24 hours under drug exposure. It also showed delayed early-phase growth, suggesting a fitness cost. <i>In vivo</i>, the tolerant strain remained viable up to 8 hours after treatment, whereas non-tolerant strains were cleared. WGS revealed identical gene content across all isolates, but non-synonymous mutations in pilE_3, a type IV pilin gene, were exclusively present in tolerant strains. RNA-seq analysis showed upregulation of pilin-associated genes and downregulation of zinc-independent ribosomal paralogs (<i>rpmE2</i> and <i>ykgO</i>), suggesting a combined mechanism of surface remodeling and translational suppression associated with the tolerant phenotype. Ceftriaxone tolerance enables prolonged survival of <i>N. gonorrhoeae</i> despite apparent susceptibility by standard MIC-based testing. This phenotype may contribute to treatment failure, recurrent infection, and ongoing transmission, indicating the need for revised diagnostic and therapeutic strategies.IMPORTANCECeftriaxone remains the last reliable option for gonorrhea therapy, yet recurrent infections can occur despite isolates being classified as susceptible by MIC testing. One possible explanation is antibiotic tolerance, a phenotype that allows survival during drug exposure without changes in MIC. Although tolerance has been described in other pathogens, its role in gonococcal infection has remained poorly defined. In this study, we provide the first detailed characterization of a ceftriaxone-tolerant <i>Neisseria gonorrhoeae</i> clinical isolate associated with repeated treatment failure. By combining <i>in vitro</i> killing assays, an <i>in vivo Galleria mellonella</i> infection model, whole-genome sequencing, and transcriptomic profiling, we demonstrate that tolerance enables prolonged survival under ceftriaxone and is linked to pilin gene variation and ribosomal remodeling. These findings illustrate how a clinically observed phenomenon can be mechanistically dissected and emphasize tolerance as a hidden factor contributing to gonococcal persistence and potential treatment failure.