Mechanistic Effects of E-Liquids on Biofilm Formation and Growth of Oral Commensal Streptococcal Communities: Effect of Flavoring Agents.

Background: Vaping has become a global health concern. As research continues, more studies are beginning to question the relative safety of E-liquid flavoring additives. The oral cavity is the first site of exposure to E-liquid aerosol, making it critical for investigation. Because of the importance of commensal bacterial biofilms for oral health, we sought to explore the effects of E-liquids ± flavors on the formation and growth of single- and multi-species biofilms and to investigate the mechanism of inhibition. Methods: Quantitative and confocal biofilm analysis, death curves, and colony-forming units (CFU) were evaluated with flavorless and flavored (tobacco, menthol, cinnamon, strawberry, blueberry) E-liquids using four strains of oral commensal bacteria (Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis, and Streptococcus oralis). Results: All flavoring agents show a dose-dependent inhibition in the growth of single-species and multi-species biofilms. Furthermore, CFUs, death curves, and light microscopy show that flavoring agents have a bactericidal mode of inhibition on the growth of these oral streptococci. Conclusions: These results show that flavored, rather than unflavored, E-liquids are more detrimental to biofilm formation and growth of oral commensal bacteria. Consequently, E-liquid flavorings agents could pose risks to the oral microenvironment, and by extension, to systemic health.

Flavorless vs. Flavored Electronic Cigarette-Generated Aerosol and E-Liquid on the Growth of Common Oral Commensal Streptococci.

INTRODUCTION: Electronic cigarette (ECIG) use or vaping has become popular globally. While the question "Is vaping safer than smoking?" continues, it is becoming clearer that one of the most dangerous components of E-liquids are the flavorings. Since the oral cavity is the first anatomical site to be assaulted by ECIG aerosol, the aim of this study is to test the hypothesis that flavored ECIG aerosols or E-liquids pose a more detrimental effect on the growth of commensal oral streptococcal bacteria compared to flavorless aerosols or E-liquids. METHODS: Kirby Bauer assays and 24-h planktonic growth curves were used to compare the effects of flavorless vs. flavored (tobacco, menthol, cinnamon, strawberry and blueberry) ECIG-generated aerosols and E-liquids on the growth of four common strains of oral commensal bacteria (Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis and Streptococcus oralis). RESULTS: Kirby Bauer assays revealed inhibition of growth for all bacteria tested when exposed to 100% menthol, cinnamon or strawberry flavors. In contrast, 5% flavor in E-liquid had no effect. When exposed to 100 puffs of ECIG-generated aerosol ± flavors (≈ 0.05% flavor in brain heart infusion media) or an equivalent amount of E-liquid ± flavors, twenty-four hour planktonic growth curves indicated no effect on growth for all streptococci tested. Subsequent twenty-four hour planktonic growth curves testing the effects of E-liquid ± flavors (0.0625, 0.125, 0.25, 0.3125, 0.625, and 1.25% flavor in brain heart infusion media) revealed dose-dependent inhibition of growth, particularly for menthol, cinnamon and strawberry), for all bacteria tested. CONCLUSION: These results support the hypothesis that flavored E-liquids are more detrimental to the growth of oral commensal bacteria than unflavored E-liquids. The streptococci tested in this study are early colonizers and part of the foundation of oral biofilms and dental plaque. Disturbances in the composition and growth of these primary colonizers is crucial to the development of a healthy dental plaque and host-bacteria interactions. E-liquids and their aerosols containing flavoring agents alter the growth of these bacteria. Such perturbations of pioneering oral communities pose a potential risk to the health of the oral cavity and, ultimately, health in general.

Oral Pathogen Porphyromonas gingivalis Can Escape Phagocytosis of Mammalian Macrophages.

Macrophages are phagocytic cells that play a key role in host immune response and clearance of microbial pathogens. Porphyromonas gingivalis is an oral pathogen associated with the development of periodontitis. Escape from macrophage phagocytosis was tested by infecting THP-1-derived human macrophages and RAW 264.7 mouse macrophages with strains of P. gingivalis W83 and 33277 as well as Streptococcus gordonii DL1 and Escherichia coli OP50 at MOI = 100. CFU counts for all intracellular bacteria were determined. Then, infected macrophages were cultured in media without antibiotics to allow for escape and escaping bacteria were quantified by CFU counting. P. gingivalis W83 displayed over 60% of the bacterial escape from the total amount of intracellular CFUs, significantly higher compared to all other bacteria strains. In addition, bacterial escape and re-entry were also tested and P. gingivalis W83, once again, showed the highest numbers of CFUs able to exit and re-enter macrophages. Lastly, the function of the PG0717 gene of P. gingivalis W83 was tested on escape but found not related to this activity. Altogether, our results suggest that P. gingivalis W83 is able to significantly avoid macrophage phagocytosis. We propose this ability is likely linked to the chronic nature of periodontitis.

