Genomic Epidemiology of Azithromycin-Nonsusceptible Neisseria gonorrhoeae, Argentina, 2005–2019

Azithromycin-nonsusceptible Neisseria gonorrhoeae strains are an emerging global public health threat. During 2015–2018, the prevalence of azithromycin-nonsusceptible gonococcal infection increased significantly in Argentina. To investigate the genomic epidemiology and resistance mechanisms of these strains, we sequenced 96 nonsusceptible isolates collected in Argentina during 2005–2019. Phylogenomic analysis revealed 2 main clades, which were characterized by a limited geographic distribution, circulating during January 2015–November 2019. These clades included the internationally spreading multilocus sequence types (STs) 1580 and 9363. The ST1580 isolates, which had MICs of 2–4 μg/mL, had mutations in the 23S rRNA. The ST9363 isolates, which had MICs of 2–4 or >256 μg/mL, had mutations in the 23S rRNA, a mosaic mtr locus, or both. Identifying the geographic dissemination and characteristics of these predominant clones will guide public health policies to control the spread of azithromycin-nonsusceptible N. gonorrhoeae in Argentina.

Whole-genome sequencing (WGS) provides higher resolution and accuracy than other typing methods, making it an ideal method to study the dissemination and transmission dynamics of N. gonorrhoeae strains on a national and international level (21,22). Furthermore, WGS data offer insights into AMR determinants, thereby enabling prediction, enhanced detection, and characterization of high-risk clones (22,23). Several studies have found N. gonorrhoeae lineages and clones driving AMR transmission among N. gonorrhoeae strains within local, national, and international networks (16,17,(24)(25)(26). Genomic surveillance provides information on current and emerging trends of circulating strains. Phenotypic, epidemiologic, and genomic surveillance data are critical for designing public health interventions and treatment strategies. Genomic approaches, including molecular epidemiology and detection of AMR determinants, are crucial for monitoring resistance to firstline drugs. We examined the genomic background of azithromycin-resistant N. gonorrhoeae isolates with MICs >2 μg/mL collected throughout Argentina during 2005-2019.

Materials and Methods
We examined 96 azithromycin-resistant N. gonorrhoeae isolates (MICs >2 μg/mL) from male and female patients treated at STI hospitals throughout Argentina. We selected 95 isolates from 8,002 consecutive isolates collected through the Gonococcal Antimicrobial Susceptibility Surveillance Programme-Argentina during January 2005-November 2019; we also included an isolate with high-level azithromycin resistance cultured in 2001 (20). We confirmed the N. gonorrhoeae species by culture on selective agar media, microscopic analysis using Gram staining, rapid oxidase positivity, superoxol test, carbohydrate utilization test, and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (microflex LT/SH; Bruker Daltonik, https://www.bruker. com) (27). The study was approved by the Research Ethics Committee of the Hospital General de Agudos "Bernardino Rivadavia" (Buenos Aires, Argentina). MIC determinations and whole-genome sequencing for all isolates were conducted using methods previously described (Appendix, https://wwwnc.cdc. gov/EID/article/27/9/20-4843-App1.pdf).
For phylogenetic analysis, we identified singlenucleotide polymorphisms (SNPs) in sequence reads mapped against the WHO P reference genome using the variant calling tool Snippy version 4.4.5 (https:// github.com/tseemann/snippy). We identified and filtered recombinant regions using Gubbins version 2.1.0 (Sanger, https://sanger-pathogens.github.io/ gubbins); the resulting core SNP alignment consisted of 9,415 sites. We used IQ-tree version 1.6.1 (http:// www.iqtree.org) to infer a maximum-likelihood tree from the whole-genome SNP alignment with a generalized time-reversible model of evolution using gamma correction for among-site rate variation with 4 rate categories; branch support was estimated by bootstrap analysis of 10,000 replicates (32). We visualized the resulting phylogeny with Figtree version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and phandango (33). We clustered sequences using RAMI with a branch length threshold of 0.01 (34). For comparison, we selected international isolates and publicly available genomic data on the basis of MICs, MLST STs (i.e., ST9363 and ST1580), and NG-MAST genogroups (i.e., G470 and G12302) from the National Center for Biotechnology Information (https://www. ncbi.nlm.nih.gov), European Molecular Biology Laboratory (https://www.embl.org), and the DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp). We found 17 genomes from the United Kingdom, 3 from Canada, 3 from Scotland, 17 from Australia, 28 from the United States, 7 from Brazil, and 11 from Norway (16,17,24,25,(35)(36)(37). We generated a phylogenetic tree of 86 international and 96 isolates from Argentina as described for domestic isolates and visualized the tree in Figtree version 1.4.4. Sequence reads are available from the European Nucleotide Archive (accession no. PRJEB41007).

