# Abstract

 # 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. # Results The flow chart of the study is shown in [Figure 1](#). Forty-seven patients, 16 in the probiotic group, 16 in the antibiotic group and 15 in the placebo group were analyzed. All patients entering the study also completed it. No compliance problems were noted, all patients followed the protocol of the study. Only one subject from the antibiotic group reported an adverse event (nausea). <figure> <p><img src="" /></p> <figcaption>Flowchart of the study design</figcaption> </figure> 

## 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](#) and [3](#)). ::::table-wrap

::: caption



##

##

## Baseline data of patients in the treatment groups

:::

Variable Treatment Group p- value

______________________________________________________________________

```
                              Probiotic group (n=16)   Antibiotic group (n=16)   Placebo group (n=15)   
```

Age[1](#) (years) 46.5 ± 9.3 49.0 ± 7.9 52.8 ± 7.5 0.1171
Gender (M/F)[2](#) 8 / 8 10 / 6 8 / 7 0.8150
Smokers[2](#) 7 (43.8%) 3 (18.7%) 6 (40.0%) 0.3440
CAL (mm)[3](#) 3.8 ± 0.7 4.4 ± 0.9 4.7 ± 1.5 0.0824
PPD (mm)[3](#) 2.7 ± 0.6 2.9 ± 0.4 3.1 ± 0.9 0.2437
BOP (%)[1](#) 49.3 ± 18.1 57.4 ± 10.2 52.5 ± 12.6 0.0850
Plaque accumulation (%)[3](#) 54.5 ± 18.8 58.6 ± 18.8 56.1 ± 9.4 0.5256

ANOVA (p\<0.05);

Fisher's exact test (p\<0.05);

Kruskal Wallis test (p\<0.05).

CAL: Clinical attachment level; PPD: Probing pocket depth; BOP: Bleeding
on probing.:::: ::::table-wrap
::: caption



##

##

## Intra- and intergroup comparisons of clinical and microbiological parameters (mean ± SD or median, IQ score)

:::

```
                                                           Probiotic group                                             Antibiotic group                                     Placebo group                                                                                                                     
```

______________________________________________________________________

```
                                                           (n=16)                                                      (n=16)                                               (n=15)                                                                                                                            
                                             Baseline      3 months            6 months            9 months            P                  Baseline      3 months            6 months            9 months            P               Baseline      3 months            6 months            9 months            P
```

CAL (mm) 3.8 ± 0.7 3.4±0.6[†](#) 3.3 ±0.6 3.4±0.6[†](#) 0.0001[\*](#) 4.4 ±0.9 4.0 ± 1.0[†](#) 3.9 ± 1.0[†](#) 4.1 ±1.0 0.0001[\*](#) 4.7 ± 1.5 4.1 ± 1.4[†](#) 4.1 ± 1.4[†](#) 4.3 ± 1,4[†](#) 0.0001[\*](#)
PPD (mm) 2.7 ± 0.6 2.1 ±0.3[†](#) 2.3 ± 0.4[†](#) 2.2±0.3[†](#) 0.0001[\*](#) 2.9 ±0.4 2.3 ± 0.4[†](#) 2.3 ± 0.3[†](#) 2.3 ± 0.3[†](#) 0.0004[\*](#) 3.1 ± 0.9 2.4±0.5[†](#) 2.4±0.5[†](#) 2.5 ± 0.6[†](#) 0.0001[\*](#)
BOP(%) 49.3 ± 18.1 39.2 ± 14.8 42.1 ± 13.6 42.4 ± 14.6 0.0010[\*](#) 57.4 ± 10.2 43.6 ± 12.5[†](#) 44.1 ± 13.5[†](#) 48.1 ± 14.1 0.0128[\*](#) 52.5 ± 12.6 40.7 ± 13.3[†](#) 41.5 ± 14.5[†](#) 45.9 ± 12.9 0.0002[\*](#)
Plaque accumulation (%) 54.5 ± 18.8 24.7 ± 11.3[†](#) 25.2 ± 13.1[†](#) 28.1 ± 14.6[†](#) 0.0023[\*](#) 58.6 ± 18.8 28.0 ± 17.5[†](#) 32.6 ± 15.7[†](#) 31.8 ± 14.8[†](#) 0.0001[\*](#) 56.1 ± 9.4 32.4 ± 13.9[†](#) 28.7 ± 13.0[†](#) 27.4 ± 13.0[†](#) 0.0119[\*](#)
Total cultivable microbiota (CFU)[\*](#) 105 7.6, 31.8 0.5, 1.3 0.8, 1.7[†](#) 0.6, 2.9 0,0619 10.2, 23.5 1.1,3.0[†](#) 1.9, 4.0[†](#) 2.2, 5.9[†](#) 0,0937 10.0, 20.2 2.9, 25.4 0.5, 8.1 2.8, 24.7 0,1445

