Sustained Transmission of Neisseria gonorrhoeae Strains with High-Level Azithromycin Resistance (MIC ≥ 256 μg/mL) in Argentina, 2018 to 2022

ABSTRACT Azithromycin combined with ceftriaxone is the recommended dual therapy for uncomplicated gonorrhea in many countries. Nevertheless, the increasing prevalence of azithromycin resistance compromises the effectiveness of this treatment strategy. From 2018 to 2022, we collected 13 gonococcal isolates with high-level azithromycin resistance (MIC ≥ 256 μg/mL) across Argentina. Whole-genome sequencing revealed that these isolates were mainly represented by the internationally spreading Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroup G12302, containing the 23S rRNA A2059G mutation (in all four alleles) together with mosaic mtrD and mtrR promoter 2 loci. This information is important to develop targeted public health policies to control the spread of azithromycin-resistant N. gonorrhoeae in Argentina and internationally. IMPORTANCE Azithromycin resistance in Neisseria gonorrhoeae has been increasing in numerous populations worldwide, which is of concern, as azithromycin is part of the recommended dual treatment in many countries. Here, we report 13 N. gonorrhoeae isolates with high-level azithromycin resistance (MIC ≥ 256 μg/mL). This study observed that high-level azithromycin-resistant gonococcal strains have shown sustained transmission in Argentina and are related to the successful international clone NG-MAST G12302. Genomic surveillance together with real-time tracing and data-sharing networks will be crucial in controlling the spread of azithromycin resistance in gonococcus.

line treatment (2,7,8). In Argentina, a significant increase in azithromycin-resistant isolates (MICs $ 2 mg/mL) was recently observed (9), and high-level azithromycin-resistant (AZM-HLR) isolates (MIC $ 256 mg/mL) were also identified (9). This increasing azithromycin resistance threatens the effectiveness of the ceftriaxone-azithromycin dual antimicrobial treatment for gonorrhea. Here, we investigated the epidemiological and genomic characteristics of AZM-HLR N. gonorrhoeae isolates in Argentina from 2018 to 2022.
All patients (n = 13) were male, and 10 were men who have sex with men (MSM) ( Table 1). The median age was 32 years (range, 20 to 54 years). Four males were HIVpositive and three had previous gonorrhea. Of the patients, 12 had urogenital infections and 1 had an extragenital infection (perianal abscess). Data regarding previous or current treatment was not available. The isolates were identified in Ciudad Autónoma de Buenos Aires (CABA; n = 9) and Buenos Aires (n = 2) and Córdoba (n = 2) provinces, respectively. All isolates were AZM-HLR but were susceptible to ceftriaxone, cefixime, and spectinomycin, and all except one were susceptible to ciprofloxacin ( Table 1). All isolates had the 23S rRNA A2059G mutation in all four 23S rRNA gene alleles together with mosaic-like 2 mtrR promoter and mtrD sequences (5, 12) (Fig. 1). All isolates belonged to MLST ST9363, and nine were also characterized as NG-MAST ST3935 and NG-STAR type 1993 (CC213) ( Table 1). The mean SNP difference between isolates was 24.1 (range, 0 to 78). The Argentinean isolates were closely related to AZM-HLR isolates from Norway, Indianapolis, USA, and Barcelona, Spain ( Fig. 1) (16-18), i.e., with mean pairwise SNP differences of 26.6 (range, 12 to 64) compared to Norwegian isolates, 26.7 (12 to 64) for Barcelona isolates, and 28.9 (14 to 67) for Indianapolis isolates.
In the present study, phylogenomic analysis showed that many AZM-HLR isolates have a high degree of genomic relatedness, suggesting a recent and sustained transmission within a sexual network mainly composed of MSMs. All isolates contained the 23S rRNA A2059G SNP, in all four alleles, which causes AZM-HLR, and an mtrR promoter/mtrD mosaic 2 encoding the MtrRCDE efflux pump system (5,9,12). These isolates were mainly represented by MLST ST9363 and NG-MAST ST3935, and this AZM-HLR strain appears to have been in sustained transmission at least since 2018 in Argentina (9). NG-MAST ST3935 is  (17). The first four columns next to the tree represent the country or city of origin/reporting. The following two columns describe azithromycin resistance determinants and the last three columns represent the MLST ST (all ST9363), NG-MAST genogroup (all except three were G12302), and NG-STAR clonal complexes (all except two were CC213). more than 99% similar to ST12302, which also carries an mtrR promoter/mtrD mosaic 2 sequence and has been responsible for the recent expansion of azithromycin-resistant gonococcal strains (MICs, 2 to 4 mg/mL) in Europe, Canada, and the United States (5,6,21). Previous gonococcal evolution studies have estimated that approximately 4 (range, 0 to 14) SNPs occur per genome per year, allowing the relationship between strains to be determined (22). Our Argentinean AZM-HLR isolates showed a high level of genomic similarity with AZM-HLR isolates reported in the United States, Spain, and Norway (mean, 26 to 29 SNPs), indicating that importation and subsequent dissemination has occurred (16)(17)(18).
In Argentina, dual therapy (ceftriaxone 500 mg plus azithromycin 1 g) is recommended as a first-line empirical treatment of uncomplicated gonorrhea (4). Nevertheless, the significant increase of low-level azithromycin resistance (MICs, 2 to 16 mg/mL) (9), and the spread of AZM-HLR isolates, may threaten the efficacy of this treatment strategy. Due to the increase of azithromycin-resistant N. gonorrhoeae isolates, some countries, such as the United States, United Kingdom and some other European countries, have returned to first-line treatment with ceftriaxone high-dose (0.521 g) monotherapy (2,7,8). High-dose ceftriaxone monotherapy for gonorrhea might be of benefit in Argentina to mitigate the selection of azithromycinresistant isolates, which could also be reasonable because ceftriaxone resistance has been rare (23,24). In general, there is an urgent need for improved access to N. gonorrhoeae diagnostics, antimicrobial resistance detection, and genomic epidemiological data. This information will be critical in preventing the dissemination of gonococcal clones resistant to azithromycin (and/or resistant to ceftriaxone) and preserving the current available therapeutic option for gonorrhea.
In conclusion, AZM-HLR gonococcal strains have shown sustained transmission in Argentina and are related to the successful international clone NG-MAST G12302 (5,(16)(17)(18). Enhanced antimicrobial resistance surveillance, including genomic monitoring using individual-level epidemiologic data, is necessary for the development of bettertargeted public health policies to prevent the spread of these gonococcal isolates.
Data availability. Sequence reads for all Argentinean isolates used in this study are available at the European Nucleotide Archive (https://www.ebi.ac.uk/ena/browser/ home) under the BioProject accession number PRJEB55521.