To elucidate the genome-based epidemiology and phylogenomics of azithromycin resistant Neisseria gonorrhoeae strains (MIC>2 mg/L) in 2009-2014 in Europe and clarify the azithromycin resistance mechanisms.. Azithromycin resistant N. gonorrhoeae in Europe

Objectives: To elucidate the genome-based epidemiology and phylogenomics of azithromycin resistant Neisseria gonorrhoeae strains (MIC>2 mg/L) in 2009-2014 in Europe and clarify the azithromycin resistance mechanisms. Methods: Seventy-five azithromycin resistant N. gonorrhoeae isolates (MICs: 4->256 mg/L) collected in 17 European countries during 2009-2014 were examined with antimicrobial susceptibility testing and whole genome sequencing. Results: Thirty-six N. gonorrhoeae multiantigen sequence typing (NG-MAST) STs and five major phylogenomic clades, including 4-22 isolates from several different countries per clade, were identified. The azithromycin target mutation A2059G (Escherichia coli numbering) was found in all four alleles of the 23S rRNA gene in all isolates with high-level resistance to azithromycin (n=4; MIC≥256 mg/L). The C2611T mutation was identified in 2-4 alleles of the 23S rRNA gene in the remaining 71 isolates. Mutations in mtrR and its promoter were identified in 43 isolates, comprising isolates within the whole azithromycin MIC range. No mutations associated with azithromycin resistance was found in the rplD or rplV gene and none of the macrolide resistance-associated genes mefA/E, ereA, ereB, ermA, ermB, ermC, and ermF was identified in any isolate. Conclusions: Clonal spread of relatively few N. gonorrhoeae strains accounts for the majority of the azithromycin resistance (MIC>2 mg/L) in Europe. The four isolates with high-level resistance to azithromycin (MIC≥256 mg/L) were widely separated in the phylogenomic tree and did not belong to any of the major clades. The main azithromycin resistance mechanisms were the A2059G mutation (high-level resistance) and C2611T mutation (low and moderate level of resistance) in the 23S rRNA gene.