The adjunctive effect of probiotics to nonsurgical treatment of chronic periodontitis: A randomized controlled clinical trial.

BACKGROUND: Probiotics catch more attention as adjunctive periodontal therapy. This study was conducted to assess the benefit of locally delivered Lactobacillus reuteri (L. reuteri) probiotic as an adjunctive to scaling and root planing (SRP) in the treatment of chronic periodontitis clinically and microbiologically.
MATERIALS AND METHODS: Bacterial cultures and clinical evaluation were recorded in 20 sites of chronic periodontitis in 12 patients and followed up at 3 and 6 months from the start of intervention using clinical attachment level, probing pocket depth, plaque index (PI), bleeding on probing, and microbiologically for Porphyromonas gingivalis (P. gingivalis) load. Patients meeting the inclusion criteria were scheduled within 1 week for two sessions of SRP. After SRP, oral hygiene measures were reassured, and sites were divided randomly into two groups 10 sites each. Group I received SRP only, while Group II received SRP and subgingival delivery of 1 ml of probiotic L. reuteri suspension at baseline and 1, 2, and 4 weeks using a blunt syringe. A periodontal pack was applied after the placement of the drug.
RESULTS: We found noticeable variation between the two groups in all evaluation aspects at 3 and 6-month follow-up periods except PI at 6 months in which there was no significant difference between both groups.
CONCLUSIONS: The results proved the antimicrobial benefit of L. reuteri probiotic as a promising adjunctive therapy in improving periodontal parameters. However, further long-term studies with large sample size are needed to evaluate the extent of the added value of L. reuteri suspension. Copyright:

INTRODUCTION

The goal of periodontal treatment is to alter the microbial etiology, arresting the disease progression and resolve inflammation.[1] Several treatment modalities are available to achieve these goals and they can be broadly classified into nonsurgical and surgical treatment.[2]

Many studies described the effectiveness of scaling and root planing (SRP) as a nonsurgical treatment modality.[34] However, recolonization of pathogenic bacteria toward pretreatment levels occurs within weeks.[5]

Different therapeutic approaches have been established to inhibit bacterial growth and colonization such as antibiotics and LASER beam therapy.[678] Recently, probiotics were suggested as a potential treatment by inhibiting pathogenic bacterial colonization and overgrowth by competition over nutrition, decreasing adhesion, and producing antimicrobial substances and immunity modulation.[9]

Probiotics are live bacteria that include a large spectrum of bacteria such as Lactobacillus spp. and Bifidobacterium spp., which are the most famous types.[10]

Lactobacillus reuteri (L. reuteri) withdrew attention. Its modes of action include beneficial alteration of local ecology, antibiotic substance production, and host-immunity modulation.[11]

Many studies describe the ability of Lactobacilli to inhibit periodontal pathogen's growth.[1213] Several trials proved the synergistic effect of L. reuteri and SRP in CP treatment.[1415]

Hence, the goal of this research is to determine the added benefit of L. reuteri probiotic to SRP in treating CP cases clinically and microbiologically.

MATERIALS AND METHODS

This blinded interventional prospective study (randomized controlled trials [RCT]) was done in agreement with principles guidelines declared in Helsinki experimentation on human subjects and after the approval of the ethics committee. This research was done in the Periodontology Department, Faculty of Dentistry.

A total of 20 sites limited to posterior teeth from 12 systemically healthy patients of both sexes (three males and nine females) enrolled in the study after fulfilling inclusion criteria which were

Systemically healthy patients of both sexes between 35 and 55 years, suffering moderate chronic periodontitis according to Armitage criteria[16]

Having at minimum two teeth with one proximal site with (clinical attachment level [CAL]) of 3–4 mm, and (probing pocket depth [PPD]) of 5–6 mm

Horizontal bone erosion by panoramic radiography [Figure 1]

Figure 1

Panoramic radiograph for diagnosis

Patients who are compliant, with no history of missed clinic appointments.

The following criteria were also excluded:

Previous use of systemic or local antibiotic, anti-inflammatory drugs, probiotics, or periodontal therapy in the previous 6 months

Smokers

Patients who are lactose intolerant were excluded.