Patient Data
The 96 N. gonorrhoeae isolates were collected from male (90.6%) and female (6.3%) patients; sex was unreported for 3.1% of patients. Patient age was reported for 88 (91.7%) isolates. Patients were 4-47 years of age (mean 24.3 years of age); 79.5% were <30 years of age. In total, 72 isolates were cultured from the urethra, 11 from urine, 3 from the cervix, 2 from the vagina (in children 4 and 6 years of age), 1 from the pharynx, and 7 from an unreported site.

Molecular AMR Determinants
All 5 isolates with MICs of >256 μg/mL had the A2059G mutation in all 4 23S rRNA alleles, whereas none of the 91 isolates with MICs of 2-16 μg/mL had this SNP (Table 1). Most (72; 75%) isolates with MICs of 2-16 μg/ mL had the 23S rRNA C2611T mutation. Nearly all (70; 97.2%) of these isolates had the C2611T mutation in all 4 23S rRNA alleles, except for 2 isolates: 1 with a single mutated allele that had a MIC of 4 μg/mL and 1 with 3 mutated alleles that had a MIC of 8 μg/mL. Interspecies mosaics in the mtr locus (which encodes the tripartite MtrCDE efflux pump), as well as mutations in the mtrR promoter, coding region, or both, have been associated with increased azithromycin MICs (38)(39)(40). Among the 80 (83.3%) isolates with mtrR mutations, 17 (17.7%) had an mtrR-35A promoter deletion, 44 (45.8%) had an MtrR G45D amino acid mutation, 1 (1.0%) had an mtrR-35A deletion and MtrR G45D substitution, and 18 (18.8%) had a mosaic N. meningitidis-like mtrR promoter. We did not identify any isolates with the mtr 120 mutation. Eighteen isolates, all of which had MICs of 2-4 μg/mL, had no 23S rRNA mutations; however, 13 contained a mosaic mtrR promoter and 5 had a mtrR-35A deletion. Among 18 isolates with mosaic mtrR promoters, 100% also had mosaic sequences in the mtrD, 100% in mtrC, and 94.4% in mtrE loci. Fifteen isolates with a mosaic mtrD allele had sequences identical to the N. meningitidis-like mosaic previously described (39,40); 2 isolates had sequences sharing 97.8% identity and 1 had a sequence sharing 97.3% identity with the N. meningitidislike mosaic (Appendix Figure 1). Isolates containing a mosaic-like mtr locus had MICs of >2 to >256 μg/mL. Isolates with MICs of >256 μg/mL also contained the 23S rRNA A2059G mutation.
We did not find any mutations associated with macrolide resistance in the rplD gene, which encodes ribosomal protein L4, or the rplV gene, which encodes ribosomal protein L22 (23). In addition, we did not find AMR mutations in macAB, which encodes the MacA-MacB efflux pump, or the acquired macrolide resistance genes, ere, mef, erm, mph, and msr (38). An isolate that had a MIC of 2 μg/mL had an unclear resistance mechanism.

