Effect of Cigarette Smoke on Oral Biofilm Formation

Objectives: Smoking is a known risk factor for oral cancer and periodontal disease. Dysbiotic biofilms cause diseases such as caries and periodontitis, while normal microflora are important in maintaining health. Previously, we showed that cigarette smoke significantly increases coaggregation among the primary colonizers but does not affect the secondary and tertiary colonizers. The purpose of the present study was to investigate the effects of cigarette smoke on oral biofilm formation.
Methods: Primary biofilms were created by seeding glass slides with Actinomyces naeslundii, Streptococcus mitis, S.sanguis, S.oralis, and Veillonella parvula, standardized to an optical density (OD) of 1.2 at 650 nm. Glass slides were seeded with 1 ml of each culture. Secondary biofilms were created by further seeding the primary biofilms with Fusobacterium nucleatum, Capnocytophaga ochracea, Porphyromonas gingivalis, Parvimonas micra, and Tannerella forsythia. The test group was conditioned with 1% cigarette smoke extract. The primary and secondary biofilms were stained with a live/dead stain and imaged using the Olympus FV1000 Multiphoton microscope. Five large colonies were imaged per plate, and the biomass, smoking thickness, and surface area were analyzed using Comstat 2. Statistical comparisons were done using 2-sample t-tests.
Results: Smoke conditioning resulted in a decreased biomass of primary biofilms, while increasing the biomass of secondary biofilms. In both primary and secondary biofilms, smoke conditioning also led to an increase in surface area of dead bacteria. The biofilm thickness was not different between smoke-free and smoke-rich primary or secondary biofilms.
Conclusions: Taken together with our previous work on coaggregation, the present study demonstrates that smoke conditioning leads to increased coaggregation among primary colonizers that results in structurally unsound biofilms with low biomass and large numbers of dead bacteria. This suggests that smoking may increase the risk for disease by preventing colonization by a commensal-rich community.