Genotypic and phenotypic analysis of Neisseria gonorrhoeae for the identification of resistance to various antibiotics in pregnant women attending antenatal care at the King Edward VIII Hospital in Durban, KwaZulu-Natal, South Africa.

Abstract

Introduction: Worldwide antimicrobial resistance (AMR) is making the clinical management of sexually transmitted infections (STIs) increasingly challenging with a particular emphasis on the emergence of antibiotic resistant strains of Neisseria gonorrhoeae (N. gonorrhoeae). In the current study, the detection and emerging patterns of drug resistant clinical isolates of N. gonorrhoeae to previous and current antibiotics to treat cervicitis pathogens as per syndromic management guidelines was investigated. Methodology: This cross-sectional study was conducted at King Edward VIII Hospital and included 307 antenatal attendees. Two endocervical swabs were collected from each enrolled woman. Each enrolled women also provided data on socio-demographic, behavioural and clinical factors. The first swab was placed in Amies Charcoal media immediately after collection. This swab was used to confirm the identification of N. gonorrhoeae clinical isolates using culture based assays. Culture confirmed isolates were grown in Mueller Hinton Broth and were each adjusted to have a bacterial suspension turbidity of a 0.5 McFarland Standard. These isolates were then subjected to antibiotic susceptibility testing to ceftriaxone, tetracycline, spectinomycin, azithromycin, ciprofloxacin, penicillin G and cefixime using the Etest™ method. The second swab was processed for molecular based assays. Extracted DNA from the second swab was subjected to the TaqMan quantitative Polymerase Chain Reaction (qPCR) assay, an in-house 16S ribosomal RNA (rRNA) PCR and PCR detection of the opacity (opa) gene. DNA extracted from the endocervical swabs and cultured isolates were used for the detection of specific targets (genes/plasmids/mutations) associated with resistance to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, azithromycin and ceftriaxone. All statistical analysis performed in this study was conducted in RS Studio. Results: The prevalence of N. gonorrhoeae was 7.8% (24/307) when detected by the TaqMan qPCR assay and 1.9% (6/307) by culture. When compared to culture, PCR for the opa gene and PCR for the 16S rRNA, the TaqMan qPCR assay was a more superior assay demonstrating a diagnostic accuracy of 94.5%. Susceptibility testing of the six isolates obtained after culturing showed resistant phenotypes for penicillin G (12 - 64 mg/L), tetracycline (1.9 - 32 mg/L) and ciprofloxacin (1.16 - 3 mg/L). Isolates displayed either dual or triple resistance to these 3 antibiotics. However, all isolates showed susceptibility to spectinomycin (>64mg/L), azithromycin (1mg/L), ceftriaxone (>0.125 mg/L) and cefixime (>0.125 mg/L). This study also detected the resistance determinants associated with penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, ceftriaxone and azithromycin from the molecular level using the primary endocervical swab sample. Gene mutations and plasmids associated with resistance to tetracycline (tetM gene carried on a plasmid), penicillin G (penicillinase producing plasmid) and ciprofloxacin (Ser-91 mutation) were detected confirming the results obtained with the susceptibility assays. Resistance mutations associated with the remaining antibiotics were not detected. There was a 100% correlation of cultured isolates and endocervical swabs for detecting the specific AMR determinants conferring resistance to tetracycline, penicillin G, and ciprofloxacin. Conclusion: The TaqMan qPCR assay has the ability to serve as a future diagnostic assay for the detection of N. gonorrhoeae. Despite the lack of resistance to spectinomycin, cephalosporins and azithromycin in our study population, continuous surveillance for emerging patterns of resistance to these antibiotics is still required since they form part of the current South African treatment guidelines. The detection of resistance determinants from the molecular level without the need for culture may prove to be more feasible for future epidemiological investigations focused on tracking antimicrobial susceptibility or resistance patterns in N. gonorrhoeae.