Intra-group comparison by

Friedman test (p\<0.05) and intra- group multiple comparison by

Bonferroni- corrected Wilcoxon signed rank test (p\<0.017). Significant
values were given in bold formatting.

Inter-group multiple comparison by

Bonferroni- corrected Kruskal Wallis test and ANOVA (p\<0.017)

CAL: Clinical attachment level; PPD: Probing pocket depth; BOP: Bleeding
on probing; CFU: Colony forming units:::: ::::table-wrap
::: caption



##

##

## Intra- and intergroup comparisons of microbiological parameters (mean ± SD or median, IQ score at days 0 and 270

:::

Variable Time point Treatment group p- value[\*\*](#)

______________________________________________________________________

```
                                        Probiotic group (n=16)   Antibiotic group (n=16)   Placebo group (n=15)   For mean       For delta                                    
                                        Mean ± SD                Delta ± SD                Mean ± SD              Delta ± SD     Mean ± SD           Delta ± SD               
```

% *P. gingivalis*\
Day 0 14.2 ± 17.6 -13.1 ± 18.6 17.8 ± 19.3 -15.6 ± 17.9 14.7 ± 15.7 -14.4 ± 15.9 0.97738 0.99028
Day 270 1.1 ± 2.6† 2.1 ± 6.6† 0.3 ± 1.0† 0.5882\
Number of subjects with\
*P. gingivalis*\
Day 0 15 (93.7%) 14 (87.5%) 13 (86.6%) 0.8590\
Day 270 9 (56.2%) 9 (56.3%) 3 (20.0%)† 0.0740\
*A. actinomycetemcomitans*\
Day 0 3 (18.7%) 3 (18.7%) 0 (0%) 0.2200\
Day 270 1 (6.3%) 6 (37.5%) 5 (33.3%) 0.0860\
*T. forsythia*\
Day 0 16 (100%) 16 (100%) 15 (100%) 1.0000\
Day 270 12 (75.0%) 11 (68.7%) 10 (66.6%) 0.9240

Intra- group comparison by

Wilcoxon signed rank test and Mc Nemar test. p\<0.05. Significant values
were given in bold formatting. Inter-group comparison by

Kruskal Wallis test, ANOVA and Fisher's exact test. p\<0.05:::: 

## Intragroup analysis The comparison of CAL, PPD, BOP, plaque accumulation values for the baseline and 3-, 6-, and 9-month time points for all groups are presented in [Table 2](#). In the probiotic group we observed a significant reduction of the clinical attachment loss at 3 and 9 months and of the PPD and plaque accumulation at all times of the follow-up. In the antibiotic group, we perceived a significant reduction of the CAL and BOP at 3 and 6 months, as well as a significant reduction of the PPD and plaque accumulation at all times of the follow-up. Finally, in the placebo group, the CAL, PPD and plaque accumulation decreased significantly at all times and the BOP at 3 and 6 months. 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](#), we analyzed the variation of the microbiological variables between the basal time and the 9-month follow-up. Percentage of *T. forsythia* and *A. actinomycetemcomitans* is not reported, because the development of these microorganisms was not observed at any time of the analysis. The percentage of *P. gingivalis* (p\<0.05) decreased in all the groups, compared with baseline. The reduction of the number of subjects with *P. gingivalis* was significant only in the placebo group (p\<0.05).


Extracted trial result:
Outcome: reduction of PPD (continuous)
Intervention: antibiotic
Comparator: placebo
Intervention group size: 16
Intervention mean: 2.3
Intervention standard deviation: 0.3
Comparator group size: 15
Comparator mean: 2.5
Comparator standard deviation: 0.6
Notes: found in table 2