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ORIGINAL ARTICLE
Year : 2022 | Volume
: 21
| Issue : 4 | Page : 395-402
Investigation and comparison of the effects of two probiotic bacteria, and in reducing mutans streptococci levels in the saliva of children
Vijaya Lakshmi Bolla1, Munnangi S Reddy2, Namineni Srinivas3, Ch Sampath Reddy3, Pradeep Koppolu4
1 Asst Professor, Department of Periodontics, Government Dental College and Hospital, Hyderabad, India
2 Professor, Department of Pedodontics and Preventive Dentistry, SVS Institute of Dental Sciences, Mahbubnagar, Telangana, India
3 Professor, Department of Pedodontics and Preventive Dentistry, Sri Sai College of Dental Surgery, Vikarabad, Telangana, India
4 Asst Professor, Department of Preventive Dental Sciences, College of Dentistry, Dar Al Uloom University, Riyadh, KSA
Correspondence Address:
Vijaya Lakshmi Bolla
Asst Professor, Department of Periodontics, Government Dental College and Hospital, Hyderabad, Telangana
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_133_21
Abstract
Background: Probiotic organisms Lactobacillus reuteri UBLRU-87 and Bifidobacterium bifidum UBBB-55 were proven to be acting against the caries causing organisms. Aims: This study aims to evaluate the influence of Lactobacillusreuteri, Bifidobacterium bifidum and their blend on Mutans streptococci count in the saliva of children, and also to appraise the sustainability of their action. Materials and Methods: A randomized, double-blind and placebo-controlled study with 60 subjects (15 in each group) in 6–14 years of age group. The children consumed curd containing Bifidobacterium bifidum (UBBB 55, MTCC 5398) and Lactobacillus reuteri (UBLRu 87, MTCC 5403), and their blend once daily for 14 days. The control group received curd with no Probiotic in it. The saliva samples were collected just before the curd (T0) administration to establish baseline levels of mutans streptococci and after a day of the final consumption of the curd (T14). The follow-up samples at 21 days (T21) and 28 days (T28) after the baseline were also collected to know the sustainability of action probiotics on mutans streptococci if any. Results: Statistically significant reduction of mutans streptococci is observed in the group administered with Lactobacillus reuteri and the effect lasted up to a minimum of 21 days. Mixed cultures are seemed to be not effective against the oral microorganisms. Conclusions: The probiotic organism Lactobacillus reuteri in Indian curd is effective on salivary mutans streptococci, and the effect was sustained for some period after the administration.
Abstract in French Résumé
Contexte: Il a été démontré que les organismes probiotiques Lactobacillus reuteri UBLRU-87 et Bifidobacterium bifidum UBBB-55 agissaient contre les organismes responsables des caries. Objectifs: Cette étude vise à évaluer l'influence de Lactobacillus reuteri, Bifidobacterium bifidum et de leur mélange sur le nombre de streptocoques Mutans streptocoques dans la salive des enfants, et également d'évaluer la durabilité de leur action. Conception de l'étude: Une étude randomisée, en double aveugle et placebo avec 60 sujets (15 dans chaque groupe) dans le groupe d'âge 6-14 ans. Les enfants ont consommé du lait caillé contenant du Bifidobacterium bifidum (UBBB 55, MTCC 5398) et Lactobacillus reuteri (UBLRu 87, MTCC 5403), et leur mélange une fois par jour pendant 14 jours. Le groupe témoin a reçu du lait caillé sans probiotique. Les échantillons de salive ont été prélevés juste avant l'administration du lait caillé (T0) pour établir les niveaux de base des streptocoques mutans. Les échantillons de salive ont été prélevés juste avant l'administration du lait caillé (T0) pour établir les niveaux de base de streptocoques mutans et un jour après la consommation finale du lait caillé (T14). Les échantillons de suivi à 21 jours (T21) et 28 jours (T28) après la ligne de base ont également été collectés pour connaître les niveaux de streptocoques mutans. la ligne de base ont également été collectés pour connaître la durabilité de l'action des probiotiques sur les streptocoques mutans, le cas échéant. Résultats: Réduction statistiquement significative statistiquement significative des streptocoques mutans est observée dans le groupe administré avec Lactobacillus reuteri et l'effet a duré jusqu'à un minimum de 21 jours. Les cultures mixtes ne semblent pas être efficaces contre les micro-organismes oraux. Conclusions: L'organisme probiotique Lactobacillus reuteri dans le caillé indien est efficace sur la salive Indian curd est efficace sur les streptocoques mutans salivaires, et l'effet a été maintenu pendant une certaine période après l'administration.
