Australian Dental Journal
The official journal of the Australian Dental Association
Australian Dental Journal 2015; 60: 225–232 doi: 10.1111/adj.12225
Subgingival bacterial recolonization after scaling and root planing in smokers with chronic periodontitis M Feres,* MAC Bernal,* F Matarazzo,† M Faveri,* PM Duarte,* LC Figueiredo* *Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos, S~ao Paulo, Brazil. †Department of Periodontology, State University of Maringa, Maringa, Parana, Brazil.
ABSTRACT Background: The aim of this study was to compare subgingival bacterial recolonization patterns after scaling and root planing in current smokers and non-smokers. Methods: 15 smokers and 15 non-smokers with chronic periodontitis received scaling and root planing in six visits lasting one hour each, over a period of 21 days. Clinical monitoring was performed at baseline and 180 days, and microbiological monitoring was performed at baseline, immediately after scaling and root planing (Day 0) and at 42, 63 and 180 days post-therapy. Subgingival plaque samples were analysed by checkerboard DNA–DNA hybridization. Results: An improvement in clinical condition was observed for smokers and non-smokers; however, non-smokers showed a greater reduction in mean clinical attachment level in intermediate sites in comparison with smokers (p < 0.05). At Day 0, there was a significant reduction in the mean counts of the three pathogens from the red complex, Eubacterium nodatum and Parvimonas micra only in non-smokers (p < 0.05). There was a significant increase in the proportion of host-compatible species in non-smokers and smokers from baseline to 180 days posttherapy (p < 0.05). However, a significant decrease in the pathogenic species was observed only in non-smokers. Conclusions: Smokers were more susceptible to the re-establishment of a pathogenic subgingival biofilm than nonsmokers. Keywords: Chronic periodontitis, dental scaling, microbiology, smoking. Abbreviations and acronyms: BOP = bleeding on probing; CAL = clinical attachment level; MB = marginal bleeding; PCR = polymerase chain reaction; PD = probing depth; PI = plaque accumulation; SRP = scaling and root planing; SUP = suppuration. (Accepted for publication 20 September 2014.)
INTRODUCTION There is a body of evidence suggesting that the onset, progression and severity of periodontal tissue destruction may be modulated by some environmental and systemic factor modifiers, such as diabetes mellitus and smoking. There is consistent agreement about the existence of a strong positive correlation between cigarette smoking, deeper periodontal pockets and increased alveolar bone loss, clinical attachment and teeth.1 In contrast, conflicting results have been published about the influence of smoking on subgingival biofilm and periodontal tissue breakdown. While some investigations2–8 have found little or no difference in the composition of the subgingival biofilm between smokers and non-smokers, others have found that cigarette smoking is associated with a higher prevalence and/or levels of pathogenic species.9–12 © 2015 Australian Dental Association
Several studies have shown that smoking is related to an unfavourable clinical response to both nonsurgical and surgical periodontal therapy.13,14 In regards to the microbiological data, some investigations have suggested that smokers harbour higher levels of periodontal pathogens than non-smokers after scaling and root planing (SRP),4,7,15,16 whereas others have demonstrated no influence of smoking on the microbiological outcomes of treatment.8,17 The hypothesis that smoking could contribute to a lower reduction and faster re-establishment of subgingival pathogenic biofilm after periodontal therapy may explain the frequently observed negative clinical response of smokers.17 Therefore, the aim of the present study was to compare the patterns of subgingival bacterial recolonization in moderate to deep sites right after, and up to 6 months post-SRP in current smokers and non-smokers with chronic periodontitis. 225
M Feres et al. MATERIALS AND METHODS The ideal sample size, to ensure adequate power for the microbiological data of this study, was calculated considering differences of at least 6.6 percentage points between groups for the proportion of the red complex species and a standard deviation of 5.0 percentage points.18 Based on these calculations it was determined that 13 subjects per group would be necessary to provide 80% power with an a of 0.05. Fifteen non-smokers (control group) and 15 current smokers (test group) with moderate to severe chronic periodontitis19 were selected from patients referred to the Periodontal Clinic of Guarulhos University, Brazil. The Clinical Research Ethics Committee of Guarulhos University previously approved this study protocol. All eligible subjects were informed of the nature, potential risks and benefits of their participation in the study and signed a consent form. Inclusion and exclusion criteria The frequency and duration of cigarette consumption was self-reported by the subjects by means of a questionnaire. All current smokers included in the study had smoked at least 10 cigarettes per day within the past five years.20 No attempt was made to validate smoking history by measures such as serum cotinine levels. Subjects in the non-smokers group had never smoked before. All subjects were >30 years old, had at least 15 teeth, excluding third molars and teeth with advanced decay indicated for extraction, and a minimum of six teeth with at least one site each