Additional clinical benefits of probiotics as an adjunctive therapy to nonsurgical periodontal treatment of periodontitis: a systematic review and meta-analysis

Background: With more and more concerns concentrated on the novel therapy applying probiotics, we challenge its trustworthy clinical efficacy as an adjuvant to scaling and root planning (SRP) as compared with SRP alone or combined with placebo or other nonsurgical treatment applied as the initial therapy of periodontitis. Methods: Electronic databases retrieval was performed until October 2017 under certain screening condition. Clinical randomized controlled trials (RCTs) to assess the efficacy of SRP + probiotic versus SRP were included. Primary outcome variables were PPD (pocket probing depth) reduction and CAL (clinical attachment level) gain. Nine publications were eligible for the systematic review and three were evaluated in the meta-analysis. Results: Results demonstrated statistically significant bleeding on probing (BOP) percentage reduction (5.34, p< 0.00001) at short-term, but not a significant differences of overall PPD reduction (0.23mm, p=0.25) for SRP + probiotic treatment versus SRP at short-term, and there was only a tendency (p = 0.08) with regard to overall CAL gain. However significant reduction of PPD and gain of CAL were observed when stratified for deep pockets (0.61mm, p< 0.00001) and for moderate pockets (0.37mm, p=0.006) at short-term respectively. Conclusions: Within the ranges of this study, the adjunctive use of probiotics seem to achieve short-term clinical benefits in the treatment of periodontitis. Conclusions must be treated with caution and future long-term RCTs are needed to testify the clinical application value of probiotics.

. The target of this conventional treatment is to remove adherent and unattached bacterial biofilms as well as deposits of calculus thus reducing inflammation and pocket depths, and promoting periodontal reattachment [5][6][7]. Unfortunately, such therapeutic approaches are frequently observed with the outcomes of recolonization of treated sites by periodontopathogens, causing to the recurrence of the periodontitis [8]. To increase the efficacy of non-surgical periodontal treatment, adjunctive treatments have been applied such as the use of chlorhexidine, antibiotics, antiseptics or photodynamic therapy to achieve a better decontamination of the periodontal environment [9][10][11].
Although these non-surgical treatments have been confirmed to have some certain clinical effectiveness, efforts to improve periodontal therapies through complementary treatments are at research. Recently, probiotic therapy has gained increasing interests among the medical researchers.
Probiotics are living microorganisms, principally bacteria which, when administered in adequate amounts, confer a health benefit to the host [12]. Clinical advances have already been made in the prevention and treatment of systemic gastrointestinal diseases in which polymicrobial intestinal transplants have shown its potential in restoring a balance between the intestine and its microbial inhabitants [13,14]. Probiotics can exert clinical benefits on the host with the promise to improve and maintain the symbiosis of polymicrobial communities through prevention of adhesion of pathogenic species, inhibition of bacterial growth, modulation of the mucosal immune system or the cell proliferation and improvement of intestinal barrier integrity [15]. Probiotics have been employed as effective adjuncts to control periodontal inflammation [16][17][18][19]. The therapeutic effects on the periodontitis have been highlighted in vitro and in vitro models [20][21][22]. However, this is a relatively new field, and data regarding the probiotics therapy in treating periodontitis is scarce.
The objective of this systematic review aims to analyze the available scientific evidence answering the following focused question developed in accordance with the recognized Patient, Intervention, Comparison and Outcome (PICO) format: 'What is the clinical efficacy of probiotic as an adjunctive therapy of SRP, in terms of PPD reduction and CAL gain, when compared with SRP alone or in combination with placebo or other adjunctive treatments in the management of periodontitis in humans?'

Protocol
This systematic review was prepared in accordance with the PRISMA guidelines [23], and the protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO, registration number CRD42017083840).

Search strategy
The following databases: MEDLINE, Cochrane Central Register of Controlled Trials and Science Direct databases were searched from January 2012 through October 2017. The following strategy was used in the search using Boolean operators and an asterisk symbol (*) as truncation, was employed to identify papers using MesH, keywords and other free terms: (((Periodontitis OR Chronic periodontitis OR Aggressive periodontitis OR Periodontal disease OR Periodont* OR probing pocket depth OR periodontal pocket)AND (Intervention OR Therapy OR Treatment)) OR (Scaling and root planning OR SRP OR nonsurgical periodontal therapy OR non-surgical therapy OR Periodontal treatment OR Periodontal therapy) AND (Probiotic OR Probiotic* OR Probiotic therapy OR Probiotic effect OR Probiotic treatment)).
Only articles published in English language have been considered.

Inclusion criteria
A study was considered eligible for inclusion in this systematic review if it met the following criteria:

1.
Type of studies: Randomized clinical trials 2. Subjects: Anyone who received probiotics as a preventive or treatment agent for periodontitis.

3.
Type of treatment intervention: Oral probiotic administration compared with placebo, or another active intervention. Randomized clinical trials were included when they (1) tested one or more probiotic agents as an adjunct to scaling and root planing (SRP) alone or with a placebo and (2) had a control group that received the same SRP as the treatment group. We considered any type of probiotic with any type of administration method.

