TITLE:
Analysis of the Human Oral Microbiome of Smokers and Non-Smokers Using PCR-RFLP and Ribotyping
AUTHORS:
Shreyasee Chakraborty, Verneshia Persaud, Sonia Vanegas, Gloryanne Gautier, Nwadiuto Esiobu
KEYWORDS:
Oral Microbiome, PCR-RFLP, 16S rDNA Sequence, Restriction Enzymes
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.10,
August
27,
2014
ABSTRACT: Recent advances in the
field of microbial and medical ecology emphasize the critical role played by
oral bacteria in the delicate dynamic equilibrium of human health and disease,
creating the need to define the bacterial communities associated with healthy
and non-healthy conditions and to capture shifts in community structure germane
to diagnosis. Employing PCR-RFLP of the 16S rDNA gene from metagenomes and
plate-wash (cultured) bacteria of oral wash from 10 volunteers, this study
evaluated the stability of oral bacteria in healthy subjects and documented
community shifts in smokers. Sequence analysis of selected 16S gene amplicons
cloned with the Gene Hunter PCR-Trap vector and pCR 4-TOPO cloning kits was
conducted to determine the bacteria identity and diversity indices of the two
groups. Ribopatterns generated by the restriction enzymes HaeIII and Sau3AI were
significantly (p AluI using the GelCompare II software cluster analysis. A stable
core of bacteria DNA fingerprint was detected in all healthy subjects, and
remained unchanged over the study period of 3 months. Signature bands (1500 bp
with HaeIII) in smokers and in
non-smokers (800 bp and 700 bp with Sau3A1) were evidently suggesting the
presence of potential biomarkers of healthy and non-healthy states. There was
no significant difference in the DNA fingerprints of cultured and metagenomic
extracts. The genera Xanthomonas, Streptococcus and phylum Candidatus
occurred in large numbers in both groups, however, a major shift in composition
with the dominance of gram-negative bacteria in smokers compared to healthy
subjects was quite remarkable. Taxonomic diversity in smokers was quite high,
including members of the genera Rothia, Synechococcus, Neisseria, Thiomargarita and
Pyrobaculum. These data highlight the presence of a stable core microbiome
amidst a wide diversity, identify a distinct smokers’ cluster and open the way
for the search for potential biomarkers for specific diseases.