with a probing depth (PD) of between 5–7 mm and a clinical attachment level (CAL) of between 5–10 mm. Exclusion criteria were pregnancy, lactation, periodontal or/and antibiotic therapies in the previous six months, continuous use of mouthrinses containing antimicrobial agents, long-term administration of anti-inflammatory and immunosuppressive medications, systemic conditions that could affect the progression of periodontal disease, endodontic– periodontal lesions, extensive prosthetic rehabilitations and orthodontic appliances. Experimental design During the initial phase of this prospective, single, blinded clinical study, smokers and non-smokers received clinical and microbiological monitoring, fullmouth supragingival plaque and calculus removal, as well as extractions, provisional restorations and removal of overhangs of fillings were performed as necessary. Subsequently, SRP was performed by the same periodontist, who was blinded to the smoking 226
habit of the subjects. The SRP was performed using manual instruments (Hu-Friedy; Chigago, IL, USA) in six visits lasting one hour each, over a period of 21 days. All participants were given toothbrushes, dental floss and a dentifrice containing triclosan (Colgate Palmolive Co.; S~ao Bernardo do Campo, SP, Brazil). Microbiological monitoring was repeated immediately after the last SRP appointment (Day 0) and at 42, 63 and 180 days after completion of therapy. Clinical monitoring was repeated at 180 days after therapy. During the experimental period all subjects were monitored to ensure they observed good oral hygiene practices. All clinical examinations were performed by one calibrated examiner (variability was 0.19 mm for PD and 0.23 mm for CAL). The clinical parameters recorded dichotomously were calculated by KappaLight test and the intra-examiner agreement was >0.92. The following parameters were assessed at six sites of all teeth, excluding third molars, using a manual periodontal probe (Hu-Friedy; Chicago, IL, USA): visible plaque accumulation (PI), marginal bleeding (MB), bleeding on probing (BOP), suppuration (SUP), PD and CAL. Microbiological examination Sample collection After the removal of supragingival biofilm, subgingival samples were collected from the bottom of six interproximal periodontal pockets preferentially located in different sextants. These previously selected sites were non-contiguous and presented PD of 5–7 mm and CAL of 5–10 mm at baseline and no furcation involvement. Mini-Gracey #11/12 curettes (Hu-Friedy; Chicago, IL, USA) were inserted into the base of the pocket, and the subgingival biofilm was collected and immediately placed in separate Eppendorf tubes containing 0.15 ml of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). One hundred microlitres of 0.5M NaOH was added to each tube and the samples were dispersed using a vortex mixer. Checkerboard DNA–DNA hybridization Counts of 38 bacterial species were determined in each sample, using the checkerboard DNA–DNA hybridization technique.21 The entire microbiological analysis was performed at the Laboratory of Microbiology of Guarulhos University. The samples were boiled for 10 minutes, and neutralized using 0.8 ml of 5 M ammonium acetate. The DNA released was then placed into the extended slots of a Minislot 30 apparatus (Immunetics; Cambridge, MA, USA), concentrated on a 15 x 15 cm positively charged nylon © 2015 Australian Dental Association
Subgingival recolonization in smokers membrane (Boehringer Mannheim; Indianapolis, IN, USA) and fixed to the membrane by baking it at 120 ○C for 20 minutes. The membrane was placed in a Miniblotter 45 (Immunetics; Cambridge, MA, USA) with the lanes of DNA at 90○ to the lanes of the device. Digoxigenin-labelled whole genomic DNA probes for 38 bacterial species were hybridized in individual lanes of the Miniblotter 45. After hybridization, the membranes were washed at high stringency and the DNA probes were detected using the antibody to digoxigenin conjugated with alkaline phosphatase and chemiluminescence detection. The last two lanes in each run contained standards at concentrations of 105 and 106 cells of each species. Signals were converted to absolute counts by comparison with the standard lanes on the membrane. The sensitivity of the assay was adjusted to permit the detection of 104 cells of a given species by adjusting the concentration of each DNA probe. Statistical analysis The primary outcome variable was the difference between groups for the proportion of the red complex species. The secondary outcome variables were proportions of the Actinomyces species and of the other microbial complexes, as well as clinical findings. Each individual clinical parameter, as well as the mean counts (x105) of each bacterial species evaluated and the percentage of the total DNA probe counts were computed per subject and then across subjects in both groups. The significance of differences between the two groups for age, clinical and microbiological parameters was calculated by Mann–Whitney U test. Friedman and Wilcoxon tests were used to detect significant differences within each group over the course of the study. Adjustments for multiple comparisons were performed when the 38 bacterial species were evaluated simultaneously. Chi-square test was used to compare the differences in the frequency of gender. The level of significance was established at 5% and the data were analysed using software SPSS® 17.0 (IBM Company, Chicago, IL, USA). The biostatician was unaware of the smoking status of the subjects.
