Background: Neisseria gonorrhoeae, second most common bacterial STI and a WHO priority pathogen, is increasingly exhibiting resistance to azithromycin, threatening current treatment protocols in India. Reliance on syndromic management exacerbates this issue, with limited diagnostic capacity for detecting AMR in such fastidious organisms. This study aimed to identify resistance mechanisms, sequence types, and phylogenetic relationships among azithromycin resistant N. gonorrhoeae strains and compare Minimum inhibitory concentration (MIC) values to mutations.
Methods: A cross-sectional study was conducted at National Reference Centre for STD, India. Total 1,816 clinical samples (urethral, endocervical, oropharyngeal, anorectal swabs) were processed, and culture-confirmed N. gonorrhoeae isolates were subjected to MIC strip method. Using Illumina based NGS, SNPs were detected in 23S rRNA gene and resistance-associated loci were detected for azithromycin, and cephalosporins in isolates phenotypically resistant to azithromycin. MLST, virulence gene profiling (VFDB), phylogenetic analysis were performed.
Results: Out of 146 N. gonorrhoeae isolates, 13% showed azithromycin resistance, including 7 with high-level resistance .Three azithromycin resistance genetic mechanisms were identified- 84.2% of resistant isolates carried the mtrA gene (efflux pump overactivation), 73.7% had the C2599T mutation in the 23S rRNA gene (structural modification in binding site), and mosaic mtr D alleles in 68.4% isolates.
58% isolates possessed triple resistance mechanisms simultaneously, and had MICs ≥12 µg/mL.
5 Sequence Types were identified major being ST7363 (68%).
Total 3 Clades were obtained in phylogenetic tree, most high-MIC strains clustered in Clade A and belonged to ST7363, a high-risk clone. Two isolates were extensively drug resistant- XDR (decreased susceptibility to cefixime/ceftriaxone – penA I312M) and three were MDR. A positive correlation was observed between resistance mutations and virulence factor load. As many as 68 virulence genes were identified in some strains, including those related to pili formation, LOS sialylation, and efflux pump mechanisms.
Conclusion: The findings indicate an ongoing clonal expansion (ST7363) of AMR N. gonorrhoeae strains, highlighting the urgent need for molecular surveillance, antimicrobial stewardship, and diagnostic strengthening in this LMIC. HLR and XDR strains underscore the need for accelerated efforts in vaccine development and the discovery of new therapeutic agents.