Respiratory Tract Microbiota in Electronic Nicotine Delivery System Users: Insights into Microbial Shifts and Health Implications

Abstract

Introduction: The respiratory microbiota is an important component of airway health and immune defense. Although the effects of conventional, tobacco smoking on respiratory microbial communities are well documented, the impact of electronic nicotine delivery systems (ENDS), such as e-cigarettes and heated tobacco products, remains less clear. Given the growing use of these products in Lebanon, this study investigated the respiratory microbiota of Lebanese ENDS users compared with healthy controls.

Methods: In this study, expectorated sputum samples were collected from adult healthy never-smokers and electronic nicotine delivery systems (ENDS) users residing in Lebanon following the Institutional Review Board approval and written informed consent. Bacterial genomic DNA was extracted from sputum using the QIAamp UCP Pathogen Mini Kit, purified with the ZymoBIOMICS cleanup kit, and assessed for concentration and purity by NanoDrop spectrophotometry. The V3-V4 hypervariable region of the 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. Raw paired-end reads were quality filtered and denoised using TrimGalore and the DADA2 plugin in QIIME2, and taxonomic classification of amplicon sequence variants was performed using a naïve Bayes classifier trained on the Greengenes 16S rRNA database.

Results: Alpha diversity did not differ significantly between ENDS users and non-smokers for Chao1 (H = 1.033, p = 0.310), Shannon (H = 0.314, p = 0.576), Pielou’s evenness (H = 0.314, p = 0.576), or Simpson diversity (H = 0.589, p = 0.442). Jaccard beta diversity was likewise not significantly different (PERMANOVA pseudo-F = 1.118, p = 0.062). Both groups showed similar phylum-level profiles dominated by Bacillota. In contrast, significant differences were detected at lower taxonomic levels, with Actinomyces (p < 0.0001) and Nanosynbacter (p = 0.0329) differing at the genus level and Rothia sp001808955 differing at the species level (p = 0.0002). These results indicate selective taxonomic shifts in ENDS users despite broadly preserved overall community diversity and structure.

Conclusion: In this Lebanese cohort, ENDS use was associated with selective, fine-scale shifts in the expectorated sputum microbiota rather than community-wide disruption. While overall microbial diversity and community structure remained largely preserved, differences at the genus and species levels suggest that ENDS exposure may subtly influence specific respiratory microorganisms. ENDS usage may foster a microflora environment with increased biofilm-forming capacity and pro-inflammatory status, with reduced colonization resistance. Longitudinal. Larger studies with higher-resolution analyses are needed to clarify the biological and clinical significance of these changes.