Development and validation of a human 2D in vitro model of oral Neisseria gonorrhoeae infection

Objectives The global rise in Neisseria gonorrhoeae (NG) infections, particularly oropharyngeal cases, drives treatment failures from antibiotic resistance. However, infection dynamics within oropharyngeal sites remain unclear. We developed an in vitro model using three human oropharyngeal epithelial cells to investigate infection dynamics and evaluate treatment strategies. Methods Tonsillar, floor of mouth (FOM) and gingival cell lines were infected with NG strains: antimicrobial-susceptible FA1090 and antimicrobial-resistant WHO-R. Oral commensal Neisseria oralis served as a bacterial negative control. Infected cells were treated with antibiotics known to cure NG strains (ie, ciprofloxacin/azithromycin/ceftriaxone/cefixime) and an antimicrobial negative control that does not cure NG strains (ie, tetracycline) at 1×, 2× and 3× the minimum inhibitory concentration for 30, 60 and 120min. Post-treatment, cells were treated with gentamicin to eliminate extracellular bacteria, lysed and internalised NG quantified. Results NG invasion for both strains was highest in tonsillar cells and lowest in FOM cells. Gingival cells only demonstrated high invasion by FA1090. Validation experiments confirmed FA1090 clearance was highest with azithromycin, ceftriaxone and ciprofloxacin, while cefixime and tetracycline showed variable efficacy. No tested antibiotics cleared WHO-R from all cell lines. Gentamicin consistently failed to clear infections. There was minimal invasion of N. oralis across all cell lines. Conclusions NG demonstrates site-specific and strain-specific invasion of oral cells, targeting tonsils and gingiva. The model’s validity is supported by drug efficacy results aligning with clinical data and limited invasion by N. oralis. This model provides a basis for developing a three-dimensional system to better understand oropharyngeal NG infections and identify and evaluate novel treatments.