Introduction High-level azithromycin (Azi) resistance (HL-AziR) threatens gonorrhoea dual therapy (ceftriaxone 500 mg and Azi 1g) as it renders Azi ineffective. Between November 2014–2016, 58 cases of HL-AziR (MIC >256 mg/L) N. gonorrhoeae (NG) were detected in England. Whole genome sequencing (WGS) revealed that most HL-AziR isolates were from a single clade (NG-MAST ST9768) with an A2059G mutation in 3/4 or all 4 alleles of the 23S rRNA gene. Lower-level AziR (MICs 1.0–32 mg/L) is commonly associated with a C2611T 23S rRNA gene mutation and mtrR promoter mutations. We performed WGS of ST9768 isolates with Azi susceptibility (MICs).

Methods WGS was performed on 7 non-HL-AziR ST9768 isolates from Scotland isolated in 2014. A phylogeny was constructed using the maximum likelihood algorithm based on whole genome variants. Genetic resistance determinants were analysed by mapping the WGS short reads to the 23S rRNA gene.

Results All ST9768 isolates with Azi MICs of 0.12–1.0 mg/L were part of the same WGS clade as the ST9768 HL-AziR isolates. One susceptible isolate (MIC 0.12 mg/L) had 0/4 mutated (A2059G) 23S rRNA alleles, five susceptible isolates (MICs 0.25 mg/L) had 1/4 mutated alleles and one low-level resistant isolate (MIC 1.0 mg/L) had 2/4 mutated alleles. No isolates carried the C2611T mutation.

Conclusion This is the first report of the A2059G mutation in NG isolates with Azi MICs of 0.25–1.0 mg/L. The phylogeny suggested that the HL-AziR ST9768 isolates are descendants of the low-level AziR isolates, which are in turn, descendants of the susceptible isolates. We hypothesise that azithromycin exposure provided selection pressure for one or two mutated copies of the 23S rRNA gene to recombine with wild-type copies, leading to 3 to 4 mutated copies in HL-AziR isolates. Greater understanding of the prevalent mechanisms of lower level AziR is required as HL-AziR could emerge in isolates with A2059 mutations and eliminate the effectiveness of dual therapy.