All participants gave consent after a clear explanation of the goal, nature, and health-care benefits in this study was given to all subjects. The study protocol was performed between May 2018 and February 2019.

The sample size considered a standard deviation of 0.61 mm and a difference between the test and control group of 0.82 mm for the primary outcome variable which was PPD, based on Vivekananda et al.[17] It was calculated that 10 sites were needed to provide 80% power with an α of 0.05 using StatsDirect (StatsDirect, Cheshire, UK) program.

A thorough gingival assessment was performed and complete medical and dental history was taken. Periodontal probing was done after fabrication of a customized-made acrylic stent to mark the probe depth at every evaluation point [Figure 2]. The primary outcome variable was PPD[18] and CAL.[18] The secondary outcome variables were bleeding on probing (BOP)[19] and plaque index (PI).[20]

Figure 2

Measurement of probing pocket depth using UNC-15 probe through a customized made acrylic stent with groove

A calibration exercise was performed to determine intra-examiner reproducibility. Six patients not enrolled in the study who exhibited at least 7 teeth with a Pocket depth (PD) of ≥5 mm on at least one aspect of each tooth were evaluated by the examiner on two separate visits 48 h apart. The PD and CAL were measured. Calibration was accepted if the measurements at baseline and 48 h were consistent (in mm) in ≥90% of the measurements.[21]

Investigator GH K screened the patients and randomly assigned them to test and control groups and performed the treatment procedures for all participants. Investigator M. Sh recorded the clinical parameters and she was masked to the randomization for the extent of the study.

Oral hygiene instructions including regular tooth brushing twice daily for a minimum of 2 min by modified bass brushing technique and interdental flossing was explained to the patients. Randomization of the sites was accomplished through sealed envelopes into two groups ten each by a masked supervisor.

Patients were scheduled within 1 week for two SRP appointments, performed by scalers and hand instrumentation. Group I had SRP alone. Group II patients had SRP and subgingival delivery of 1 ml of probiotic suspension using a blunt syringe (consists of L. reuteri DSM 17938 [1 × 108 CFU]); sunflower oil, medium-chain triglyceride oil, and anti-caking agent (silicon dioxide) (BioGaia, Lund, Sweden) were applied to these sites at baseline (after SRP) and 1, 2, and 4 weeks [Figures 3 and 4]. Periodontal pack was applied after drug application [Figure 5].

Figure 3

Lactobacillus reuteri probiotic suspension

Figure 4

Application of Lactobacillus reuteri probiotic suspension using a blunt syringe

Figure 5

Periodontal dressing pack after application of the drug

Patients were advised not to harsh food, avoid dental manipulation near the treated areas, and not to use any interdental aids for 24 h. No antiplaque agents, systemic antibiotics, anti-inflammatory drugs, or any other type of probiotics were allowed during the period of the study.

The gingival crevicular fluid (GCF) was collected initially, at 3–6 months using a paper point. The supragingival plaque was removed with sterile cotton pellets. Two sterilized paper points (size 35) were applied to the maximum depth of the periodontal pocket and kept for 10 s [Figure 6], then they were placed in one ml phosphate-buffered saline in an Eppendorf tube.

Figure 6

Gingival crevicular fluid sample collection using sterile paper point size 35

Samples were dispersed using a vortex mixer and preserved at −20°C until analysis. The frozen samples were sent to the central laboratory on dry ice and kept at − 80°C at once. After defrosting, 400 μl of each sample was centrifuged at 13,000 g. The gained pellet was dispersed in 200 μl InstaGene. DNA was retrieved with QIAamp DNA Mini Kit following the manufacturer's instructions. Five microliiter of the purified DNA was used for the quantification of P. gingivalis. The steps were done according to the manufactures' instructions (Primer design, UK).

The data were analyzed using version 19 (SPSS) created by IBM, Illinois, Chicago, USA. For numerical values, the range, mean, and standard deviations were calculated. The differences between the two mean values were tested using Mann–Whitney (Z) test. For comparison of series of observations measured during follow-up, Friedman Chi-square (X2) was used, and when found significant, pairwise comparison by Mann–Whitney (Z) test was used. Data were considered statistically significant when P > 0.05.