4.
Types of outcome measures: reported results in terms of PPD.

Exclusion criteria
Studies were excluded if they included patients with the habit of smoking and systemic disease or if they were duplicated or affiliated studies.

Article review and data extraction
Potential publications' titles and abstracts were scanned by two blinded authors independently (G.H.Q and C.X) and further defined whether or not consistent to inclusion. When studies appeared to meet the inclusion criteria or information in the title and abstract was insufficient to reach an explicit decision, full texts were reviewed.
Disagreements were resolved through discussions between authors and when still couldn't reach a consensus, a third examiner was consulted. Lack of pertinent data, the relevant details were provided by contacting the authors of the identified articles. Kappa values, reflecting an almost perfect interauthor agreement, were equal to 0.89 (p < 0.001).
Basic information extraction from papers included the year and authors of publication, the type of study design, the population demographics, the definition of the periodontitis, the probiotic bacterial strains used, also their mode of administration such as the frequency and duration, and other associated treat models, the follow-up procedures, the adverse effects and the key parameters recorded, and finally, the outcome assessments. (Fig. 1 and table 1)

Search outcomes and evaluation
PPD and CAL parameters were evaluated as the primary outcomes. Secondary outcomes concluded BOP changes, plaque index (PI), gingival index (GI) or gingival bleeding index (GBI), oral malodor parameters, microbiological effects, the progression and prognosis of disease. Analysis and recording was implemented by one author (G.H.Q) and verified by another author (C.X).

Risk of bias assessment
Quality analysis according to the Cochrane Reviewers' Handbook [24] was performed to assess the risk of bias assessment. Discussing and resolving the differences within the reviewers.

Data items
The meta-analysis integrated the average difference between baseline and follow-up (3 months and 1 year) of PPD reduction (overall, stratified for moderate and deep pockets), CAL gain (overall, stratified for moderate and deep pockets) and reduction of percentage of sites with BOP.

Data synthesis
First, Q and I 2 test was conducted to check the heterogeneity between studies. P value of Q statistic<0.1 or I 2 value higher than 40% was defined as an indicator of heterogeneity. Weighted mean differences (WMD) and 95% confidence interval (CI) were regarded as the differences of continuous outcomes between SRP + probiotic and SRP group for using either fixed or random models. Each included studies' mean differences and standard errors were enrolled. The mean difference can also be estimated by standard deviation using rd = sqrt (r1 2 /n1 + r2 2 /n2) formula since data were not presented in the form of mean differences. The meta-analysis were performed using Review Manager (Version 5.2. The Cochrane Collaboration, 2013, Oxford, UK).

Study selection
Quickly browsing the search items, 185 potentially relevant papers were extracted. After looking through the titles and abstracts, 153 articles were excluded owing to duplicates. Then evaluating carefully the 14 remaining articles, five were excluded for not fulfilling the inclusion criteria (Appendix S1). Besides, since two studies [25,26] were performed at the same centre and on the same date and after discussion, it came to the conclusion that the population enrolled in the study of İnce et al. was a subgroup of the population of the study of Tekce et al. Finally, a total of nine articles fulfilled the inclusion criteria and were concluded in this systematic review [27][28][29][30][31][32][33][34][35] and three were further conducted a meta-analysis. [27,28,36] (Fig. 1).

Study characteristics
The studies were all RCT published in the English language between 2013 and 2017. All of them were designed for comparison between groups and were conducted at a single centre.  (Table 1). All studies demonstrated the sample size calculation and all patients recruited were with no systemic diseases and habit of smoking and also with no history of antibiotic administration within 6 months before entering the experiment.

Risk of bias across studies
All included studies included in this systematic review were performed quality analysis according to the Cochrane Reviewers' Handbook (Higgins & Green 2011) ( Table 2), and risk of bias assessment showed that all the RCT were defined as a low risk of bias ( Table 3).

The effect of probiotics in periodontitis PPD reduction
There studies [29,33,35] showed a significant difference on overall PPD reduction (p <0.05) favoring the superior clinical efficacy of the SRP + probiotic treatment when compared to the SRP. One study [30] clarified that there only was significantly greater PPD reduction in teeth with an initial PD ≥ 4mm in favor of the SRP+probiotic group (p < 0.05). Oppositely, four researches [27,29,34,36] 38) in control group at the end of follow-up visit [25,29,39]. Additionally, the study of Piyush Shah et al found no significant differences between SRP+ pro biotic, SRP+ probiotic+ doxycycline, SRP+ doxycycline [19]. In four studies [27][28][29]36], the results of treatment were analyzed on the basis of initial PPD of which the depth was further stratified as moderate or deep accordingly. In show more favorable results than the control group.

BOP, GBI, GI and PI changes
Bleeding on probing (BOP), GBI reduction, Gingival index and PI were more important in the SRP + probiotic group in all the included studies but with no significant inter-group differences [27,[29][30][31][32][33]39], except two studies [28,35]. BOP, GI and PI were significantly lower in the SRP + probiotic group than in the SRP + placebo group at all time points in the two studies [28,35].

