Chakraborty, Shreyasee

Defining the bacterial communities associated with healthy status will permit rapid
detection of shifts associated with disease and foster effective probiotic intervention. This study
compares the PCR-RFLP of oral metagenomes and cultured bacterial community, as well as the
bacterial diversity profile of smokers, non-smokers and oral disease patients. The goal was to
evaluate the stability of bacteria associated with health and capture possible shifts potentially
diagnostic of smokers and oral disease.
Oral wash samples were obtained from 5 healthy and 5 smokers twice, 3 months apart. Samples
from 5 oral disease patients were also collected. Metagenomic and the genomic DNA of a
cultured subset were amplified using primer 1492R and 27F.The generated 16SrRNA gene
amplicons were used for arestriction digestion assay and bcloning with the Gene Hunter PCRTrap
vector and pCR 4- TOPO cloning kits. The restriction fingerprints were statistically tested
using Gel Compare II. The cloned 16S genes were sequenced using the ABI GeneAmp 9700
Thermal cycler. Sequences were analyzed by BLAST on the RDB II database and the HOMD.
Sau 3AI and Alu I produced the best distinctive markers of 300 bp in healthy nonsmokers and
500bp in smokers in plate wash communities. The 16S sequence data suggest the presence of a
core microbiota in all subjects mostly Streptococcus, Gamella, Candidatuse spp and confirmed
that the smokers harbored highly diverse and distinct community Neisseria pharynges, Rothia
mucilaginosa. Remarkably, there was a high stability of the fingerprints and diversity profile for
smokers and nonsmokers after 3 months.

This study evaluated the stability of oral bacteria in healthy subjects and documented
community shifts in smokers and oral/periodontal disease by employing PCR-RFLP,
DGGE and sequence analysis of the 16S rDNA gene from metagenomes and plate-wash
(cultured) bacteria of oral wash from 15 participants,. A stable core of bacterial DNA
fingerprint was detected within and between subjects and did not change over time when
analyzed in smokers and healthy non-smokers. Signature bands in smokers, non-smokers
and periodontal disease subjects were evident suggesting the presence of potential
indicators of health and poor oral health. Taxon diversity was higher in smokers
including members of the genera Rothia, Synechococcus, Neisseria, Thiomargarita and
Pyrobaculum but highest in periodontal disease. The two techniques successfully aligned
the subjects within appropriate categories (based on their oral microbial genetic
patterns)confirming their diagnostic suitability.