one of the first ecosystems to come into contact with tobacco smoke , and to be affected by it , is the oral microbiome . smokers present a high - at - risk cohort for periodontitis ( tomar and asma , 2000 ) , a microbially driven oral disease that affects two - thirds of adult humans ( eke et al . , 2012 ) . we have previously demonstrated that smoking is associated with pathogen enrichment in periodontal disease ( shchipkova et al . , 2010 ) and that smokers acquire these pathogens within 24  h of biofilm formation ( kumar et al . , 2011b ) . the goal of the present study , therefore , was to investigate whether smoking has a role in the etiopathogenesis of periodontitis by creating an at - risk - for - harm subgingival microbiome in states of clinical health . the study was approved by the office of research at the ohio state university ( 2008h0122 ) and the nhs national research ethics service northeast ( 09/h0904/46 ) . subjects who reported diabetes , hiv , pregnancy , immunosuppressants , bisphosphonates , steroids , current orthodontic therapy , antibiotics or professional dental cleaning within 3 months , and those requiring pretreatment antibiotic coverage were excluded . subjects had least 20 natural non - carious teeth , 3  mm probing pocket depths at all sites , plaque index of 0.9 ( loe et al . , 1965 ) and gingival index of 1.2 ( loe and silness , 1963 ) . both groups were frequency - matched for age , gender , race / ethnicity , education and socioeconomic status ( supplementary table 1 ) . subgingival plaque was collected by inserting endodontic paper points into the interproximal gingival sulci of 10 randomly selected teeth and pooled . bacterial dna was isolated ( kumar et al . , 2012 ) and 16s pyrotag sequencing was performed ( dowd et al . , 2008 ) in a commercial facility ( research and testing laboratories , lubbock , tx , usa ) . for each sample , v1v3 and v7v9 regions of the 16s gene were sequenced and combined to create a composite data set ( kumar et al . , 2011a ) and compared with a locally hosted version of greengenes ( desantis et al . , 2006 ) using the quantitative insights into microbial ecology pipeline ( caporaso et al . , 2010 ) and the sequences are deposited in the sequence read archive database of ncbi ( accession number srs590909 ) . in all , 1  581  383 denoised and chimera - depleted sequences were classified with 97% similarity to 572 species - level operational taxonomic units ( s - otus ) ; with 15527 s - otus in each individual . smokers demonstrated a remarkable similarity in microbial composition ( figure 1a ) , as evidenced by significant lineage - based clustering using principal coordinate analysis ( pcoa ) ( p<0.001 , analysis of similarity ( anosim ) ) . we have previously shown that ethnic origin may be a significant determinant of subgingival microbial composition ( mason et al . , 2013 ) . however , when a subset of 50 caucasians ( figure 1b ) and 34 african americans ( figure 1c ) was analyzed by pcoa , both ethnic groups clustered by smoking status . in contrast , when a subset of 17 african - american and 25 caucasian smokers ( figure 1d ) was compared by pcoa no clustering was observed , indicating that smoking supersedes the influence of genotypic factors such as ethnicity on the microbiome . a principle trait of a health - compatible community is niche saturation , an ecological phenomenon where a few species dominate the community , and resist colonization by pathogenic organisms ( brockhurst et al . , 2007 ) , thus maintaining mucosal health ( abt and pamer , 2014 ) . increase in species diversity has been observed in periodontal disease ( loe et al . , 1965 ; listgarten , 1976 ; loesche and syed , 1978 ) , and high diversity drives further speciation ( emerson and kolm , 2005 ) , leading to perpetuation of disease . shannon diversity index incorporates both the number of s - otus ( richness ) and relative abundance of each s - otu ( evenness ) into a single value . whereas a diversity index of zero represents a mono - species community , a higher value may result either from the presence of several species or from equitable distribution of a few species . smokers demonstrated a significantly higher shannon diversity index ( 4.85 versus 4.35 , p=0.01 , welch analysis of variance ) , and lower variance ( as measured by the standard deviations on the diversity index ) than nonsmokers ( p=0.0002 , levene 's test ) , indicating that each smoker harbored more numbers of s - otus than a nonsmoker ; however , as a group , current smokers demonstrate less heterogeneity in the composition of the subgingival microbiome . thus , with respect to diversity , the microbiome of a clinically healthy smoker demonstrated the characteristics of a community associated