Cigarette Smoking and Oral Microbiota

The oral microbial community (the microbiota), is centrally related to nutrition, metabolism,
immunity, inflammation, and endocrine balance. Cigarette smoking is associated with serious
health outcomes including cancer, cardiovascular disease and chronic lung disease. However,
little is known about the relationship between cigarette smoking, oral microbiota and
tobacco-related health outcomes. Microbiotas in the different sites of the same oral cavity
vary widely. Recent studies indicated that the difference in microbial profiles between
buccal mucosa and gingival plaque is as distinct as the difference between tongue and stool.
To date, knowledge about the effect of smoking on oral microbiotas is lacking. As an initial
step toward understanding the role of the oral microbiota in smoking-related health outcomes,
we propose a pilot study to evaluate the effects of cigarette smoking on the microbiotas in
buccal rinse obtained from the oral cavity and across 8 different oral cavity sites/niches
(saliva, swabs from tongue dorsum, hard palate, buccal mucosa, keratinized gingiva, palatine
tonsils, supragingival plaque, subgingival plaque). We will seek to recruit up to 50
volunteers through the Eastman Dental General Dentistry Clinic, Rochester, NY, including 25
current smokers with > 5 years of smoking history and 25 never smokers as a comparison group.
Age-group, gender, and race (Caucasian/African- American) will be matched for the two groups.
In each subject, the buccal cell (mouthwash) collection will be collected according to the
PLCO buccal cell collection protocol. In addition, collections across 8 different oral cavity
sites will be done using a protocol developed for the Human Microbiome Project. These 9
collections will be used for high throughput sequencing of 16S rRNA microbial genes at the
Institute of Genome Sciences, University of Maryland School of Medicine. The findings will
provide information about the effect of smoking on microbial taxa composition and relative
abundance at each collection site. This pilot study will demonstrate the effects of smoking
on oral microbiotas across different oral cavity sites and will also demonstrate which oral
collection site is most promising for the detailed study about cigarette smoking and oral
microbiota. In addition, the data collected will provide a validation framework to use the
PLCO buccal specimens for large-scale studies. The information about the validity of using
PLCO buccal cell collection will be an invaluable asset for further large scale studies of
the microbiota and cancer risk. We will also be able to investigate if there is any taxonomy
or microbial diversity difference between heavy smokers and never-smokers. This pilot study
will provide essential information about collection, design and analyses that will enable
further studies of the relationships between cigarette smoking, the oral microbiome and
tobacco-related cancers.

Under an Amendment, the participants will be contacted to provide another set of oral swabs
and mouthwash samples. The goal is to examine the stability or oral microbiota in 3-6 months
and confirm the results based on the first time point. The collection methods, assays and
analyses will be analogous to those performed previously.

- INCLUSION CRITERIA

This study will recruit a convenience sample of 50 volunteers (25 current smokers with at
least 5 years of smoking history, 25 never smokers). Current smokers are defined as
individuals who have smoked more than 100 cigarettes in their lifetime and have smoked 5 or
more cigarettes in the last 24 hours. Recent use of other tobacco products (pipe, cigar,
snuff, cigarillos, and chewing tobacco) is an overall exclusion, but use in the remote past
(> 6 month ago) is acceptable in smokers. Never smokers are defined as individuals who have
never smoked cigarettes nor used any other tobacco products including pipe, cigar, snuff,
cigarillos, or chewing tobacco.

The ethnic mix of the clinic is roughly 50% Caucasians and 50% African- Americans with a
small number of unspecified or other racial groups. The median age is about 50 and the
gender mix consists of an equal number of men and women. We therefore will select smokers
and frequency match to non-smokers based on ethnicity (White, African-American), gender
(male, female), and age (above or below the median, estimated to be 50).

EXCLUSION CRITERIA

We will exclude pregnant women and other racial groups because they may represent very
small numbers and thus be difficult to match. Hormonal changes associated with pregnancy
and unique cultural habits associated with specific ethnic groups could be associated with
highly unique or variable microbiome patterns, and therefore reduce the power to detect
differences associated with smoking which is our primary goal. We will also exclude
subjects with antibiotic usage in the last three months and subjects with previous
diagnosed major periodontal disease or cancer because they might be potential confounders.