Other outcomes measures
Five studies [27,28,[32][33][34]  Interestingly, owing to different follow-up periods, discrepancies existed between Teughels et al who found that after 3 months' observation, only in deep sites and the number of patients that there was significant difference between groups. Three included studies [25,29,38] in which the periodontal risk assessment tool [41] was conducted, reported that after receiving additional probiotics treatment, more patients were classified as low risk and fewer patients as high risk for disease progression. Moreover, the study of Penala et al analyzed that there was a statistically significant reduction in halitosis scores (p < 0.05) at 1 month and 3 months in test group when compared to placebo, and Ince et al illustrated that decreased GCF MMP-8 levels and increased TIMP-1 levels were more significant important up to day 180 (p<0.05) in experimental group.

Compliance and adverse effects of probiotics
None of the RCT included in this review reported the adverse effects of probiotic administration and patients all showed good compliance with probiotics application.

Publication bias
Due to the small number of trials included in this review, it was not possible to statistically assess publication bias.

Discussion
As the mainstay of the management of periodontitis is the reduction and elimination of specific periodontal pathogens (periodontopathogens/pathobionts) and SRP always being a primary step in the treatment [42], further antimicrobial agents can also be used in conjunction with mechanical procedures to reduce the pathogenic microbial burden and provide a satisfactory clinical outcome particularly in chronic situations [43,44]. However, there is evidence that periodontopathogens, such as Tannerella forsythia and A. actinomycetemcomitans, remain in periodontal pockets after nonsurgical therapy [45,46] , and bacterial recolonization occurs even shortly after scaling and root planning and the emergence of antibiotic resistance in these pathobionts have provoked human's concerns. Under the circumstances of that, it's urgently to call for new approaches for the management of periodontitis. These limitations of conventional periodontal therapy gave rise to many attempts to introduce one rather incredible or innovative approach that has been attempted during the last few decades as an alternative for the adjuvant treatment of periodontitis through administration of probiotics to manage a number of infectious diseases, so that the disease-causing pathogens are inhibited, promoting the development of a healthy flora, thus leading to restoration of health [47][48][49]. Probiotics therapy has gained great success in conquering intestinal inflammatory diseases and increasingly occupies the dominant position in selecting therapies to treat some certain intestinal abnormities [50]. On the basis that the oral cavity is closely connected with the intestinal canal and they share plenty of similarities, it's well-founded to postulate that the probiotics can also be applied to treat dental inflammatory diseases.
This systematic review and meta-analysis was aimed to unveil whether adjunct use of probiotics has additional clinical efficacy in the treatment of periodontitis. Our findings were in favor of administration of Lactobacillus as an adjuvant to SRP compared to SRP therapy alone. All the studies assessed had a low risk of bias and the studies incorporated in the meta-analysis share similar characteristics. Moreover, the results showed a statistically significant bleeding on probing (BOP) Interestingly, two studies unraveled that L. rhamnosus SP1 and Streptococci sp. [29,39] had no additional clinical effects as an adjuvant therapy to SRP, demonstrating the species specificity of probiotics in managing periodontitis. Based on these findings, we further guess that whether another extensively researched probiotics Bifidobacterium can be administrated to treat periodontal diseases.
Fortunately, there are some in vitro studies have evidenced that Bifidobacterium sp. can modify immunoinflammatory and microbiologic parameters and promote a protective effect against experimental periodontitis [51][52][53]. However in vivo studies are needed to purport its clinical benefits.
Considering that applying probiotics can result in the reduction of burden of periodontopathic organisms such as A.actinomycetemcomitans, P. intermedia and P. gingivalis [27,28,[32][33][34] and the concentrations of proinflammatory factors [35], however, the mechanisms underlying the observed salutary effect of probiotics are unclear as yet. According to the ecological plaque hypothesis and the changes of the periodontal microenvironment [48], the possible mechanisms may be the potential of probiotics to change the overall composition of the periodontal biofilm in favor of commensals and alleviate the dysbiosis caused by periodontopathogens (the so called 'red complex bacteria') and competitive capture of adhesion sites and nutrient marasmus [54,55]; modulation of the immune system [56,57]; modulation of cell proliferation and apoptosis [58]; production of antimicrobial substances, such as lactic acid, hydrogen peroxide, and reuterin [54,59]; WMD,Weighted mean differences; CI, confidence interval; CFUs, colony forming units

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Authors' contributions
GHQ, CX, LL, WXQ and XY adapted the economic model for this analysis. GHQ and CX wrote the first and subsequent versions of the manuscript. All the authors contributed to fruitful discussion of the results and to the review of the manuscript. XY is the guarantor of the overall content of the paper. All authors have read and approved the manuscript, and ensure the integrity and accuracy.

Ethics approval and consent to participate
No applicable

Consent for publication
No applicable

Conflict of interest
None of the authors of this review declares a conflict of interest or obtained any kind of financing or support from any company related to the production of probiotics.      Forest plot of overall percentage of BOP reduction at short-term