Genomic background and subinhibitory azithromycin exposure shape ceftriaxone resistance evolution in Neisseria gonorrhoeae

Abstract Combination antibiotic therapy is widely used to delay antimicrobial resistance, yet its evolutionary consequences remain poorly understood and may vary substantially across genetic backgrounds. Using short-term experimental evolution in six Neisseria gonorrhoeae strains spanning diverse resistance genotypes, we examined how ceftriaxone alone or combined with subinhibitory azithromycin shapes resistance trajectories. We show that adaptive outcomes under combination therapy are strongly dependent on genomic context. In a laboratory reference strain (ATCC 49226), ceftriaxone–azithromycin exposure triggered early disruption of DNA repair pathways, leading to hypermutation, rapid resistance amplification, and extensive diversification across efflux, envelope, and genome-maintenance pathways. In contrast, a clinical isolate (NG21) exhibiting the strongest ceftriaxone–azithromycin interaction among the strains tested failed to establish a sustained adaptive trajectory under dual pressure, despite robust adaptation under ceftriaxone monotherapy. Across the remaining strains, combination therapy consistently attenuated β-lactam resistance amplification relative to monotherapy, although the magnitude of suppression varied by genotype. Together, these results suggest that combination therapy does not impose a uniform evolutionary constraint: depending on the strain background examined, it was associated with either suppression of resistance emergence or increased evolutionary diversification. These findings highlight the importance of considering population structure and genomic context when evaluating the evolutionary consequences of combination therapies.