Genomic characterization and global phylogenomic placement of a high-level azithromycin (HL-AziR) and fluoroquinolone-resistant Neisseria gonorrhoeae ST9363 strain from Chile

Background: Antibiotic-resistant Neisseria gonorrhoeae (Ng) is a WHO priority pathogen and represents a serious public health threat. This study reports the first Chilean Ng ST9363 isolate (GC11/23) exhibiting dual high-level resistance to azithromycin and fluoroquinolone, recovered from a urethral discharge sample in July 2023.

Methods: Antimicrobial susceptibility was assessed using disk diffusion and MIC strip tests. Whole-genome sequencing (WGS) was performed using a hybrid approach, followed by annotation using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). In silico analyses included Staramr, MLST and NG-STAR. Phylogenetic analysis was performed on the core genome of 2417 global ST9363 Ng isolates using IQ-TREE and visualised with TreeViewer.

Results: GC11/23 exhibited resistance to ciprofloxacin, tetracycline, penicillin and azithromycin, while remaining susceptible only to ceftriaxone and spectinomycin. The isolate belonged to ST9363 and NG-STAR 3194. Mutations associated with fluoroquinolone resistance were identified in gyrA and parC, while high-level azithromycin resistance (HL-AziR) was linked to mutations in all four copies of the 23S rRNA gene, as well as in mtrD and mtrR. Additional resistance-associated alterations included an insertion (ins346D) in penA and a mutation in rpsJ, conferring resistance to penicillin and tetracycline, respectively. Phylogenetic analysis clustered GC11/23 with isolates from the USA and Europe collected in 2019.

Conclusions: GC11/23 represents a multidrug-resistant Ng strain belonging to ST9363/NG-STAR 3194 and carrying mutations conferring resistance to fluoroquinolones, azithromycin, penicillin and tetracycline. Its phylogenetic placement within a European lineage highlights the global dissemination of HL-AziR and fluoroquinolone-resistant strains.