Effects of flavorless electronic cigarette aerosol on the survival and growth of common oral commensal streptococci

The use of electronic cigarettes (ECIG) has become very common. Consequently, critical analysis of the biological effects of ECIG aerosol deserves attention. Since the mouth is the first anatomical cavity exposed to aerosol, the oral bacteria within are also exposed. We hypothesize that while cigarette smoke has a detrimental effect on the survival and growth of oral commensal streptococci, flavorless ECIG aerosol does not. Survival and growth of several strains of commensal streptococci were measured after exposure to flavorless ECIG aerosol ± nicotine and smoke. Peristaltic pumps were used to transport flavorless aerosol ± nicotine or cigarette smoke into chambers containing recently seeded colony forming units of four strains of oral commensal streptococci on agar plates. Bacterial survival and growth, based on colony counts and sizes, were determined 24 hours post-exposure. Lastly, aerosol or smoke were delivered into chambers containing the four strains of streptococci pre-adhered to plastic coverslips. Bacterial survival and growth, as indicated by biofilm formation, were determined 24 hours post-exposure via scanning electron microscopy. 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 decrease bacterial survival and growth in all parameters measured. Therefore, unlike cigarette smoke, flavorless ECIG aerosol has only a small effect on the survival and growth of oral commensal streptococci.

hour at 37°C, 5% CO 2 and the excess unbound bacteria were washed 3 times with 0.5 ml PBS.

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Excess liquid on the coverslips was removed and the 12-well plates containing the coverslips 202 were exposed uncovered to air, flavorless ECIG-generated (± 20 mg/ml nicotine) or cigarette 203 smoke for up to 75 puffs. Following exposure, 1 ml of 50% BHI broth (v/v in sterile water) was 204 added to each well of the 12-well plate ensuring that exposed coverslips were completely 205 submerged. Exposed bacteria were subsequently incubated for 24 hours at 37°C, 5% CO 2 to 206 allow for biofilm growth on the coverslips. At the end of the 24-hour incubation period, BHI 207 broth was removed from the wells and the coverslips were washed 3 times with 1 ml PBS to 208 remove excess unbound bacteria. Biofilms were fixed with 1 ml of 4% formaldehyde for at least 209 30 minutes. Coverslips were then processed for SEM imaging (described below).

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Biofilm processing and SEM imaging 213 The 4% formaldehyde was removed from each well and each coverslip was rinsed two 214 times with 1 ml of deionized water. The biofilms on the coverslips were then dehydrated using

Effects of flavorless ECIG aerosol and smoke on CFU counts
CFU counts of commensal oral streptococci seeded on agar and exposed to puffs of air and 65 and 84 for S. oralis. Bacteria exposed to flavorless ECIG aerosol ± nicotine grow similar 261 numbers of colonies as compared to those exposed to air, although significant differences (p < 262 0.01) between aerosol with and aerosol without nicotine exist for S. gordonii and S. mitis at 75 263 and 50 puffs, respectively (Fig 3). In drastic contrast, bacteria exposed to 50 or 75 puffs of Besides the obvious absence of colonies following exposure to 50 and 75 puffs of smoke, 275 colonies exposed to 25 puffs of cigarette smoke also appear to have a smaller size compared to 276 those colonies exposed to air or flavorless ECIG aerosol ± nicotine. Figure 4A   aerosol ± nicotine and cigarette smoke. As shown, each of these species is able to grow biofilms after exposure to air (control) and flavorless ECIG aerosol ± nicotine, but not cigarette smoke.

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Compared to air exposures (0 and 75 puffs), 75 puffs of flavorless ECIG aerosol ± nicotine is 304 permissive for biofilm formation and growth regardless of the overall architecture of bacterial 305 communities for all four species. This indicates that flavorless ECIG aerosol ± nicotine has little 306 to no effect on oral commensal streptococci biofilm formation and growth under these 307 conditions. The current work demonstrates that flavorless ECIG aerosol ± nicotine has little to no 319 toxic effect on the in vitro growth of the four oral commensal streptococci tested here. Our data 320 show that CFUs for all four species exposed to flavorless ECIG aerosol ± nicotine can grow 321 similar numbers and to similar sizes as compared to their untreated counterparts. Our data also 322 demonstrate that bacteria attached to coverslips and exposed to flavorless ECIG aerosol ± 323 nicotine are also able to grow biofilms like their untreated controls. However, when bacteria are 324 exposed to cigarette smoke, growth of colonies and biofilms is severely impaired or completely obliterated. Furthermore, it is evident that nicotine is not the culprit of this impairment since 326 ECIG aerosol contained a higher concentration of nicotine than cigarette smoke on a per puff 327 basis ( Figure 2). Altogether, based on these results we propose that flavorless ECIG aerosol ± 328 nicotine does no apparent harm to these four oral bacteria under the conditions tested.

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Overall, flavorless ECIG aerosol (± nicotine) appears to have little or no effect on CFU 331 number and colony size of all bacteria tested (Figures 3 and 4). S. gordonii and S. intermedius 332 exhibit a slight decrease in CFU number when exposed to 25 and 50 puffs of aerosol without 333 nicotine, respectively, and S. gordonii exhibits a slight increase in colony size when exposed to