at baseline (p < 0.05). SRP led to a statistically significant decrease in mean PD, CAL and in the percentage of sites with visible plaque and SUP for both smokers and non-smokers at 180 days post-therapy. However, the percentage of sites exhibiting MB and BOP was only significantly decreased in the nonsmoking group (p < 0.05) at 180 days post-SRP (Table 1). In addition, the full-mouth percentage of shallow, intermediate and deep sites did not differ significantly between groups at baseline (p > 0.05, data not shown). Furthermore, the percentages of deep sites (PD ≥5 mm) were 38.1 15.3% in the smokers and 35.9 14.4% in non-smokers at baseline (p > 0.05). The mean changes in PD and CAL in both groups from baseline to 180 days are presented in Fig. 1. The results are organized according to different baseline PD categories: shallow, intermediate and deep sites. The only difference observed between the groups was a greater reduction in mean CAL in initially intermediate sites of non-smokers subjects (p < 0.01). At sampled site level, the mean PD and CAL was 5.6 0.6 mm and 6.5 0.4 mm for smokers and 5.8 0.5 mm and 6.1 0.4 mm for non-smokers at baseline (p > 0.05). At 180 days, the mean PD and CAL of the sampled sites were 3.4 0.7 mm and Table 1. Demographic characteristics and mean (SD) full-mouth clinical parameters at baseline and 180 days post-therapy in non-smokers and smokers Variable
Gender (male/female) Age (years) Years of smoking Cigarette/day Probing depth (mm) Clinical attachment level (mm) % of sites with Plaque accumulation
Subject recruitment occurred from July 2012 to January 2013. There were no drop-outs during the course of the experimental period. Smokers were not significantly different from non-smokers in terms of age or gender distributions, or in the initial mean PD and CAL and mean percentage of sites with visible plaque, BOP and SUP. The percentage of sites exhibiting MB was significantly lower in the smoking group
Bleeding on probing
© 2015 Australian Dental Association
Groups Non-smokers (n = 15)
Smokers (n = 15)
Baseline Baseline Baseline Baseline 180 days Baseline
42.1 6.5 0 0 3.8 0.7a 3.1 0.4b 4.4 1.0a
180 days Baseline* 180 days Baseline
44.5 40.1 8.6 62.0
180 days* Baseline 180 days
23.6 14.1b 2.8 3.3a 0.2 0.6b
14.4b 19.4a 3.2b 19.3a
40.5 26.6 17.1 3.9 3.3 4.7
35.7 15.5 14.2 67.0
8.2 7.3 7.3 0.6a 0.6b 1.2a
19.9b 19.1a 10.2a 18.2a
53.9 24.6a 3.5 5.4a 0.4 1.1b
The significance of differences between time points was assessed using the Wilcoxon test (different small letters indicate p < 0.01). The significance of difference between groups in each time point was assessed using the Mann–Whitney U test (*p < 0.05). SD: standard deviation. 227
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Fig. 1 Bar charts of the mean changes (SD) in probing depth (PD) and clinical attachment level (CAL) at sites with initial PD ≤3 mm (shallow), 4–6 mm (intermediate) and ≥7 mm (deep) from baseline to 180 days post-therapy in smokers and non-smokers. *Signiﬁcant differences between groups; Mann–Whitney U test.
4.8 0.5 mm for smokers and 3.3 0.3 mm and 4.5 0.4 mm for non-smokers (p > 0.05).
