Neisseria gonorrhoeae 23S rRNA A2059G mutation is the only determinant necessary for high-level azithromycin resistance and improves in vivo biological fitness

Abstract

Objectives The global emergence of Neisseria gonorrhoeae isolates displaying high-level azithromycin resistance is a major concern for the currently recommended azithromycin/ceftriaxone dual therapy. N. gonorrhoeae high-level azithromycin resistance has been associated with an A2059G mutation in 23S rRNA. Here we investigated the specific contribution of this 23S rRNA A2059G mutation to high-level azithromycin resistance and its impact on biological fitness. Methods A2059G/G2059A alleles were specifically cloned into all four genomic copies of 23S rDNA of an azithromycin-susceptible isolate and a high-level azithromycin-resistant isolate. WT and mutant strains were subsequently investigated for azithromycin susceptibility using the agar dilution method. In addition, their biological fitness was studied by comparative liquid growth in the presence of hydrophobic and amphipathic compounds, by competition assays in a mouse vaginal tract infection model and by competition assays for invasion and intracellular survival. Results Azithromycin susceptibility analyses showed that the 23S rRNA A2059G mutation is the only genetic determinant required for N. gonorrhoeae to display the high-level azithromycin resistance phenotype. Further analysis of biological fitness showed that strains containing 2059G outcompeted isogenic strains containing 2059A for colonization in the mouse vaginal tract infection model and for invasion of HeLa cervical epithelial cells. Furthermore, the A2059G mutation enhanced growth in the presence of lithocholic acid or Triton X-100. Conclusions Our findings that the 23S rRNA A2059G mutation is sufficient for high-level azithromycin resistance and that this mutation generally enhanced the biological fitness of N. gonorrhoeae have important implications for the currently recommended treatment policies and antimicrobial stewardship programmes.