Evidence for a Constrained Mutational Pathway to High-Level Spectinomycin Resistance in Neisseria: RpsE Loop 2 Mutations and Associated Growth Costs

Antimicrobial resistance in Neisseria gonorrhoeae is a global concern. Spectinomycin is unusual in that resistance can emerge rapidly during localized outbreaks yet often disappears from clinical populations after drug withdrawal, suggesting an associated growth cost. To investigate evolutionary routes to spectinomycin resistance, we performed in vitro selection on two N. gonorrhoeae strains and two commensal Neisseria species. Derived cell lines were characterized by minimum inhibitory concentration (MIC) determination, whole-genome sequencing, growth-kinetics analysis, and molecular modelling of the RpsE (ribosomal protein S5) interface with the ribosome. All high-level resistant isolates (MIC > 2048 mg/L) acquired substitutions or deletions in loop 2 of RpsE. Modelling showed that these mutations perturb the conserved network of stabilizing contacts between RpsE residues Lys25 (Lys23 in E. coli numbering) and Lys28 (Lys26), as well as helix 34 nucleotides G922, A923, and C1069 of 16S rRNA, potentially altering the architecture of the spectinomycin-binding site. These mutations were associated with high-level resistance but reduced growth rates, with the resulting growth costs depending on the specific pattern of contact rearrangements. Convergent evolution towards loop 2 mutations supports the existence of a constrained mutational pathway to high-level spectinomycin resistance in the strains and species examined here. This constraint may help explain the rapid decline of resistant variants in the absence of drug pressure and underscores the importance of genomic surveillance.