RESULTS

The patient demographics showed no difference in both groups. No subjects were lost or excluded over the following 6 months. The performed treatment was well tolerated by all the patients without any side effects [Table 1 and Figure 7 flowchart].

Table 1

Comparison between the two studied groups according to biographic data

	Control group (n=6), n (%)	Test group (n=6), n (%)	Test	P FE
Gender
Male	1 (16.7)	2 (33.3)	χ2=0.444	1.000
Female	5 (83.3)	4 (66.7)
Age
Mean±SD	39.33±3.20	39.33±3.20	t=0.0	1.000

Level of significance P>0.05. SD – Standard deviation; χ2: Chi-square test, FE: Fisher’s exact, t: Student’s t-test, P: P value for comparison between the two studied groups; n: number of patients;

Figure 7

Flowchart of patient inclusion and follow-up

No difference was observed between the two groups at baseline regarding the clinical and bacteriological parameters as evident by their mean baseline values P > 0.05.

For clinical results, Group II exhibited a statistically significant decrease in the mean PI and BOP scores at 3–6 months as compared to their mean baseline value P < 0.05. Group I recorded statistically significant reductions of the mean PI score at 3–6 months as compared to their mean baseline value P < 0.05. However, Group I recorded no difference in BOP score at 3 and 6 months in comparison to baseline value P > 0.05 [Table 2]. PI intergroup results showed a statistically significant difference between the two groups at 3-month follow-up period in favor of Group II (P < 0.05) and no difference between the two studied groups after 6 months P > 0.05, While for the BOP intergroup, interpretation recorded a statistically significant difference between them at 3 and 6 months (P < 0.05) follow-up periods in favor of Group II [Table 2].

Table 2

Comparison of plaque index and bleeding on probing percentage between the studied groups at different periods of follow-up

Variable	Time point	Treatment groups		P0
		Control group, n (%)	Treatment group, n (%)
PI	Baseline
	1	0 (0.0)	0 (0.0)	1.000
	2	0 (0.0)	0 (0.0)
	3	10 (100.0	10 (100.0)
	At 3 months
	1	5 (50.0)	10 (100.0)	0.033*
	2	5 (50.0)	0 (0.0)
	3	0 (0.0)	0 (0.0)
	At 6 months
	1	0 (0.0)	0 (0.0)	0.650
	2	5 (50.0)	7 (70.0)
	3	5 (50.0)	3 (30.0)
Friedman’s χ2		18.571	18.865)
P		0.001*	0.001*
Baseline versus 3 months		0.004*	0.002*
Baseline versus 6 months		0.025*	0.008*
BOP	Baseline
	Negative	0 (0.0)	0 (0.0)	1.000
	Positive	10 (100.0)	10 (100.0)
	At 3 months
	Negative	3 (30.0)	10 (100.0)	0.003*
	Positive	7 (70.0)	0 (0.0)
	At 6 months
	Negative	0 (0.0)	6 (60.0)	0.011*
	Positive	10 (100.0)	4 (40.0)
Friedman’s χ2		6.000	15.200
P		0.050	0.001*
Baseline versus 3 months		0.083	0.002*
Baseline versus 6 months		1.000	0.014*

* P – Statistically significant at P<0.05. χ2 – Friedman’s Chi-square Chi-square test for comparison follow-up periods to baseline; P0 – P0 value for comparison between the two studied groups using Mann-Whitney (Z) test; PI – Plaque index; BOP – Bleeding on probing; n – number of patients;

Intragroup results showed a statistically significant reduction in the mean PPD and CAL scores at 3–6 months (P < 0.05) in comparison to the mean baseline for both groups. Intergroup results recorded a slight statistically significant difference in PPD reduction CAL gain in favor of Group II at 3 months (P < 0.05), whereas we found a highly statistically significant difference at 6 months in favor of Group II (P = 0.001) [Table 3].