significant differences between groups (p > 0.05). Species from the red and orange complex showed a progressive trend toward recolonization to pretreatment levels at 42, 63 and 180 days post-therapy in the smoker group, although without statistical significance at any time point (p > 0.05). In non-smoker subjects, immediately after SRP (Day 0) there was a significant reduction in the mean counts of the three pathogens from the red complex, and Porphyromonas gingivalis levels remained reduced until 180 days post-therapy (p < 0.05). Two species from the orange complex (Eubacterium nodatum and Parvimonas micra) showed reduction at day 0 and at 63 days posttherapy (p < 0.05). Prevotella intermedia was only reduced at 180 days after SRP. Figure 3 summarizes the proportions of microbial complexes at each time point. At baseline and Day 0, the proportions of the different microbial complexes were similar between groups (p > 0.05). The orange complex at 42 and 180 days and the red complex at 63 and 180 days post-therapy were present in significantly higher proportion in smokers than in nonsmokers (p < 0.05). SRP led to a significant reduction in the red complex for smokers and non-smokers (p < 0.05). From baseline to 180 days post-therapy the proportion of the red complex was reduced (34.3% to 19.9%) and the proportions of beneficial purple (4.0% to 7.9%), yellow (3.8% to 14.6%) complexes and Actinomyces species (5.9% to 19.4%) were significantly increased in non-smokers (p < 0.05). The proportions of yellow complex (4.7% to 14.2%) and Actinomyces species (3.8% to 14.2%) were increased in the smokers at 180 days post-therapy (p < 0.05). However, no significant reduction was observed in the proportion of the red complex (37.9% to 26.1%) at this time point (p > 0.05). There was a significant increase in the proportion of host-compatible species (20.1% to 50.8%) and a decrease in the proportion of pathogenic species (72.6% to 45.0%) from baseline to 180 days in the non-smokers (p < 0.05). Although smokers showed an increase in the host-compatible species (24.1% to 35.3%; p < 0.05), no significant reduction was observed in the proportion of pathogenic species (69.0% to 55.3%; p > 0.05) at 180 days post-therapy (Fig. 4).
Microbiological results Figure 2 presents the mean counts (x105) of the 38 species evaluated in non-smokers (Fig. 2a) and smokers (Fig. 2b) at each experimental period. The species were grouped according to the microbial complexes described by Socransky et al.22 Despite some apparent differences in the microbial profile related to the bacterial counts between smokers and non-smokers at baseline, data analysis showed no statistically 228
DISCUSSION The clinical relevance of studying the dynamics of subgingival bacterial recolonization in smokers is because this group responds poorly to SRP, and this may be attributed to the critical regrowth of pathogenic species after treatment. Furthermore, acknowledgment of the subgingival bacterial recolonization pattern of smokers after SRP may be useful in © 2015 Australian Dental Association
Subgingival recolonization in smokers (a)
Fig. 2 Mean counts (x105) of the 38 test species evaluated at baseline, 0, 42, 63 and 180 days post-therapy in non-smokers (Fig. 2a) and smokers (Fig. 2b). The species were ordered according to the microbial complexes.22 The signiﬁcance of differences within each group between baseline and each time point (#day 0, £42 days,✚63 days and ∞180 days) was assessed using the Friedman and Wilcoxon tests (*p < 0.05) and adjusted for 38 comparisons. © 2015 Australian Dental Association
M Feres et al.
Fig. 3 Pie charts of the mean proportion of each microbial complex at baseline, 0, 42, 63 and 180 days post-therapy in smokers and non-smokers. The colours represent different microbial complexes.22 The signiﬁcance of differences within each group between baseline and 180 days was assessed using the Wilcoxon test (#p < 0.05). The signiﬁcance of differences between groups at each time point was assessed using the Mann–Whitney U test (**p < 0.01, *p < 0.05).
Fig. 4 Bar charts of the mean changes in the proportion of host compatible species (Actinomyces species, purple, yellow and green complexes) and periodontal pathogens (orange and red complexes) between baseline and 180 days post-therapy in smokers and non-smokers. The signiﬁcance of differences within each group between baseline and 180 days was assessed using the Wilcoxon test (*p < 0.05).
