Nicotine-free and Nicotine-containing E-cigarettes Differentially Alter Microbial Colonization Dynamics

Objectives: We have previously shown that e-cigarettes (ENDS) increase virulence of the oral microbiome, however, reasons for this pathogenicity are unknown. The present study investigated the effects of e-cigarette aerosol on colonization dynamics in oral biofilms.
Methods: Saliva-conditioned hydroxyapatite discs were seeded with six pioneer species to create commensal biofilms. Pathogen-rich biofilms were created by further seeding commensal biofilms with Fusobacterium nucleatum, followed 24 hours later by eight periodontal pathogens. Biofilms were created under exposure to either nicotine-containing e-cigarette aerosol, nicotine-free aerosol or clean air. RNA was isolated, and mRNA sequenced. Sequences were annotated using the KEGG database and Megan6 pipeline. Biofilms were imaged, surface topography quantified and analyzed with Imaris.
Results: Significant differences (Dunn’s test, p<0.001) were observed in surface area and volume following e-cigarette exposure. Nicotine-free ENDS increased area and volume of commensal biofilms. Addition of F.nucleatum plateaued this growth, while colonization with pathogens resulted in greater surface area than controls. Nicotine-containing ENDS, on the other hand, decreased commensal biofilm surface area. While inclusion of F.nucleatum reversed this effect, pathogen colonization led to a further decrease in surface are. This topographical shift parallels transcriptional activity related to cell wall and capsule synthesis. Significant differences (Adonis, p<0.05) in gene regulation were observed between ENDS and controls at all levels of biofilm development. A large number of these genes encoded cell wall and capsule synthesis. Specifically, downregulated cell wall and capsule synthesis genes ranged from 59 to 100 in nicotine-containing and nicotine-free biofilms, while upregulated genes ranged from 62 to 104. Cell wall and capsule genes that were induced only upon ENDS exposure varied from 119 to 168 for both nicotine-containing and nicotine-free ENDS.
Conclusions: E-cigarette aerosol differentially regulates bacterial colonization dynamics in commensal and pathogen rich biomes. This has important implications for host-bacterial homeostasis in periodontal health and periodontitis.