Mots-clés: Caillé, probiotique, mutans salivaires
Keywords: Curd, probiotic, salivary mutans
Introduction 
At the start of the 20th century, Ukrainian-born Russian Nobel Prize laureate Elie Metchnikoff reported that Bulgarians lived longer than people in other nations and imagined that this was because they consumed fermented milk products containing viable bacteria. The concept was, the bacteria in the fermented products competed with microorganisms injurious to health.[1]
Various studies on probiotic organisms have already shown a positive effect on the general health[2] but their possible action in the mouth is restricted.[3] The lack of probiotic research in Odontology is also apparent with limited randomized controlled trials with caries as an endpoint measure.[4],[5]
Lactobacillus reuteri and Bifidobacterium bifidum were proven to be acting against the caries causing organisms.[5],[6],[7],[8],[9],[10],[11]
Lactobacillus reuteri is an obligate heterofermentative resident in the gastrointestinal tracts (GITs) of humans, and it was reported to produce those compounds that can exhibit antagonistic activity such as reuterin and reutericyclin, and which are water-soluble besides having broad-spectrum antimicrobials features thus effective over a wide pH range and resistant to proteolytic and lipolytic enzymes.[6]
Bifidobacterium bifidum, one of the first species to be introduced into probiotic research, was the predominant anaerobic bacteria naturally occurring within the small intestinal lumen of healthy, breastfed children. They remain vital due to playing a critical role in maintaining equilibrium among normal intestinal flora.[12]
Mixed cultures might contain bacteria that complement each other's health effects and thus have synergistic probiotic properties. Studies have already confirmed the potential benefit of multistrain probiotic supplements in the management of Irritable Bowel Syndrome, Ulcerative Colitis and Pouchitis.[13],[14]
The relationship between the mutans streptococci levels in saliva and the caries risk in the individuals got evaluated earlier.[15],[16]
Curd, a type of yoghurt and an essential component of the conventional Indian diet, was used in this study to make the research done applicable and readily available to the community.
Materials and Methods Study design
Our study is a randomized, double-blind and placebo controlled study with 60 subjects.
Resident children belonging to Mary A. Knotts hostel, Vikarabad, India falling under the same socioeconomic status, were chosen for the study. The residents, their parents/guardians, and hostel authorities were already explained the study's nature and purpose through an educative lecture.
This study got approval from the Institutional Review Committee for the study protocol on Ethical Issues of Sri Sai College of Dental Surgery and Research Centre, Vikarabad, India.
We obtained informed consent from the hostel authorities besides oral permission from the children.
Furthermore, we recorded a briefcase history in a specifically designed pro forma, which included information regarding the patient's overall medical status, drug history, general health, and well-being.
Children in the age group of 6–14 years, who had not taken antibiotics or probiotics in any form within 2 weeks before the study, and who have Decayed, Missing, and Filled surfaces (DMFS) /defs ≥5 (caries active) were included in the study.
Children with systemic diseases, immune-compromising conditions, special children and those who were not willing to eat curd or participate in the study were excluded.
