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Tobacco smoking has been identified as one major risk factor for destructive periodontal disease. Scaling and root planing have been shown to be less effective in smokers with periodontitis. The aim of the present study was to compare the subgingival microbial flora of treated and untreated smokers and non-smokers.Four independent adult patient groups with periodontitis were included in this investigation: 88 untreated smokers (U-S); 90 untreated non-smokers (U-NS); 119 treated non-smokers (T-NS); and 171 treated smokers (T-S). Clinical variables included cumulative plaque index (CPI), probing depth (PD), clinical attachment level (CAL), cumulative bleeding index (CBI), and cumulative suppuration index (CSI). Paper point samples from the deepest bleeding pocket in each quadrant of the dentition were analyzed for the presence and levels of 6 periodontal bacterial pathogens using anaerobic culture techniques.U-S showed a higher mean cumulative plaque index than U-NS (3.5 versus 2.7). Mean PD and mean CAL were higher in the T-S in comparison to the T-NS group (7.0 versus 6.6 mm and 5.6 versus 4.7 mm, respectively). Microbiological characteristics of U-S were a higher prevalence of Prevotella intermedia/nigrescens and higher mean levels of Peptostreptococcus micros (Pm) and Fusobacterium nucleatum (Fn). T-S patients were characterized by higher prevalence of Bacteroides forsythus (Bf), Pm, and Campylobacter rectus (Cr) and higher mean levels of Pm and Fn. The mean percentage of B. forsythus tended to be higher in the T-S group than in the T-NS group (6.9% versus 5.6%). The relative risk to be infected with Bf, Pm, and Cr was statistically higher in smokers (odds ratios: 1.9, 1.9, and 1.6, respectively). The chance to findor =10% of Bf, Pm, and/or Fn was 3.3 higher in smokers when A. actinomycetemcomitans and P gingivalis were absent. Detection ofor =20% Pm/Fn in treated patients was strongly associated with smoking (odds ratio 13.8, P= 0.002).Smoking is a determining factor for the composition of the subgingival microflora in adult patients with periodontitis and may select for a specific cluster of periodontal pathogens, notably Bf, Pm, Fn, and Cr. On the basis of these observations, smoking, among other criteria, may be one parameter to use in deciding to treat refractory periodontitis in smokers with a systemic antibiotic therapy directed against smoking-associated periodontal bacteria.