Effects of Oral Commensal Streptococci on Porphyromonas gingivalis Invasion into Oral Epithelial Cells.

The objective of this study was to determine if the interaction between common oral commensal bacteria and oral epithelial cells would provide protective effects against the invasion of periodontopathogen Porphyromonas gingivalis. Oral epithelial OKF6/Tert cells were used in co-cultures with Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, and Streptococcus intermedius. The viability of OKF6/Tert cells following a bacterial challenge was evaluated by trypan blue exclusion. The adherence of commensal species was determined by CFU counts. P. gingivalis invasion in OKF6/Tert cells was assessed before and after exposure to commensal species according to CFU counts. Viability assays show that only S. gordonii and S. intermedius display low toxicity toward OKF6/Tert cells. Both commensals adhere to OKF6/Tert cells at an average ratio of 1 CFU to 10 cells. P. gingivalis invasion into host cells is significantly reduced by 25% or 60% after exposure to S. gordonii or S. intermedius, respectively. The results suggest that these commensal species bind to host cells and diminish P. gingivalis invasion. This is important in the context of periodontal disease since P. gingivalis primarily acts on the host by invading it. Therefore, efforts to decrease invasion will eventually lead to future therapies harnessing the mechanisms employed by oral commensal bacteria.

A Comparison of Flavorless Electronic Cigarette-Generated Aerosol and Conventional Cigarette Smoke on the Planktonic Growth of Common Oral Commensal Streptococci.

Background: Smoking is the number one predictor for the development of periodontal disease. Consequently, electronic cigarette (ECIG) use has prompted investigations into the health-related risks induced by ECIG-generated aerosol on oral commensal bacteria as compared to cigarette smoke. Since E-liquid contains fewer constituents than smoke, we hypothesize that growth media containing E-liquid or aerosol has less impact on oral commensal streptococci than cigarette smoke. Methods: Eight-hour growth curves were generated for three strains of streptococci following exposure of growth media to nicotine alone (0.05, 0.1, 0.2 mg/mL), E-liquid ± nicotine (2.3, 4.7, 7.0 µL/mL), ECIG-generated aerosol ± nicotine (25, 50, 75 puffs), or cigarette smoke (2, 5, 10, 25, 50, 75 puffs). Nicotine and E-liquid were added to the media at concentrations equivalent to vaporized amounts of 25, 50, or 75 puffs. Absorbance readings were taken at 0, 2, 4, 6, and 8 h of bacterial growth. Results: Both E-liquid and aerosol (±nicotine) had little to no effect on eight-hour streptococcal growth. In contrast, five puffs of smoke inhibited streptococcal growth. Conclusions: Smoke-treated growth media, but not E-liquid or ECIG-generated aerosol, inhibits the growth of oral commensal streptococci. A possible implication is that aerosol may induce less periodontitis than smoke.

A Comparison of Flavorless Electronic Cigarette-Generated Aerosol and Conventional Cigarette Smoke on the Survival and Growth of Common Oral Commensal Streptococci.

Background: The use of electronic cigarettes (ECIG) has become very common. Consequently, critical analysis of the biological effects of ECIG aerosol deserves attention. Flavorless ECIG aerosol is known to comprise fewer harmful constituents than cigarette smoke. Therefore, we hypothesize that aerosol has less immediate effect on the viability of oral commensal streptococci than smoke. Methods: Survival and growth of four strains of commensal streptococci were measured after exposure to flavorless ECIG aerosol ± nicotine and smoke. Peristaltic pumps were used to transport aerosol or smoke into chambers containing recently seeded colony-forming units (CFUs) of the oral commensal streptococci on agar plates. Bacterial survival and growth, based on colony counts and sizes, were determined 24 h post-exposure. Additionally, aerosol or smoke were delivered into chambers containing pre-adhered streptococci to plastic coverslips and biofilm formation was determined 24 h post-exposure via scanning electron microscopy. Results: The results suggest that flavorless aerosol ± nicotine has a modest effect on bacterial growth both as colonies on agar and as biofilms. In contrast, smoke dramatically decreased bacterial survival and growth in all parameters measured. Conclusion: Unlike cigarette smoke, flavorless ECIG aerosol has only a small effect on the survival and growth of oral commensal streptococci.

Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid.

Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation.