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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 1  |  Page : 57-63

A double-blind, placebo controlled study to assess the clinical and microbiological effects of a probiotic lozenge as an adjunctive therapy in the management of chronic periodontitis


Department of Periodontics, Mahatma Gandhi Post Graduate Institute of Dental Sciences, Puducherry, India

Date of Submission22-May-2018
Date of Decision10-Jun-2018
Date of Acceptance24-Jun-2018
Date of Web Publication14-Feb-2019

Correspondence Address:
Grace Tara Paul
No. 26, II Cross, Pothigai Nagar, Navarkulam, Lawspet P.O, Puducherry - 605 008
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjhr.cjhr_71_18

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  Abstract 


Background: The study was conducted to determine the effect of a probiotic lozenge as an adjunct to scaling and root planing in patients having chronic generalized periodontitis in a double blinded, placebo-controlled design. Materials and Methods: A total of 30 chronic generalized periodontitis patients, in the age group of 25–60 years were recruited for the study. Clinical parameters of plaque index (PI), bleeding index (BI), gingival index (GI), probing pocket depth (PPD), clinical attachment level (CAL) and microbiological levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were assessed at baseline, 3, 6, and 12 weeks. All patients underwent scaling and root planing (SRP) at baseline and were recalled after 3 weeks. At 3 weeks, the patients were prescribed either probiotic or placebo lozenges for a 3-week period. Statistical analysis was conducted for clinical and microbiological parameters. Results: The PI, PPD, and CAL significantly reduced in both the groups from baseline to 12 weeks. GI and BI reduced significantly at 3 and 6 weeks in the probiotic group and at 3, 6, and 12 weeks in the placebo group. There was a significant decrease in the assayed periopathogens in both the probiotic and placebo groups at all-time intervals. Conclusions: The present study did not show a significant difference of using probiotic over SRP. As the duration of the study was limited to a period of 3 months, the need to conduct large multicenter studies is warranted to substantially validate the use of probiotics in the management of chronic periodontitis.

Keywords: Chronic periodontitis, Lactobacillus brevis CD2, lozenge, placebo, probiotic, scaling and root planing


How to cite this article:
Paul GT, Gandhimadhi D, Kennedy Babu S P. A double-blind, placebo controlled study to assess the clinical and microbiological effects of a probiotic lozenge as an adjunctive therapy in the management of chronic periodontitis. CHRISMED J Health Res 2019;6:57-63

How to cite this URL:
Paul GT, Gandhimadhi D, Kennedy Babu S P. A double-blind, placebo controlled study to assess the clinical and microbiological effects of a probiotic lozenge as an adjunctive therapy in the management of chronic periodontitis. CHRISMED J Health Res [serial online] 2019 [cited 2019 Oct 23];6:57-63. Available from: http://www.cjhr.org/text.asp?2019/6/1/57/252297




  Introduction Top


The current understanding of periodontal diseases clearly establishes them as infections caused by specific microbiota, residing in subgingival biofilms on the nonshedding surfaces of the oral cavity. The main etiological factors of periodontal disease seem to be the absence of beneficial bacteria, the presence of pathogenic bacteria, and a susceptible host.[1],[2] There has been a major paradigm shift in treating periodontal diseases from a nonspecific to a more specific targeted approach. Present treatment options propose the modification of ecological niches, from pathogenic organisms to a biofilm of commensals.[3]

The World Health Organization defines probiotics as viable microorganisms, which when administered in sufficient doses, confer a health benefit on the host.[4] These probiotic nanosoldiers can alter, delay, or halt pathogenic microorganisms. This mechanism of action can be utilized in periodontics, to modify plaque, manage halitosis, and improve probing pocket depth and clinical attachment loss.[3]

Probiotics, for oral use, have been used as tablets, gels, chewing gum and lozenges. A study in dogs, by Teughels et al. have shown that the administration of beneficial bacteria, as an adjunct to scaling and root planing (SRP), could inhibit recolonization of pathogens in periodontal pockets.[5] Shimauchi et al. revealed that the regular use of probiotic tablets improved plaque index (PI) and probing pocket depth (PPD) and also reduced the prevalence of moderate-to-severe gingival inflammation.[6] Another study showed a reduction in proinflammatory cytokines in patients with gingivitis, when they consumed probiotic chewing gum, for 2 weeks.[7]

More studies on the use of probiotics in various formulations are warranted as the need of the hour, to substantiate and recommend routine use of probiotics as a part of the periodontal treatment plan and modify the existing treatment strategy from a nonspecific to a more specific and targeted approach. Until date, three studies have been conducted using probiotic lozenges in chronic periodontitis patients[8],[9],[10] and one in patients with aggressive periodontitis.[11] Of the three studies conducted in chronic periodontitis, only one has been done on the Indian population (Vivekananda et al.). In the study mentioned, a split-mouth design was employed and the lozenge used was a strain of Lactobacillus reuteri. Our study was conducted with a double-blind randomized placebo-controlled trial design, to study the role of probiotics in patients with chronic periodontitis. The probiotic used was Lactobacillus brevis.


  Materials and Methods Top


Design

This was a double-blind, placebo-controlled, randomized trial conducted at a single center (Department of Periodontics, Mahatma Gandhi Post Graduate Institute of Dental Sciences, Puducherry) in India from June 2013 to July 2014.

Participants

Patients of both sexes, in the age group of 25–60 years, with mild-to-moderate chronic periodontitis, as defined by PPD 5–7 mm in >30% of the probing sites, having a minimum of 16 remaining natural teeth (minimum of at least four teeth per quadrant) were included. The patients were otherwise in good general health and had not participated in any clinical trial during the previous 4 weeks, had not taken antibiotics in the past 2 months or were on ongoing antibiotic treatment. Pregnant women, lactating mothers, smokers, alcoholics, or subjects who had undergone surgical or nonsurgical therapy within 6 months before start of the study were excluded. The sample size was calculated using a target for the power of the statistical test to be applied once the sample is collected.

The study was conducted according to the Institute Ethics Committee and the Declaration of Helsinki and adhered to good clinical practice guidelines. All participants provided signed informed consent.

Study drugs and randomization

The investigational drug used was a strain of L. brevis CD2, supplied by CD Pharma India Pvt., Ltd. Each lozenge contained 1 billion colony forming unit of the bacterial strain. Eligible patients were randomly assigned in a 1:1 ratio to receive two lozenges twice daily, of either the probiotic or the placebo, for 3 weeks. Both lozenges were supplied in similar packaging and were identical in color, shape, texture, and flavor. The patients were instructed to suck the lozenges, two in the morning and two at night time, and to refrain from eating or drinking hot beverages for at least 1 h before and after lozenge intake.

Sequence generation

The random numbers were generated by computerized random number, in permuted blocks of 6. The randomization list and numbered packing of the intervention were prepared by a person not involved in the study.

Randomization-allocation concealment

All the randomization numbers were concealed in separate envelopes and marked by patient number on the outer envelope.

Randomization implementation

The randomization was performed by staff not involved with the study.

Blinding

The individually sealed envelope method was used to maintain blinding of the investigators and study participants.

Assessments

At entry to the study (screening visit), each patient's demographic characteristics, medical history, and current medications were recorded.

Clinical parameters include PI by Silness and Loe,[12] gingival index (GI) by Loe and Silness,[13] Bleeding index (BI) by Ainamo and Bay,[14] PPD and clinical attachment level (CAL). PPD and CAL were assessed using University of North Carolina– 15 (UNC-15) periodontal probe.

At baseline, after recording the clinical parameters and collection of samples for microbiological analysis, the patients were subjected to full mouth SRP. Scaling was done using ultrasonic scalers (EMS™) and root planing with Gracey curettes (Hu Friedy ™). The SRP procedure was completed within 2 days. Oral hygiene instructions were provided and the patients called for follow-up visit after 3 weeks. Clinical and microbiological parameters were evaluated and the lozenges (either probiotic or placebo) prescribed for 3 weeks, and the patients were advised to return after 3 weeks. No intervention was done after 6 weeks. Clinical and microbiological assessments were repeated at the end of 6 weeks and 12 weeks. Compliance was measured by pill count and regular telephone calls. Any patients with <85% drug compliance were excluded from final analysis.

Microbiological assessment

After recording the clinical parameters, at least 10 sites with 5–7 mm PPD were selected for subgingival pooled plaque sample collection, at baseline, 3 weeks, 6 weeks and 12 weeks. The sample site was isolated with sterile cotton rolls and supragingival plaque removed using cotton rolls. A sterile curette was introduced to the base of the pocket and plaque removed. The curette with the collected plaque was dispensed in separate vials containing reduced transport media and sealed to avoid contamination. They were transported within 2 days to the laboratory (Department of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka). The vials were vortexed briefly for 30 s to properly disperse the samples, and then diluted in 1:10 proportionate, i.e., 100 μl of sample diluted with 900 μl of the transport fluid. A volume of 10 μl aliquots from this were inoculated using a sterile loop onto petri plates with the following media: For the evaluation of Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi), blood agar, which was enriched with hemin and Vitamin K was used.[15] They were incubated at 37°C for 72 h in anaerobic jars. Kanamycin blood agar, as an anaerobic selective medium was also used. The samples were again incubated at 37°C for 72 h in anaerobic jars.

For Aggregatibacter actinomycetemcomitans (Aa), dentaid agar was used. They were incubated at 37°C in 5% CO2 for 72 h, in an anaerobic jar, with modified Gas-Pak.[16] After this period, the plates were examined and the numbers of different colonies were noted.

Identification of individual species was done using various morphological and biochemical characters. For whitish transparent colonies 2–3 mm in diameter, adherent to agar surface, Gram-negative coccobacilli, catalase positive, oxidase negative, indole negative, and nitrate reduction fermentation of glucose, maltose, mannitol and xylose were identified as Aa. Pg was identified by Gram-negative bacilli producing black pigmented colonies, hemolytic, lack of fluorescence under ultraviolet light, catalase negative, indole positive and lack of ability to ferment any sugars. Black pigmented colonies of Pi were identified due to brick red fluorescence under ultraviolet light, catalase negative, positive indole test, and fermentation of glucose and sucrose with acid production, but not cellobiose and lactose. Quantification of organisms, identified by the above-mentioned procedure, was expressed as colony forming units/ml.

Statistical analysis

The descriptive statistics (mean ± standard deviation) was presented for continuous variables under each group. Since the data did not follow a normal distribution, nonparametric Mann–Whitney test was used to compare the variables between the two groups. Within group comparisons of quantitative variables were done using Wilcoxon signed rank test. For all tests, a P < 0.05 were considered as statistically significant. The Statistical analysis was performed using SPSS statistical software version 19.0 (SPSS Inc., Chicago, IL, USA).


  Results Top


A total of 45 patients were selected following the inclusion and exclusion criteria. Of these, 12 patients were excluded (4 patients had periodontal abscesses, requiring antibiotics, 3 patients were unwilling to participate, 3 patients were occasional smokers, and 2 patients were found to be diabetic during routine screening). Three patients showed inactive sites [Figure 1].
Figure 1: Consort diagram for the patient disposition

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Thirty, systemically healthy, chronic periodontitis patients (13 males and 17 females; mean age of 38.1 years) were finally included. Age, gender distribution, clinical and microbiological parameters were similar in both groups at baseline. A total of 25 patients (14 in the probiotic and 11 in the placebo groups) successfully completed the study. No adverse events of the investigational drugs were mentioned by the patients or observed by investigators.

Clinical parameters

The results indicate that SRP led to a significant improvement in all clinical parameters in both probiotic and placebo groups. There was a significant reduction in PI, PPD and CAL from baseline to 3 weeks, 6 weeks, and 12 weeks in both groups [Table 1] and [Graph 1],[Graph 2],[Graph 3]. Within the groups; the reduction was significant at all-time interval Intergroup differences were not significant at any point. Similarly, the GI and BI too reduced, significantly at 3 and 6 weeks in the probiotic group, and at 3, 6 and 12 weeks for the placebo arm, when compared with baseline. No statistically significant difference was seen between the two groups [Table 1] and [Graph 4], [Graph 5].
Table 1: Between group comparisons of clinical and microbiological parameters at different time points

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Microbiological parameters

Though SRP led to a statistically significant improvement in clinical parameters at 3 weeks in both groups, changes in microbiological parameters were comparable at 3 weeks when compared with baseline. Decrease in counts of analysed periodontopathogens was not significantly changed between groups at 3, 6, and 12 weeks [Table 1] and [Graph 6],[Graph 7],[Graph 8].



Within group comparisons in levels of Aa showed a significant reduction at 6 and 12 weeks in the probiotic groups and at 6 weeks in the placebo group. Changes in levels of Pg were not significantly changed at 6 weeks when compared to baseline, but a significant reduction was found at 12 weeks in the probiotic group. On the other hand, levels of Pg were significantly reduced at 6 and 12 weeks in the placebo arm. Pi reduced significantly in the probiotic group at week 12 in the probiotic group, but the changes were comparable at 6 and 12 weeks in the placebo group.


  Discussion Top


Periodontitis has two distinct but interconnected etiologic components, periodontopathic bacteria adjacent to the periodontal tissues, and host-mediated connective tissue-destructive responses to the causative bacteria.[17]

The current new model proposed to understand periodontal disease is the Polymicrobial Synergy and Dysbiosis. This model proposes that there is an imbalance in the abundance or influence of a microbial community. A dysbiotic state promotes inflammation and periodontal disease.[18] Hence, treatment strategies against periodontal diseases have evolved with the aim of eliminating specific pathogens or suppressing destructive host response.

The characteristics of periodontal diseases such as inflammation and attachment loss are associated with proteolytic events. Oral damage can result from the release of an array of proteolytic enzymes by colonizing bacteria as well as host-derived proteases.[19],[20],[21],[22]

Since periodontitis is a localized disease condition, a local drug delivery system should be preferred. Probiotics are one such option. A literature search provided us with a list of studies using probiotics in the form of powder, capsule, chewable tablets and lozenges in chronic periodontitis and other inflammatory conditions of the oral cavity.[8],[23]

Riccia et al. demonstrated that treatment of patients with L. brevis CD2 lozenges led to the total disappearance or amelioration of all analyzed periodontal disease-associated clinical parameters. The clinical beneficial effect of the treatment was paralleled to a significant decrease of salivary nitric oxide synthase activity as well as prostaglandin E2, matrix metalloproteinase 2, and interferon-γ levels. The findings in our study using the same product showed a statistically significant reduction in PI, GI, BI, PPD, and CAL in both the probiotic and placebo group; the difference between the groups was however not significant.[24]

We did a literature search, to find studies that did not show any added clinical benefits on using probiotics. It yielded two such studies, namely, Iniesta et al. 2012[25] and Hallström et al.[26] 2013. Iniesta et al. used Lactobacillus containing tablets in patients suffering with gingivitis in a placebo controlled trial for 8 weeks. The results did not show any improvement in any of the clinical variables. Hallström et al. conducted a study on eighteen females, with experimental gingivitis. The patients were given L. reuteri lozenges and were evaluated for PI, GI and BI. The gingival crevicular fluid was assayed for the concentration of seven inflammatory mediators and bacterial samples were checked for DNA-DNA hybridization. Daily intake of probiotic lozenges did not seem to significantly affect the plaque accumulation, inflammatory reaction or the composition of the biofilm during experimental gingivitis.

Teughels et al.[9] conducted a randomized placebo controlled trial on patients with chronic periodontitis and evaluated the clinical and microbiological effects using L. reuteri probiotics. No statistically significant inter-group differences were observed in the amount of full-mouth PPD reduction between the two groups at 12 weeks. When analyzing the data in detail, by looking at PPD specific subgroups, there was a significant (P < 0.05) lower mean PPD at the end of the study in the SRP + probiotic group for deep pockets when compared to the SRP alone group.

In the present study, we compared full mouth PPD in both the SRP + Probiotic group and the SRP+ placebo group; across all time intervals and found the reduction to be comparable between the two groups. This was similar to the results in Teughels et al. study. We did not sub classify into deep and moderate pockets.

Our study showed improvements in all clinical parameters with reduction in microbial load of periopathogens, but intergroup comparisons did not yield any statistically significant results.

The authors are of the opinion that a larger sample size could have probably given us more scope to conclude the results effectively. Randomised controlled trials that have low power generally generate false-negative results. This study was conducted in a small sample size of patients and hence did not yield statistically significant results between the groups.

The probiotic used was L. brevis, which is a Gram-positive rod, found in the normal gut microbiota of the human intestines, vagina, and feces. Probably, a probiotic, with specific action in the oral cavity would have been warranted.


  Conclusion Top


Although the current study did not show any advantage of probiotics over SRP alone, it nevertheless does not undermine its use. This has served as a hypothesis generating trial. More randomized controlled trials of a larger sample size and longer duration are required to completely understand and exploit the benefits of probiotics in the management of periodontal diseases.

Financial support and sponsorship

The study was an investigator initiated effort. The study drugs (probiotic and placebo lozenges) for the study were provided free of cost by CD Pharma India Pvt. Ltd., New Delhi, India. However, the microbiological analysis was funded by CD Pharma India Pvt. Ltd., India. The company was not involved in protocol writing, data collection, data analysis or data interpretation.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Teughels W, Newman MG, Coucke W, Haffajee AD, Van Der Mei HC, Haake SK, et al. Guiding periodontal pocket recolonization: A proof of concept. J Dent Res 2007;86:1078-82.  Back to cited text no. 5
    
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Twetman S, Derawi B, Keller M, Ekstrand K, Yucel-Lindberg T, Stecksen-Blicks C, et al. Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory mediators in gingival crevicular fluid. Acta Odontol Scand 2009;67:19-24.  Back to cited text no. 7
    
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Vivekananda MR, Vandana KL, Bhat KG. Effect of the probiotic Lactobacilli reuteri (Prodentis) in the management of periodontal disease: A preliminary randomized clinical trial. J Oral Microbiol 2010;2. doi: 10.3402/jom.v2i0.5344.  Back to cited text no. 8
    
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Korostoff JM, Wang JF, Sarment DP, Stewart JC, Feldman RS, Billings PC, et al. Analysis of in situ protease activity in chronic adult periodontitis patients: Expression of activated MMP-2 and a 40 kDa serine protease. J Periodontol 2000;71:353-60.  Back to cited text no. 21
    
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Krasse P, Carlsson B, Dahl C, Paulsson A, Nilsson A, Sinkiewicz G, et al. Decreased gum bleeding and reduced gingivitis by the probiotic Lactobacillus reuteri. Swed Dent J 2006;30:55-60.  Back to cited text no. 23
    
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Riccia DN, Bizzini F, Perilli MG, Polimeni A, Trinchieri V; Amicosante G, et al. Anti-inflammatory effects of Lactobacillus brevis (CD2) on periodontal disease. Oral Dis 2007;13:376-85.  Back to cited text no. 24
    
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