with disease . thus , smoking appears to create a microenvironment that selects for a large and specific group of micro - organisms ; and by doing so , may decrease the niche saturation abilities of this community . this loss of microbial protection may be one mechanism by which smoking increases the risk for disease . smokers demonstrated higher abundances of anaerobes and lower levels of aerobes when compared with nonsmokers ( p=0.02 , t - test ) ( figure 2a , innermost circle ) . also , 67 of the 128 genera were significantly different in abundances between groups , as were 172 s - otus . the subgingival microbiome of smokers was enriched for periodontal and systemic pathogens fusobacterium nucleatum , f. naviforme , filifactor alocis , dialister microaerophilus , desulfobulbus sp . clone r004 , megasphaera sueciensis , m. geminatus , m. elsdenii , m. micronuciformis , acinetobacter johnsonii , a. guillouiae , a. schindleri , a. baumannii , a. haemolyticus , pseudomonas pseudoalcaligenes and pseudoramibacter alactolyticus ( figure 2a outermost circle , figure 2b , and supplementary table 2 ) , with significantly lower levels of several health - compatible commensals , for example , streptococcus sanguinis , s. parasanguinis , s. oralis , granulicatella elegans , g. adiacens , actinomyces viscosus , a. israelii , a. dentalis , neisseria subflava and hemophilus parainfluenzae ( figure 2a outermost circle , figure 2c and supplementary table 2 ) . f. nucleatum ( figure 2b ) acts as a bridging species ' , enabling co - adhesion between early ( commensal ) and late ( pathogenic ) colonizers . it has recently been implicated in the etiology of colorectal cancer ( rubinstein et al . , 2013 ) , another disease for which smokers present a high - at - risk cohort ( giovannucci , 2001 ) . the high abundance of this species in healthy smokers along with several pathogens suggests yet another mechanism by which smoking creates pathogen - rich oral biofilms . the high levels of this species in smokers may also suggest a mechanism by which smoking increases the risk for colorectal cancer , and warrants further investigation . another interesting observation was the high levels of f. alocis ( figure 2b ) in periodontally healthy smokers . earlier investigations by our group have identified f. alocis as a putative periodontal pathogen ( kumar et al . , 2005 ) and several lines of evidence have emerged since , supporting this hypothesis ( griffen et al . , 2012 , shaddox et al . , 2012 ) . f. alocis is an obligate anaerobe whose growth in vitro has been shown to be stimulated by oxidative stress ( aruni et al . , 2011 ) , which may explain the high levels observed in the present study . ( figure 2a and supplementary table 2 ) are usually abundant in supragingival plaque of caries - active individuals ( gross et al . , 2010 ) . we observed a significant elevation of s. mutans and lactobacillus salivarius in the subgingival community of smokers . various studies have demonstrated an increased risk of caries in smokers ( benedetti et al . , 2013 ) . the elevation of these cariogenic species could explain the high caries - risk in smokers , and requires further investigation . in summary , clinically healthy smokers possess a highly diverse , pathogen - rich , commensal - poor , anaerobic microbiome that is more closely aligned with a disease - associated community . this work suggests that smoking has a role in creating an at - risk - for - harm microbiome , thus priming the oral environment for a downstream  ecological catastrophe ' ( marsh , 2003 ) .
dysbiotic oral bacterial communities have a critical role in the etiology and progression of periodontal diseases . 
 the goal of this study was to investigate the extent to which smoking increases risk for disease by influencing the composition of the subgingival microbiome in states of clinical health . 
 subgingival plaque samples were collected from 200 systemically and periodontally healthy smokers and nonsmokers . 
 16s pyrotag sequencing was preformed generating 1  623  713 classifiable sequences , which were compared with a curated version of the greengenes database using the quantitative insights into microbial ecology pipeline . 
 the subgingival microbial profiles of smokers and never - smokers were different at all taxonomic levels , and principal coordinate analysis revealed distinct clustering of the microbial communities based on smoking status . 
 smokers demonstrated a highly diverse , pathogen - rich , commensal - poor , anaerobic microbiome that is more closely aligned with a disease - associated community in clinically healthy individuals , suggesting that it creates an at - risk - for - harm environment that is primed for a future ecological catastrophe .