proposing more effective periodontal therapies for this risk group. With reference to clinical results, in the present study smokers showed significantly lower MB than nonsmokers at baseline, confirming previous observations in the literature.1,8 This finding may be related to the ability of some cigarette compounds to mask this clinical sign of inflammation by impairment of the vascular and immune responses.23 Smokers and non-smokers presented improvements in the majority of the clinical parameters evaluated at 180 days post-therapy. 230
However, it is important to note that non-smokers showed a greater reduction in mean CAL in initially intermediate sites (4–6 mm) as well as in mean levels of MB and BOP in comparison with smokers. In the present study, oral hygiene, as measured by PI, was similar between smokers and non-smokers before and after SRP, as suggested by several previous studies.1,8,15–17 Therefore, it could be implied that the unfavourable subgingival recolonization pattern observed in smokers did not originate from differences in the supragingival plaque control between groups. © 2015 Australian Dental Association
Subgingival recolonization in smokers In the present study, despite some apparent differences between smokers and non-smokers in the bacterial counts at baseline, these differences did not achieve statistical significance (Fig. 2). In addition, the subgingival biofilm profile did not differ considerably between those who had never smoked, and current smokers before therapy when considering the proportions of DNA probes of the individual species and the proportion of the microbial complexes. These results are in agreement with other studies that have also demonstrated that cigarette smoking has no substantial effect on the composition of the pathogenic subgingival microbiota.2–6 On the contrary, Zambon et al.9 demonstrated by immunofluorescence that higher proportions of smokers harboured Tannerella forsythia, P. gingivalis, Aggregatibacter actinomycetemcomitans than non-smokers. van Winkelhoff et al.,10 using culture technique, showed that P. intermedia and Prevotella nigrescens were more often detected in untreated smokers, and the mean percentage of total count of P. micra and Fusobacterium nucleatum were higher in both untreated and treated smokers. These contradictory findings may be explained by differences among the studies regarding sample size, subject demographics, history of periodontal treatment, time and frequency of smoking, number and type of bacterial species examined, microbiological techniques used (culture, DNA probe, polymerase chain reaction (PCR), immunoassay), method of data expression (count, proportion, prevalence) and number of sample evaluated. The majority of previous investigations evaluating the microbiological effects of SRP on smokers are cross-sectional or retrospective evaluations, or prospective studies observing the microbiological changes in few time points and at a maximum of 2 months post-therapy. In addition, most studies have targeted a limited number of bacterial species. Therefore, to the authors’ knowledge this is the first prospective controlled clinical study that systematically and sequentially evaluated the pattern of subgingival microbial recolonization in smokers after SRP. The dynamics of subgingival microbiota shifts after SRP in nonsmokers were evaluated by early studies using dark-field microscopy or culture techniques.24–26 In agreement with our results from both smokers and non-smokers, they observed an abrupt reduction in the subgingival microbiota at 1–2 weeks after SRP, followed by gradual bacterial re-establishment towards pretreatment levels over time.24–26 The present study showed that smokers had the worst microbiological response and recolonization pattern after SRP when compared with non-smokers. Smokers presented no significant reduction in pathogenic species immediately after SRP and a greater regrowth of potentially pathogenic species at time intervals of 42, 63 and © 2015 Australian Dental Association
180 days post-therapy. In contrast, SRP had an immediate effect on the mean levels of the three species from the red complex and E. nodatum and P. micra from the orange complex in non-smokers. In addition, the levels of some pathogenic species from the red and orange complexes remained low at 42, 63 and/or 180 days post-therapy in non-smokers. Our results are in contrast to those from Preber et al.27 who found no effect of smoking on the prevalence of P. gingivalis at 2 months after SRP. Furthermore, our data differed from those of Renvert et al.17 who, using culture technique to compare the influence of SRP on the microbiota of smokers and nonsmokers, suggested that microbiological changes were consistent with clinical changes rather than being the effect of the smoking habit at 6 months post-therapy. Moreover, contrary to our results, Apatzidou et al.8 observed reductions in the frequency of detection of putative periodontal pathogens by PCR at 6 months, with no significant differences between smokers and non-smokers. However, data from the present investigation are in agreement with those found by Grossi et al.,15 who showed that current smokers had a lower reduction in the subgingival levels of T. forsythia and P. gingivalis than non-smokers at 3 months after SRP. The present findings are also in agreement with those from Haffajee et al.,4 who demonstrated that the prevalence of P. gingivalis, T. forsythia and Treponema denticola decreased significantly in nonsmokers but not in smokers after SRP. Similarly, Darby et al.16 observed by PCR that some periodontal pathogens, especially T. forsythia, were detected more frequently in smokers than non-smokers at 2 months post-SRP. The poor effect of SRP in reducing individual levels and/or proportions of periodontal pathogens in smokers has been confirmed by various authors.7,10,18,28–30 van der Velden et al.,7 in a retrospective study using the culture technique, demonstrated that smoking increased the probability of subjects remaining positive for P. intermedia, F. nucleatum and P. micra after SRP. van Winkelhoff et al.,10 in a cross-sectional investigation, showed that the mean proportions of the total anaerobic count and the prevalence of P. micra and F. nucleatum were higher in treated smokers than treated non-smokers. In addition, there is evidence that the growth of anaerobic bacterial species in smokers could be related to the remarkable increase in anaerobiosis that occurs in the oral cavity of smokers.31 Overall, the most important findings of this study were that SRP was not effective in significantly reducing the levels of some suspected and known periodontal pathogens in smokers, and this risk group presented a subsequent re-establishment of a more pathogenic subgingival biofilm than non-smokers. Since the clinical outcomes after therapy are dependent on 231
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Address for correspondence: Dr Luciene Cristina Figueiredo Universidade Guarulhos Centro de P os-Graduacß~ao e Pesquisa – CEPPE Pracßa Tereza Cristina, 229 Centro 07023-070 Guarulhos, SP Brazil Email: [email protected]
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