Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy

Abstract Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. Objectives The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. Material and Methods Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. Results Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). Conclusion The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA.

Previous studies have evaluated the association between the frequency of fimA genotypes and periodontal health status in adults 5,6 . P. gingivalis was detected in 36.8% of the healthy subjects and in 87.1% of the patients with periodontitis. Among the P. gingivalis-positive healthy adults, the most prevalent fimA was genotype I (76.1%), followed by genotype V (29.7%). In contrast, most patients with periodontitis carried fimA genotype II (66.1%), followed by genotype IV (28.9%) 6 . These findings indicate that there are both disease-associated and non-disease-associated strains of P. gingivalis, and their infectious traits, which influence periodontal health status, could be differentiated based on the clonal variation of fimA genes 6 .
Tobacco consumption is a risk factor for periodontal disease. Smoking is associated with higher clinical attachment loss and gingival recession, reduced bone height and density, and, consequently, with increased tooth loss 5 . The mechanism by which tobacco affects the periodontal tissue is related to toxic substances such as nicotine and cotinine, which has been associated with various cellular changes that may contribute to the onset and subsequent progression of periodontal disease 7 . Tobacco use promotes several adverse effects, such as reduced gingival blood flow 8 , oxidative stress and alterations in immunoinflammatory responses, reducing the functional activity of neutrophils such as chemotaxis, glycolytic activity and phagocytosis 9 ; it also impairs wound healing 10 and interferes on bacterial acquisition and host response to colonization in biofilms 11 . The frequency of periodontopathogens was previously investigated in smokers and nonsmokers. P. gingivalis has been detected more frequently in the periodontal pockets of smokers (66.7% -pocket depth of 3-5 mm) in comparison with nonsmokers (52.2%), and it has been found in high levels in sites with periodontitis 12 .
Scaling and root planing is the most common and Considering the presence of different genotypes of P.
gingivalis fimA, Teixeira, et al. 16 (2009) suggested an association between the genotype fimA IV and disease severity in smoker-chronic periodontitis subjects.
However, no study was found evaluating the influence of periodontal treatment on the frequency of different fimA genotypes in smokers and nonsmokers with periodontitis. The aim of this study was to compare the frequency of different genotypes of P. gingivalis fimA in smokers and nonsmokers with periodontitis, before and after 3 months of nonsurgical periodontal therapy.
The null hypotheses are: 1) there is no difference in the periodontal status and frequency of P. gingivalis fimA genotypes between smokers and nonsmokers; and 2) smoking did not interfere in the response to periodontal treatment and levels of P. gingivalis fimA genotypes.

Clinical results
There was no statistical difference between SM and NS regarding age, gender and race (Table 1).
Median cigarette consumption was 20 cigarettes per day, and the duration (median) of the smoking habit was 23 years. Comparing SM and NS, at baseline, both groups had a similar periodontal status ( Table 2). gingivalis at baseline and 3 months after periodontal therapy, as described in Table 3. Genotype I was detected in the majority of SM (60%) and NS (87.5%) at baseline, and the frequency did not diminish after 3 months of treatment (SM -60% and NS -93.8%).
The frequency of genotypes II, III and V fimA was higher in SM than NS at baseline; however, a

Discussion
In this study, we confirmed that P. gingivalis is a common microorganism present in periodontal disease in SM and NS, corroborating other studies 16,20 . The Some studies have shown that SM have higher prevalence and severity of periodontal disease, and worse results after periodontal therapy than NS 7,25 . In this study, SM and NS were matched by periodontal parameters (PI, BOP, PPD, GR and CAL) at the baseline, since no statistical difference was observed between the groups. Besides, instruction on oral hygiene was given to all patients, regardless of the group, in order not to bias data collection. However, higher levels of PI were observed after 3 months of periodontal treatment only for the SM group. One hypothesis is that smokers may be less motivated to keep a high-quality oral hygiene throughout the period of study 26 . Due to plaque accumulation, smokers showed a higher GI mean over  16,20,31 . This determined that fimA genotypes I and II were the most prevalent at baseline Some studies have established that fimA genotype IV is considered an important virulence factor for the pathogenesis of periodontal disease 6,32 . However, in our study, there was no detection of type IV in the groups, disagreeing with some authors, who found not only genotypes I and II, but also genotype IV among the most frequently detected 30,31 . It is important to emphasize that genotyping was performed exclusively among participants with chronic periodontitis, and there was a variation in P. gingivalis genotypes regarding other types of periodontitis, a distinction that could change the course and evolution of the pathology.
In this study, we selected samples of P. gingivalis from our previous study 33 , in which the authors detected P. gingivalis in 50% of SM and 70.8% of NS, both groups with periodontal disease. The high frequency of P. gingivalis in patients with periodontitis is according with the results of Amano, et al. 6 (2000), which detected P. gingivalis in 87.1% of patients with periodontitis. Type II fimA is associated with deeper pockets, whereas genotypes III and V seem to be involved in periodontitis to a lesser extent 12 . The results of this study corroborated with these findings, because a strong correlation was found between probing depth (>5 mm) and presence of type II fimA. Likewise, when associations between the fimbriae were tested in SM and NS at baseline and after 3 months, statistical analysis revealed significant differences between SM and NS, when II and V fimA genotypes were associated. This may be attributed to deeper pockets in SM compared with NS. Results obtained by Darby,

Conclusion
Within the limitation of the relatively small sample size, this study concluded that the most prevalent P. gingivalis fimA genotypes detected in periodontal participants were genotypes I and II. However, the presence of fimA genotype II was higher in SM.
Periodontal treatment was effective in controlling periodontal disease and in reducing P. gingivalis fimA type II and V. The authors suggest that more longitudinal studies are necessary to establish whether genotypes of P. gingivalis fimA can be maintained for long periods of time, and whether they influence the evolution of periodontal disease over time.