Molecular epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in men at risk in Gauteng South Africa

Abstract

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae has emerged worldwide and treatment failures of ceftriaxone and azithromycin; the last remaining empirical first-line therapy for gonorrhoea, are reported. However, there is little information about the situation in South Africa where syndromic management is used to treat sexually transmitted infections (STIs). The purpose of this PhD study was to investigate the occurrence of AMR and molecular epidemiology of N. gonorrhoeae infections in high-risk men from the public and private healthcare sectors in South Africa. The study included specimens from two study groups of participants: (1) core transmission groups of men-who-have-sex-with-men and men with recurrent discharge accessing sexual health services in Johannesburg; (2) N. gonorrhoeae and Mycoplasma genitalium isolates from patients accessing the private healthcare services. Urine and urethral swabs were collected from men for N. gonorrhoeae culture followed by antimicrobial susceptibility testing (AST). Molecular diagnostics for curable STIs was performed, including M. genitalium as an important coinfection, and whole genome sequencing (WGS) of gonococcal isolates to identify genetic resistance mutations and describe gonococcal populations. Antimicrobial susceptibility testing was performed on N. gonorrhoeae isolates obtained from private sector followed by genotyping using the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method. Melting curve and sequence analysis were performed on M. genitalium strains for detection of macrolide resistance-associated mutations in the 23S rRNA. The quinolone resistance-determining regions of the parC and gyrA genes were also sequenced. Neisseria gonorrhoeae was the main aetiology (82%) of urethral discharge in male core transmission groups. High rates of AMR to tetracycline, ciprofloxacin and penicillin were detected among gonococcal isolates and azithromycin resistance was identified in 15% of the gonococcal isolates obtained from public but not in the private sector. There was no resistance to spectinomycin and cephalosporins found. Resistance to azithromycin was associated with an A39T alteration in mtrR and A deletions in the mtrR promoter. Amino acid alterations in GyrA (S91F, D95G, D95A) and ParC (D86N, S87N, E91G) were associated with ciprofloxacin resistance. Tetracycline resistant isolates harboured a tetM plasmid and had mutations in the rpsJ gene. Whole genome sequencing analysis of the gonococcal isolates revealed a wide diverse epidemic with a substantial number of novel N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) (70%) and NG-MAST (70%) sequence types (STs) identified. Neisseria gonorrhoeae strains from the private sector were genetically diverse and a substantial number of novel NG-MAST STs (83%) were identified. A high rate of azithromycin resistance was detected (19%) in the private sector but not in the public sector in M. genitalium strains harbouring mutations in the 23S rRNA. Fluoroquinolone resistance (2%) was detected in M. genitalium isolates harbouring mutations in the gryA and parC genes. The results show AMR in N. gonorrhoeae, and in the important coinfection M. genitalium, has emerged in South Africa. This work highlights that WGS can be successfully implemented in a resource-constraint setting for microbiological characterisation of gonococcal populations and their mechanisms of resistance. There is a need to urgently introduce diagnostics for STI care and scale-up surveillance for early detection of emerging AMR in STIs, in both public and the private sector in South Africa.