Adverse Effects of Electronic Cigarettes on the Disease-Naïve Oral Microbiome
October 1, 2021
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By Lindsey N. Clark, MD, and Taimur K. Mian, MD
Dr. Clark is a PGY-1 Psychiatry Resident at Community Health Network, Indianapolis.
Dr. Mian is a Psychiatry Clinical Faculty Member at Community Health Network, Indianapolis.
- A comparison study attempted to identify the effects of electronic cigarette use on oral bacterial communities by comparing the oral microbiome genetic profiles of 123 participants divided into five cohorts: electronic cigarette users, smokers, nonsmokers, former smokers, and dual users.
- Robust methodologies of deoxyribonucleic acid isolation, metagenomic sequencing, principal coordinate analysis, and linear determinate analysis identified the oral microbiome of electronic cigarette users as significantly different from that of smokers and nonsmokers.
- Results showed electronic cigarette users to have significantly more bacterial genetic clusters related to pathogenic species, bacterial virulence factors, and proinflammatory signals.
SYNOPSIS: A robust metagenomic comparison study of the effects of electronic cigarettes on oral microbiomes suggested that the unique aerosol component of electronic cigarettes poses increased risk to development of oral cavity disease.
SOURCE: Ganesan SM, Dabdoub SM, Nagaraja HN, et al. Adverse effects of electronic cigarettes on the disease-naïve oral microbiome. Sci Adv 2020;6:eaaz0108.
The human oral cavity is host to a diverse group of bacterial communities, with more than 1,000 species across seven phyla having been identified. Imbalances in the oral microbiome communities and increased inflammatory responses to oral bacteria have been associated with diseases, such as periodontitis, dental caries tooth loss, and oropharyngeal cancer.
The effects of cigarette use, chewing tobacco, and alcohol consumption on the oral cavity have been studied widely, with results showing that these habits offset the healthy balance of oral bacterial communities and create the potential for harmful bacterial species to be introduced into the oral cavity, increasing the risk for disease development.1,2 However, the effects of electronic cigarettes (e-cigarettes) on the oral microbiome, and their potential for harm, remain understudied. Considering that e-cigarettes are relatively novel by delivering nicotine in a unique heated aerosol component, more insight into their effects on the oral microbiome is needed to determine their risk for oral cavity illness.
Ganesan et al provided new evidence regarding the effects of e-cigarettes on the oral cavity in a comparison study analyzing the subgingival microbiomes across e-cigarette users, smokers, and nonsmokers. The study recruited 123 individuals considered systemically and periodontally healthy. Periodontal health, or gingival inflammation assessment, was defined as attachment loss of < 1, fewer than three sites with 4 mm of probe depths, and a bleeding index of < 20%. Systemic health was established using the American Society of Anesthesiologists Physical Status Classification. Participants were placed in one of five groups based on their current tobacco use status: smoker, nonsmoker, e-cigarette user, former smoker currently using e-cigarettes, and concomitant cigarette and e-cigarette user. Current smoking was defined as at least a five-pack-year history. Nonsmokers were defined as people having consumed fewer than 100 cigarettes in their lifetimes. E-cigarette users were defined as having used e-cigarettes daily for at least three months, with at least one cartridge per day, or 1 mL of liquid per day. Sample size was determined to have at least an 80% chance of detecting clades of bacterial genes that differed in abundance by 1%. Exclusion criteria consisted of presence of controlled or uncontrolled diabetes, human immunodeficiency virus infection, use of immunosuppressant medication, bisphosphonates, steroids, antibiotic therapy or oral prophylactic procedures within the past three months, and fewer than 20 teeth in dentition.
Subgingival plaque samples using sterile endodontic paper points and gingival crevicular fluid collections were taken from each participant at 15 sites within the oral cavity. Bacterial deoxyribonucleic acid (DNA) was isolated from the paper point samples and quantified using Qiagen DNA MiniAmp kits and Qubit fluorometers. Phylogenetic profiles for each participant’s oral microbiome were created using Kraken v1.1 software and complete genome data lists from the Human Oral Microbiome Database. Each phylogenetic profile then was tested for alpha (within-group) and beta (between-group) genetic diversity using PhyloToAST v1.4 and QIIME v1.9.
For quality control, all DNA samples were sequenced in two runs, and samples were randomly assigned to each run. Each participant’s gingival crevicular fluid samples also were used to determine cytokine assays, measuring levels of interferon-gamma (INF-gamma), interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha).
To assess and compare the genetic variability and gene clustering in the oral microbiome community between each participant cohort, Ganeson et al generated principal coordinate analysis (PCoA) plots using the software PhyloToAST (PcoA.py). The significance of identified genetic clustering in participant cohorts was determined using permutational multivariate analysis of variance (MANOVA) (adonis function, vegan package for R). Additionally, the relative abundance of functional genes across participant cohorts was assessed using linear discriminant analysis (LDA) (scikit-learn v0.18.0). Wilk’s lambda was used to test for significance of LDA identified functional gene clustering.
Overall, Ganeson et al compared oral microbiomes of 20 e-cigarette users, 25 nonsmokers, 25 current cigarette smokers, 25 former smokers, and 25 dual users. E-cigarette users were 21 to 35 years of age, predominantly Caucasian, and reported using e-cigarettes with 6 mg to 18 mg of nicotine. Interpretation of PCoA and LDA plots revealed three significantly different oral microbial profiles of e-cigarette users, smokers, and nonsmokers (P = 0.008, MANOVA/Wilks). There was no significant microbial profile difference between e-cigarette users, dual users, and former smokers who had switched to e-cigarettes (P = 0.27 and 0.35).
