Bead-Based Flow-Cytometric Analysis of Subgingival Bacterial Levels Following Smoking Cessation

Introduction: Although both bacterial plaque and smoking play important roles in periodontitis, associations between smoking and individual bacterial species or consortia have not been well elucidated. Further, there is very little evidence on the effect of smoking cessation on subgingival bacteria. Objectives: The purpose of this study was to enumerate the differences in levels of 8 bacterial species including cultivated and as-yet-uncultivated organisms following smoking cessation using bead-based flow cytometry. Methods: 16 smokers with generalized moderate to severe chronic periodontitis were treated with smoking cessation intervention and full mouth scaling and root planing. Clinical data and pooled plaque samples were obtained at baseline and 12 months from each subject using paper-points from six sites with probing depths >=5mm and attachment loss >=7mm. Genomic DNA was isolated and bacterial 16s RNA genes were amplified using polymerase chain reaction with biotin-tagged universal primers. Capture probes were prepared by covalently coupling 9 species-specific probes to 8 types of polycarboxylate microspheres with different internal fluorescence. The bead-probes were hybridized to the 16S amplicons. The probe-DNA complex was analyzed using a flow cytometer to estimate the levels of each target species based on the fluorescence from each bead-probe. One-way ANOVA was used to examine differences in levels of each species among quitters and non-quitters. Results: At 12 months post-intervention, 9 subjects had successfully quit smoking and 7 continued to smoke. No differences were observed in clinical parameters between quitters and non-quitters at any time point. The 12-month levels of Peptostreptococcus micros and Filifactor alocis were significantly higher in non-quitters than quitters (P<0.01). The 12-month levels of Veillonella parvula were higher in quitters than in non-quitters. Conclusions: Microbial recolonization following smoking cessation appears to be associated with lower levels of putative periodontal pathogens and higher levels of heath-associated species.