Table 3

Comparison of pocket depth, loss of attachment, and polymerase chain reaction between the studied groups at different periods of follow-up

Variable	Time point	Treatment groups		Z	P
		Control group	Treatment group
Pocket depth	Baseline
	Range	5-6	5-6	1.090	0.276
	Mean±SD	5.30±0.48	5.10±0.32
	At 3 months
	Range	3-5	2-3	2.291	0.022*
	Mean±SD	3.50±0.71	2.90±0.32
	At 6 months
	Range	4-6	3-4	3.227	0.001*
	Mean±SD	4.30±0.67	3.30±0.48
Friedman’s χ2		17.543	18.727
P		0.001*	0.001*
Baseline versus 3 months		0.004*	0.003*
Baseline versus 6 months		0.008*	0.004*
Loss of attachment	Baseline
	Range	3-4	3-4	1.090	0.276
	Mean±SD	3.30±0.48	3.10±0.32
	At 3 months
	Range	1-3	0-1	2.291	0.022*
	Mean±SD	1.50±0.71	0.90±0.32
	At 6 months
	Range	2-4	1-2	3.227	0.001*
	Mean±SD	2.30±0.67	1.30±0.48
Friedman’s χ2		17.543	18.727
P		0.001*	0.001*
Baseline versus 3 months		0.004*	0.003*
Baseline versus 6 months		0.008*	0.004*
PCR	Baseline
	Range	22-37	33-36	1.436	0.151
	Mean±SD	31.01±5.43	34.41±1.13
	At 3 months
	Range	23-36	19-30	2.948	0.003*
	Mean±SD	30.53±4.49	23.50±3.29
	At 6 months
	Range	31-40	20-30	3.780	0.001*
	Mean±SD	36.10±2.81	24.34±3.30
Friedman’s χ2		15.800	18.200
P		0.001*	0.001*
Baseline versus 3 months		0.202	0.005*
Baseline versus 6 months		0.005*	0.005*

* P – Statistically significant at P<0.05. χ2 – Friedman’s Chi square Chi-square test for comparison follow-up periods to baseline; P0 – P0 value for comparison between the two studied groups using Mann-Whitney (Z) test; PCR – Polymerase chain reaction; SD – Standard deviation; Z – Mann whitney test

For the microbiological results, Group I showed no statistically significant reduction in the P. gingivalis load at 3 months as compared to the mean baseline value (P > 0.05), while at 6 months, there was a statistically significant increase in P. gingivalis load (P < 0.05), whereas Group II showed a statistically significant reduction in P. gingivalis load at 3–6 months (P < 0.05) as compared to the mean baseline value. Intergroup results showed a slight statistically significant difference in P. gingivalis load at 3 months in favor of Group II P < 0.05, whereas there was a highly statistically significant difference at 6 months in favor of Group II (P < 0.05) [Table 3].

By correlating clinical and bacteriological results, Spearman rank test showed a statistically significant positive correlation between PPD, CAL, and the presence of P. gingivalis at 3–6 months (P < 0.05) in both the groups [Table 4].

Table 4

Correlation between pocket depth, loss of attachment and polymerase chain reaction results

Variable	Time point	Rho	P
Pocket depth and PCR	At 3 months	0.563	0.010*
	At 6 months	0.698	0.001*
Loss of attachment and PCR	At 3 months	0.563	0.010*
	At 6 months	0.698	0.001*

*Statistically significant at P<0.05. Significance between variables was done using the spearman rank test. P – P value for comparison between the two variables; PCR – Polymerase chain reaction; Rho – Spearman’s rank correlation coefficient

DISCUSSION

This RCT assessed the benefit of L. reuteri probiotic suspension to SRP on clinical and microbiological parameters in chronic periodontitis patients. A parallel study design was selected to overcome the shortcoming of the split-mouth design as it is prone to the “carry-across” effect of the intervention especially when it cannot be localized.[22]

Topical application of L. reuteri probiotic suspension was allowed to increase its concentration at the pocket and the direct contact with the pathogens. Moreover, the periodontal dressing was applied to remove the washout effect of the GCF and to prolong its contact time with the inflamed sites.

The clinical results were confirmed microbiologically by PCR technique to quantify the level of P. gingivalis in GCF at baseline and 3 and 6 months of treatment as P. gingivalis is known to be the main pathogen that can create dysbiosis between the host and dental plaque.[23] The probiotic application was found to be well tolerated by all patients. No side effects were reported during the study period.