Molecular Epidemiology and Phylogenomic Analysis
Among the 96 N. gonorrhoeae isolates, we observed 42 NG-MAST STs, including 21 new STs and 25 STs represented by single isolates. We found 24 isolates belonging to ST470, 7 belonging to ST20102, 6 belonging to ST696, 4 belonging to ST12302, and 4 belonging to ST20104. We found 3 NG-MAST genogroups comprising >3 isolates: 33 belonged to G470, 10 belonged to G12302, and 10 belonged to G20102. We also documented 14 MLST STs, including 2 new STs and 8 STs represented by single isolates. We found 43 isolates belonging to ST1580, 14 belonging to ST1901, 14 belonging to ST9363, and 10 belonging to ST1584. NG-STAR showed 32 types, of which 11 were new and 20 were represented by single isolates.
We found 32 isolates belonging to NG-STAR type 1038, 10 belonging to type 179, 5 belonging to type 168, and 5 belonging to type 3200.
Clade 3 was composed of 10 isolates belonging to NG-MAST G20102 and MLST ST1584. Clade 3 isolates had a mean SNP difference of 1.1 (range 0-2). Eight isolates were collected in Córdoba, 1 in CABA, and 1 in Río Negro during 2017-2019; of these, 8 were from men. All isolates required an MIC of 4 μg/mL and possessed the 23S rRNA C2611T mutation in all 4 alleles.

