Until 2006, gonorrhea was South Korea’s most prevalent bacterial sexually transmitted infection, but its incidence has since declined, primarily affecting individuals in their twenties [1]. In contrast, rates in the United States have increased, particularly among individuals aged 15–24 years [2]. Globally, ciprofloxacin resistance remains high, azithromycin resistance is increasing, and susceptibility to ceftriaxone is declining [3]. This trend has made treatment increasingly challenging and has complicated the selection of appropriate treatment regimens for gonorrhea.

Since 2014, we have analyzed Neisseria gonorrhoeae isolates from 35 hospitals in South Korea, and in 2018 and 2019, identified two strains with moderate azithromycin resistance. Azithromycin resistance and decreased susceptibility in N. gonorrhoeae have previously been reported in South Korea [4]. However, detailed whole-genome characterization of such isolates remains limited. Here, we report the genomic characterization of these two isolates in the context of evolving gonococcal treatment guidelines. These isolates represent, to our knowledge, the first azithromycin-resistant N. gonorrhoeae ST1600 strains reported in South Korea, and their N. gonorrhoeae multi-antigen sequence typing (NG-MAST) profiles differ from those of the azithromycin-resistant NG-MAST ST4207 and ST6762 lineages with elevated azithromycin minimum inhibitory concentrations (MICs) previously described in Japan [5].

We characterized these isolates as follows:

Specimens were inoculated onto modified Thayer-Martin medium and identified by MALDI-TOF mass spectrometry (Bruker) and biochemical testing using Vitek NHI cards (bioMérieux). Azithromycin susceptibility was assessed by the agar dilution method using GC II agar supplemented with 1% IsoVitaleX, according to the Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines [6,7]. Sequencing libraries were prepared from 50 ng of extracted DNA using the Twist Library Preparation EF Kit (Twist Bioscience), according to the manufacturer’s instructions. Paired-end sequencing was performed on a NovaSeq 6000 system (Illumina) using 2 × 150 bp reads. Library quantity and fragment size distribution were assessed using Qubit fluorometric assays (Thermo Fisher Scientific) and the Agilent 4200 TapeStation system (Agilent Technologies). FASTQ files were de novo assembled using Unicycler v0.4.0, and the resulting assemblies were evaluated for sequencing depth and standard assembly quality metrics before downstream analyses. Assembled FASTA files were analyzed using Pathogenwatch and the PubMLST database to determine NG-MAST and multilocus sequence typing (MLST) profiles, penA and 23S rRNA variants, and antimicrobial resistance determinants. Antimicrobial resistance determinants were identified based on curated N. gonorrhoeae reference schemes within Pathogenwatch and PubMLST and were confirmed when sequence identity and coverage relative to reference alleles were ≥ 95% and ≥ 90%, respectively.

The 2018 strain exhibited an azithromycin MIC of 32 µg/mL and resistance to penicillin G, tetracycline, and ciprofloxacin, while remaining susceptible to ceftriaxone and spectinomycin (Table 1). It carried a nonmosaic penA-109.001 allele with an A502V mutation, a -35A deletion in the mtrR promoter, and a C2611T mutation in all four 23S rRNA alleles. It was classified as ST1600 by MLST, and NG-MAST assigned it to ST16190 based on the porB 9414 and tbpB 831 alleles. The 2019 strain had a similar antimicrobial resistance pattern, with an azithromycin MIC of 32 µg/mL, but carried a non-mosaic penA-120.001 allele. It differed from the 2018 strain in the porB 9414 allele by a single-nucleotide polymorphism (A169G), yielding a novel NG-MAST type with the same tbpB 831 allele and MLST ST1600.