Microbiological and clinical effects of probiotics and antibiotics on nonsurgical treatment of chronic periodontitis: a randomized placebo- controlled trial with 9-month follow-up

ABSTRACT Objective The aim of this double-blind, placebo-controlled and parallel- arm randomized clinical trial was to evaluate the effects of Lactobacillus rhamnosus SP1-containing probiotic sachet and azithromycin tablets as an adjunct to nonsurgical therapy in clinical parameters and in presence and levels of Tannerella forsythia, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Material and Methods Forty-seven systemically healthy volunteers with chronic periodontitis were recruited and monitored clinically and microbiologically at baseline for 3, 6 and 9 months after therapy. Subgingival plaque samples were collected from four periodontal sites with clinical attachment level ≥1 mm, probing pocket depth ≥4 mm and bleeding on probing, one site in each quadrant. Samples were cultivated and processed using the PCR technique. Patients received nonsurgical therapy including scaling and root planing (SRP) and were randomly assigned to a probiotic (n=16), antibiotic (n = 16) or placebo (n = 15) group. L. rhamnosus SP1 was taken once a day for 3 months. Azithromycin 500mg was taken once a day for 5 days. Results All groups showed improvements in clinical and microbiological parameters at all time points evaluated. Probiotic and antibiotic groups showed greater reductions in cultivable microbiota compared with baseline. The placebo group showed greater reduction in number of subjects with P. gingivalis compared with baseline. However, there were no significant differences between groups. Conclusions The adjunctive use of L. rhamnosus SP1 sachets and azithromycin during initial therapy resulted in similar clinical and microbiological improvements compared with the placebo group.

Given the background in the literature, the selection of bacterial species resistant to the antibacterial treatment has been considered a global problem after the excessive use of these drugs. This leads to the search for new tools for the control of infectious diseases 8 . The use of probiotics has become more common in recent years. They are food supplements with microbial elements that have a physiologic effect on the organism that receives them.
The effect of the use of probiotics in the treatment of chronic periodontitis had been studied 13,15,16,22,26,27,29 .    actinomycetemcomitans was also primarily identified by colony morphology and catalase production.
Using direct method, total cultivable microbiota (total microbial load) was count on blood-agar, hemin-menadione medium and TSBV medium. The percentage of P. gingivalis and T. forsythia was obtained using the number of CFU/ml RTF on bloodagar hemin-menadione medium as a percentage of the total anaerobic count. The percentage of A.
actinomycetemcomitans was obtained using the number of CFU/ml RTF on TSBV as a percentage of the total anaerobic counts.
Final identification was made using PCR according to Ashimoto protocol.

Outcome variables
The primary outcome variable was change in CAL.

Results
The flow chart of the study is shown in Figure 1.

Intergroup analysis
At baseline, no significant differences in demographic, medical and clinical characteristics were found between groups (p>0.05, Table 1).
Also, there were no intergroup differences in CAL, PPD, BOP, plaque accumulation, total cultivable microbiota and percentages of P. gingivalis, A. actinomycetemcomitans and T. forsythia at 3, 6, and 9 months follow-up (Table 2    Also, we observed a variation of the total cultivable microbiota, as seen in Table 2. Compared with baseline, there was a significant reduction in the probiotic group at 6-month follow-up, while for the antibiotic group it occurred at all times (p<0.017).
In Table 3  group.
Regarding the use of azithromycin in the treatment of chronic periodontitis in our study, a significant reduction in all periodontal parameters was observed, without intergroup differences, though. This is In our study, we observed a reduction of the total cultivable microbiota at all follow-up times in the antibiotic group, but there were no significant differences with the other groups. Gomi, et al. 7 (2007) reported no intergroup differences in the prevalence of  This bacterium has been identified in oral diseases, including chronic periodontitis, and presents, among others, virulence factors associated with resistance to antibacterial treatments, becoming a reservoir of transferable elements that would favor the genetic variability associated with microbial resistance 24 .
Hence, it has been proposed that the development of commensal bacteria as probiotics is a high priority for preventive and therapeutic purposes 19 .
The use of antibiotic therapy as an adjunct to the treatment of periodontal disease is widely supported in the literature, and there is evidence that it provides additional beneficial effects to mechanical therapy 9 . However, the optimal usage protocol for antibiotic therapy and the clinical effects of the time at which antibiotic is administered during the course of the periodontal hygienic phase has not yet been clearly determined 11 . According to the literature, administration of antibiotics show higher clinical results when is accompanied by meticulous disruption and mechanical removal of the periodontal biofilm 11 . In our study, the patients started taking the probiotic, antibiotic or placebo after the last session of SRP. We The major limitation of our study is the statistical power. This study could be too small to detect the real differences between the groups. An increase of the sample size is suggested.
In conclusion, the administration of L. rhamnosus