Further nonmetric multidimensional scaling analysis of variance in the user groups showed that the duration of e-cigarette use (< 6 months vs. > 10 months) was the strongest source of variation, with nicotine concentration and type of flavoring not contributing to variations seen in the oral microbial profiles. Furthermore, 70% of the metagenome in e-cigarette users was shared by more than 80% of subjects, whereas the smoker and nonsmoker cohorts only shared 40% and 50% of their metagenomes, respectively. This presence of a large, core microbiome present in a majority of the e-cigarette users that differed significantly from the microbiome of smokers and nonsmokers suggests that the aerosol effects of e-cigarettes alters the oral cavity bacterial community via different mechanisms than traditional cigarettes. (See Table 1.)
Table 1. Comparison of Significant Variance in Oral Microbiome Profiles
Former Smokers (Current E-Cigarette Users)
P = 0.008*
P = 0.008*
P = 0.27
P = 0.35
*Statistically significant values
Furthermore, the metagenome profile of e-cigarette users showed a greater abundance of genes related to virulence factors when compared to smokers and nonsmokers (P < 0.05), including cell wall and capsular polysaccharides, peptidoglycan, and lipopolysaccharide biosynthesis, stress response, quorum sensing and biofilm formation, and resistance to antibiotics and toxic compounds. These findings suggest that the unique oral bacterial community found in e-cigarette users could increase exposure to bacterial factors that cause disease in human hosts.
Additional analysis of the cytokines assays taken from the participant’s gingival crevicular fluid showed e- cigarette users to have significantly higher levels of proinflammatory cytokines IL-2, IL-6, GM-CSF, TNF-alpha, and INF-gamma, and lower levels of anti-inflammatory cytokine IL-10 (P < 0.05, Dunn’s test). These findings suggest that the e-cigarette user microbial profile creates a higher inflammatory burden and response when compared to cigarette users and nonsmokers.
It is important to note that cigarette users showed a similar increase in proinflammatory cytokines when compared to never smokers as well with increases in cytokines IL-2, IL-6, and IL-8, TNF-alpha, and INF-gamma, and lower levels of IL-10. The authors noted that this difference may suggest that, while both e-cigarette and cigarette use increase inflammatory response cytokines in the oral microbiome, different biological pathways are involved.
Ganeson et al presented the results of a robust, rigorous comparison study combined with comparative metagenomics, adding to our knowledge of the effects of e- cigarette use on the oral cavity. Ultimately, the study shows that e-cigarette use creates the potential to shift the oral microbiome community to a state with increased exposure to bacterial virulence factors and increased host inflammatory response, both states that can predispose an individual to oral cavity diseases. The authors point out that, while none of their e-cigarette users currently had been diagnosed with periodontitis, the functional genetic profile of their oral microbiome “bore remarkable resemblance to individuals with periodontitis.”3,4 Ganeson et al hypothesized that the glycerol and glycol component of e-cigarette aerosol may serve as a nutrient source for bacteria, altering the microbial profile and biofilm structures in the oral cavity of e-cigarette users.
While e-cigarettes have been on the market for 17 years, and studies have begun to show the potential for harmful effects on the respiratory system, little remains known about their effect on the oral cavity and the oral microbiome.5,6 Because the heated aerosol contains fewer harmful chemicals than an ignited tobacco device, it has been suggested that their use is safer than cigarettes, and they often have been advertised as a smoking cessation device.7
However, studies show that the biggest user group of e-cigarettes is a young population that is taking up e-cigarette use as a new habit vs. a tool for smoking cessation. One study has even found that 20% of high schoolers in the United States admitted to trying e-cigarettes at least once a month.8 Given these numbers, physicians are incredibly likely to encounter a young patient who is using e-cigarettes recreationally.
Physicians should consider the results of the Ganesan et al study, along with the growing body of literature, which demonstrates that e-cigarettes expose users to a unique aerosol-nicotine compound that may increase the risk for oral cavity disease. Talking to adolescent and adult patients about e-cigarette use and the effects on their oral health may be beneficial for reducing the risks of developing oral cavity diseases associated with e-cigarette use.
- Börnigen D, Ren B, Pickard R, et al. Alterations in oral bacterial communities are associated with risk factors for oral and oropharyngeal cancer. Sci Rep 2017;7:17686.
- Wade WG. Oral microbiome in health and disease. Pharmacol Res 2013;69:137-143.
- Dabdoub SM, Ganesan SM, Kumar PS. Comparative metagenomics reveals taxonomically idiosyncratic yet functionally congruent communities in periodontitis. Sci Rep 2016;6:38993.
- Duran-Pinedo AE, Chen T, Teles R, et al. Community-wide transcription of the oral microbiome in subjects with and without periodontitis. ISME J 2014;8:1659-1672.
- Gotts JE, Jordt S, McConnell R, Tarran R. What are the respiratory effects of e-cigarettes? BMJ 2019;366:l5275.
- Mukhopadhyay S, Mehrad M, Dammert P, et al. Lung biopsy findings in severe pulmonary illness associated with e-cigarette use (vaping): A report of eight cases. Am J Clin Pathol 2020;153:30-39.
- Pulvers K, Nollen NL, Rice M, et al. Effect of pod e-cigarettes vs. cigarettes on carcinogen exposure among African American and Latinx smokers. JAMA Netw Open 2020;3:e2026324.
- Wang TW, Neff LJ, Park-Lee E, et al. E-cigarette use among middle and high school students — United States, 2020. MMWR Morb Mortal Wkly Rep 2020;6:1310-1312.
A robust metagenomic comparison study of the effects of electronic cigarettes on oral microbiomes suggested the unique aerosol component of electronic cigarettes poses increased risk to development of oral cavity disease.
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