A2059G (4; 5)
MtrR protein (no. isolates) A-deletion (12) †; N. meningitidis-like (14); G45D (41); mtr120 (0) ‡ A-deletion (6); G45D (3); mtr120 (0) N. meningitidis-like (4); G45D (1); mtr120 (0) Mosaic mtr locus (no. isolates) mtrC (14); mtrD (14); mtrE   resistant to benzylpenicillin, tetracycline, ciprofloxacin or some combination, but susceptible to ceftriaxone and cefixime. In Argentina, dual therapy is recommended as firstline treatment for uncomplicated gonorrhea, according to the WHO guidelines (6). The Argentine Ministry of Health and the Sociedad Argentina de Infectología recommend a single 1-g dose of azithromycin monotherapy for the treatment of Chlamydia trachomatis and Mycoplasma genitalium infections (41,42). These guidelines also recommend antimicrobial treatment for suspected infections. Azithromycin has a long half-life, resulting in detectable drug concentrations in human plasma for up to 14 days (43). Undiagnosed N. gonorrhoeae infections concurrent with the treatment of C. trachomatis and M. genitalium infections might lead to prolonged exposure to subinhibitory concentrations of azithromycin, thereby prompting the induction of or selection for resistance genes. In the United States and United Kingdom, dual therapy is no longer the first-line treatment. Instead, high-dose ceftriaxone monotherapy (500 mg in the United States or 1 g in the United Kingdom) is now recommended for treatment of uncomplicated gonorrhea (44,45). Moreover, additional treatment with doxycycline (100 mg 2×/d for 7 d) is recommended if chlamydial infection has not been excluded (44). Similar empirical antimicrobial therapies for gonorrhea and chlamydial infections might be of benefit in Argentina to reduce patient exposure to azithromycin and avoid the emergence of resistant gonococcal strains.
Azithromycin resistance (i.e., MICs of >2 μg/mL) in N. gonorrhoeae has been mainly associated with mutations in the 23S rRNA target (38). The 23S rRNA A2059G mutation causes high-level resistance (i.e., MICs of >256 μg/mL) and the C2611T mutation causes low-level resistance (i.e., MICs of 2-16 μg/mL) (38). We found that 75% of isolates had the C2611T mutation. These isolates were phylogenetically diverse; however, clade 1, which was predominated by MLST ST1580 and NG-MAST G470, comprised 38 (52.8%) isolates. NG-MAST ST470 has been associated with high-level resistance to azithromycin in Scotland (18). In addition, NG-MAST ST470 has >99% similarity to ST9768, which caused an outbreak of high-level azithromycin-resistant N. gonorrhoeae in the United Kingdom (16). Previous gonococcal evolution studies have estimated that ≈4 (range 0-14) SNPs occur per year per genome, enabling phylogenetic analysis (21). Isolates from Argentina differed from isolates from Scotland by >4 (mean 7.9) SNPs and the United Kingdom by 13 (mean 16.8) SNPs. In addition, NG-MAST ST470 isolates from the United States, Brazil, and Australia, all of which showed low-level resistance to azithromycin, were closely related to isolates from Argentina (mean 7-11 SNPs). These findings support the hypothesis that NG-MAST G470 strains from Argentina might be descended from 1 lineage of the ST470 clone, which has spread internationally and can develop high-level and low-level resistance to azithromycin. Previous research, especially that of Unemo et al. (38), hypothesized that gonococcal antimicrobial-resistant strains emerge through genetic events, such as horizontal gene transfer or spontaneous mutations; these strains can spread quickly within a geographic region through sexual networks. Furthermore, compensatory mutations or gene exchange might have preserved this lineage in Argentina. The presence of additional STs, such as the co-circulation of MLST ST1584 and MLST ST1580 (NG-MAST G470), suggests that novel introductions also have occurred.
The mtr locus recently has been described as a hotspot for genetic recombination; mosaic-like mtr loci are associated with decreased susceptibility to azithromycin (i.e., MICs of 1-4 μg/mL) and contribute to the survival and transmission of N. gonorrhoeae (39,40,46). Most clade 2 isolates were associated with MLST ST9363 and had a mosaic-like mtr locus. MLST ST9363 was the predominant strain type of isolates with MICs of 2-4 μg/mL identified in Australia during 2017 and the United States during 2014-2017 (24,25,35). We found that MLST ST9363 isolates from Argentina shared a high level of genomic similarity with the ST9363 clones reported in Australia, the United States, Canada, Norway, and Brazil, indicating that importation and dissemination has occurred. Those data further support the hypothesis that N. gonorrhoeae isolates carrying a mosaic-like mtr locus contribute to the emergence of isolates with low-level resistance to azithromycin in many countries (24,25). Isolates with MICs of >256 μg/mL have recently reemerged in Argentina (20). Those isolates belonged to clade 2 and were distinguished by the mosaic-like mtr locus and the A2059G mutation in all 4 23S rRNA gene alleles. The phylogenetic tree showed that these isolates were closely related to isolates from Norway (mean 10.2 SNPs) that also had MICs of >256 μg/mL, suggesting that strains carrying a mosaic-like mtr locus and 23S rRNA A2059G mutation can disseminate internationally. Previous studies have suggested that isolates carrying the A2059G mutation or mosaic mtr locus have enhanced fitness; elucidating the effects of both mechanisms on N. gonorrhoeae evolution might help predict the emergence and spread of azithromycin resistance (39,46,47).
Because we received a small number of isolates from some provinces, our dataset might have been limited by selection bias. In addition, we did not have access to therapy strategies and treatment success rates, which might have provided insight into the generation of resistance or the selection of azithromycin-resistant isolates. Finally, we obtained limited data regarding patients' sexual orientation and HIV status, but found that clade 2 strains were slightly more associated with male patients, including men who have sex with men, than clade 1 strains (100.0% vs. 92.1%). In addition, 3 patients who had infections caused by clade 2 strains were HIV-positive (data not shown). Increased awareness of the transmission dynamics of azithromycin-resistant gonococcal strains within sexual networks is crucial to confirming these observations. Continuing surveillance of the prevalence and distribution of azithromycin-resistant strains in addition to genomic monitoring using individual-level epidemiologic data should provide a more complete picture of azithromycin-resistant gonococcal strains. These data will inform public health strategies to control azithromycin-resistant N. gonorrhoeae.
In conclusion, the recent increase in the prevalence of azithromycin-resistant N. gonorrhoeae isolates in Argentina was mainly the result of the introduction and expansion of 2 clones belonging to MLST ST1580 and ST9363. The integration of appropriate STI diagnosis and antimicrobial prescription into health services combined with genomic, phenotypic, and epidemiologic gonococcal surveillance data will be critical in preventing the dissemination of gonococcal clones resistant to azithromycin, ceftriaxone, or both, and preserving the current available therapeutic option for gonorrhea.