Regarding PI, the more favorable results obtained in Group II can be explained as the plaque reduction brought about by SRP was enhanced by the antiplaque effect of L. reuteri through direct interactions within dental plaque due to the competition of probiotics with the colonizing bacteria on the binding sites and nutrients leading to disruption of the colonizing pathogens.[24] However, the rebound that occurred in Groups II at 6 months evaluation period reflects the best effect of probiotic that was maintained for more than 3 months.

Our results agreed with various studies on different probiotic supplements.[151725] and with the recent review which stated[24] that probiotics prevent quorum sensing and the survival of biofilm pathogens, interfere with biofilm integrity, and lead to biofilm elimination. In contrary to our results, Iniesta et al.[26] showed no difference in PI or GI at 8 weeks between interventional and control groups.

For BOP, the effect of SRP as a standard means of controlling inflammation seemed to be enhanced by the anti-inflammatory and host immune-modulatory effects of probiotics. Our results agreed with Twetman et al.,[27] who studied the benefit of a chewing gum containing two strains of L. reuteri and found that the pro-inflammatory cytokines such as interleukin (IL)-1 β, tumor necrosis factor α, and IL-8 in GCF were decreased by this probiotic therapy. However, there are opposite results, which showed that probiotics are ineffective regarding the PI and GI scores.[262829] This can be explained by the difference in the study design, patient population, probiotic bacteria, and method of introducing probiotics. SRP was not done in the majority of these studies and it is difficult for therapeutic agents to penetrate biofilms unless they are mechanically disrupted.

At 3-month follow-up period, there was a slight statistically significant difference in PPD and CAL reduction in favor of group II, whereas there was a highly statistically significant difference at 6-month follow-up period in favor of Group II. These results suggested that the effect of probiotics could be maintained for 6 months.

The favorable effect of Group II is explained by the antimicrobial and anti-inflammatory actions of L. reuteri probiotic on a wide variety of cells to modulate the host immune response.[30] The introduction of L. reuteri into the oral microbiome will shift the microbiota from pathological to commensal making the oral environment favorable for periodontal health.[31] The more CAL gain in Group II in comparison to Group I could be a result of the bone growth promoting properties of L. reuteri[32] Similarly, the results of the systematic review of Ikram et al.[33] suggested that the adjunctive use of probiotics could result in superior benefits in CAL gain in CP. Results of the present study were comparable to another study[34] which showed better clinical outcomes to the groups treated with probiotics compared to tetracycline fibers.

The favorable microbiological results in Group II can be explained by the antimicrobial property of L. reuteri as it produces reuterin, three-hydroxypropionaldehyde, within biofilms.[3536] that produces oxidative stress in pathogenic organisms which accounts for its anti-pathogenic effect.

In addition, it can be explained by microbial antagonism of the Lactobacilli strain in the test product toward P. gingivalis as described by Van Essche et al.,[37] who proved that lactic acid inhibited the growth of other bacterial strains by producing a huge amount of organic acids.

Correlating clinical and microbiological results indicates that P. gingivalis is inseparable from CP and there is a positive correlation between them. These results agreed with Kulkarni et al.,[38] who quantified P. gingivalis in CP cases and normal individuals by PCR and found a positive correlation between P. gingivalis and severity of the chronic sequel of periodontitis.

It is well known that local drug delivery in periodontal disease has shown promising results;[39] hence, the local administration of probiotics containing L. reuteri may have a better result. However, the favorable effect was shown to be short.

The main limitation of the present study is the relatively small number of participants. Nevertheless, this study could serve as a basis for future studies conducted in larger cohorts. Further studies are required to evaluate how the process of periodontal colonization works over time, substantively of probiotics, and to determine the frequency and course for optimal dose.

CONCLUSION

Our study shows that local application of L. reuteri in combination with SRP gives more favorable results than SRP alone in cases with chronic periodontitis.

Financial support and sponsorship

This research received no external funding (self-funding) except for the PCR kit supplied by our institution.

Conflicts of interest

There are no conflicts of interest.