To Örebro University

Background: Oropharyngeal Neisseria gonorrhoeae (NG) infections are common, increasing, and have a higher treatment failure compared with other infection sites. Due to antimicrobial resistance, NG has become a global public health threat as available treatments remain scarce. Little is known about where NG colonizes in the oral mucosa and therefore, where antibiotics need to be distributed to cure infection. A recent review also highlighted the lack of oral cell models available for investigating NG infection. We recently started creating an in-vitroco-culture model for NG strains with human oral epithelial cells to understand patterns of NG growth in the mouth and examine antibiotic uptake by oral cell types supporting NG growth.

Methods: NG strains were grown on Chocolate agar with IsoVitaleX and in modified Fastidious broth media in optimised conditions. NG colonies were assessed using a colony counter (Scan1200, Interscience technology). In a 2D model, NG were co-cultured with 4 human oral keratinocyte cell lines isolated from different anatomical subsites of theoral cavity. Intra- and extra-cellular NG was quantified, and intracellular spatial distribution was assessed with confocal microscopy and immunocytochemistry. Invasion into 3D spheroids was characterised with penetration depth assessed via histological analysis (haematoxylin and eosin staining) and immunocytochemistry with images taken on a Zeiss Axioscan7 slide scanner or LSM80 0confocal microscope. Real time invasion into spheroids was imaged using a MuviCyte live-cell imaging system. Lastly, host cell viability in response to NG infection was also assessed.

Results: We created the first-of-its-kind in-vitro model for NG oral infection demonstrating that it is possible to co-culture NG with oral derived cells. NG survives and infects oral cells in an in vitro setting in both 2D and 3D models. Different strains of NG infected oral cells to significantly different degrees.

Conclusion: Our presented model can be used to explore the interactions of NG with oral tissues and to investigate current and new therapeutics against oropharyngeal gonorrhoea.