High Counts and Anthracene Degradation Ability of Streptococcus mutans and Veillonella parvula Isolated From the Oral Cavity of Cigarette Smokers and Non-smokers

Abstract

The composition and metabolic functions of oral microbiota are affected by many factors including smoking leading to several health problems. Cigarette smoking is associated with changes in oral microbiota composition and function. However, it is not known if the depletion of certain bacterial genera and species is due to specific toxins in cigarette smoke, or indirectly due to competition for colonization with smoking-enriched bacteria. Therefore, the aim of this study was to determine the effect of cigarette smoking on the microbial prevalence and polycyclic aromatic hydrocarbons (PAHs) biodegradation of selected enriched and depleted oral bacteria from oral microbiota of smokers compared to that in non-smokers. Samples of oral rinse from smokers and non-smokers were collected (n = 23, 12 smokers and 11 non-smokers) and screened for oral bacterial strains of Streptococcus mutans, Lactobacillus spp., and Veillonella spp. Comparing counts, S. mutans, V. tobetsuensis, and V. dispar showed higher counts in smokers compared to non-smokers while the Lactobacillus spp. were higher in non-smokers. Lactobacillus fermentum was prevalent in smokers, representing 91.67% of the total Lactobacillus spp. isolates. The biodegradation potential of anthracene; a representative of PAHs of collected isolates, in single and mixed cultures, was assayed with anthracene as the sole source of carbon using 2,6-dichlorophenol indophenol (2,6-DCPIP) as indicator. S. mutans isolates recovered from smokers showed higher degradation of anthracene compared to those recovered from non-smokers. The anaerobic anthracene biodegradation activity of V. parvula isolates from non-smokers was the highest among all isolates of the three recovered genera from the same subject. The anthracene biodegradation potential of Lactobacillus spp. was variable. Combinations of isolated bacteria in co-cultures showed that Lactobacillus spp. interfered with anthracene biodegradation ability along with the viable counts of S. mutans and Veillonella spp. In conclusion, oral dysbiosis due to cigarette smoking was observed not only due to changes in oral bacterial relative abundance but also extended to bacterial functions such as anthracene biodegradation tested in this study. Microbe-microbe interactions changed the anthracene biodegradation potential and growth of the microbial mixture compared to their corresponding single isolates, and these changes differ according to the constituting bacteria.

Keywords: anthracene; microbial interactions; oral microbiota; polycyclic aromatic hydrocarbons biodegradation; smoking.

FIGURE 1

Prevalence of Lactobacillus spp., and Veillonella spp. in smokers and non-smokers groups.

FIGURE 2

Total viable bacterial counts of isolated identified Streptococcus mutans, Lactobacillus spp., and Veillonella spp. from oral rinse of smokers (n = 12) and non- smokers (n = 11). Statistical differences calculated using Kruskal-Wallis followed by multiple comparisons using Dunn’s corrections. Data presented as median ± interquartile range, where p < 0.05 was considered to be statistically significant.

FIGURE 3

DCPIP assay of a representative smoker sample showing dye discoloration. during anthracene biodegradation over a period of 8 days in a representative smoker sample.

FIGURE 4

Anthracene biodegradation after 8 days in smokers and non-smokers compared to standard strains, positive and negative controls. Biodegradation of anthracene by (A) S. mutans; (B) Lactobacillus spp.; and (C) Veillonella spp. represented as color intensity (%) of DCPIP. Statistical differences calculated using Mann-Whitney U-test followed by multiple comparisons using Holm Sidak’s corrections. Data presented as median ± interquartile range, where p < 0.05 was considered to be statistically significant. Bacillus subtilis (positive control one of the highest anthracene biodegrading microorganisms) and negative controls (no anthracene or no bacteria) were significantly different from the isolated species and ATCC strains. ****p < 0.00001.

FIGURE 5

Viable counts and anthracene biodegradation by selected bacteria isolated from representative smoker and non-smoker participant over a period of 10 days. S. mutans from (A) smokers and (B) non-smokers (C) L. fermentum from smoker; (D) L. salivarius from non-smoker; (E) V. tobetsuensis from smoker; (F) V. parvula from non-smoker.

FIGURE 6

Anthracene biodegradation represented as DCPIP color intensity % by S. mutans, Lactobacillus spp. and Veillonella spp. mixtures after 8 days in smokers and non-smokers. (A) Duos and triples mixtures from smokers; (B) Duos and triples mixtures from non-smokers; (C) Consortium from representative smokers and non-smokers.

FIGURE 7

Viable bacterial count of S. mutans, Lactobacillus spp., and Veillonella spp. triple assay in presence of anthracene as a sole carbon source at day 0 and day 8. (A) smokers and (B) non-smokers. For Lactobacillus spp., no colonies was detected at day 8. Statistical differences calculated using Kruskal-Wallis followed by multiple comparisons using Dunn’s corrections. Data presented as median ± interquartile range, where p < 0.05 was considered to be statistically significant.