A random selection of 60 subjects from the group of subjects satisfied the inclusion and exclusion criteria. The subjects were divided randomly into four groups named P, Q, R, and S depending on the type of Probiotic to be administered [Figure 1] and [Figure 2]:
Group P: Control group with administration of curd without any ProbioticGroup Q: The group with administration of curd with Bifidobacterium bifidum UBBB-55.Group R: The group with administration curd with a consortium of two general (Bifidobacterium bifidum UBBB-55 and Lactobacillus reuteri UBLRu 87)Group S: The group with administration of curd with Lactobacillus reuteri UBLRu 87.The probiotic organisms Bifidobacterium bifidum (UBBB 55, MTCC 5398) and Lactobacillus reuteri (UBLRu 87, MTCC 5403) were provided by Unique Biotech Limited, Hyderabad, India, in freeze-dried powder form.
Each subject was coded according to the group and the identity within the group. For example, P 9 is the ninth subject in group P.
Sample collection process
It began with the 2–3 ml of unstimulated saliva collected from each subject directly into the sterile containers with respective codes of the subjects marked on them. Saliva collected every day between 9:00 and 10:00 a.m.[6],[17] was transported to the laboratory for the microbiological culture within 2 h of their collection in the cold chain.
The saliva samples were collected before administering the curd (T0), to establish the baseline levels of mutans streptococci.
All the subjects were then given 100 g of the respective coded curd at lunchtime.
They were instructed to consume the curd entirely along with the meal and not drink or eat anything for 1 h after lunch to give sufficient time for the organisms to colonize in the mouth.
It got repeated for 14 successive days, and saliva samples were collected after a day of final consumption of the curd (T14). The follow up samples at 21 days (T21) and 28 days (T28) after the baseline were also collected to know their sustainability if any.
The saliva samples were cultured for mutans streptococci on MSB (MitisSalivarius Bacitracin) agar, a selective culture medium. The results were scored and categorized as:
Score 0 = 0–10 × 105Colony Forming unit (CFU)/ml;
Score 1 = 11–99 × 105CFU/ml;
Score 2 = 100–500 × 105CFU/ml;
Score 3 = 500 × 105CFU/ml.
The data then obtained was subjected to statistical evaluation.
Preparation of the curds
1.0 g of the freeze-dried probiotic culture of each Bifidobacterium bifidum, Lactobacillus reuteri and a consortium of both (100 billion CFU/gm) was weighed and added to 1 liter of pasteurized milk at around 37°C and mixed well for the powder to get dissolved completely and distributed evenly. Half a teaspoon of commercial curd culture was added as inoculums in all the groups and mixed well. Milk was then distributed into 100 ml cups-representing a culture of 1 × 108 CFU/ml. Each cup of curd was coded for each group, sealed with antibacterial aluminum foil and maintained at 30°C–37°C until the curd is formed. The cups were cooled, refrigerated immediately until administered.[18],[19]
Sample curds for all the groups were prepared following the same procedure before the study had started, and were sent for the analysis to check for the viability of the probiotic organisms in the curds. The analysis of the curd showed the viability of all the probiotic organisms to satisfactory levels. Once the viability was as expected, the same procedure was followed throughout the study.
Determination of mutans streptococci levels
One ml of the saliva sample was taken and transferred into a homogenizing cup utilizing a micro-pipette. It had the addition of 10 ml of sterile normal saline and homogenized at about 12,000–15,000 rpm for 7–10 min to achieve 1:10 dilution of the saliva sample.
The diluted saliva sample was mixed well using a cyclo-mixer and further diluted stepwise through a series of test tubes (size: 25 mm × 150 mm), containing 9 ml of the sterile standard saline blank solution by an appropriate decimal dilution method. One ml of the different dilutions of the saliva samples of each subject got inoculated into the MSB agar culture media through the micropipette.[6],[19],[20],[21]
MSB agar medium got sterilized, melted and cooled to 45°C–50°C. 15 ml of MSB agar medium added to each of the Petri dishes selected and autoclaved. The plates got incubated in a CO2 incubator at 37°C for 48 h, and the number of colonies counted manually in each plate.
The colonies of Streptococcus mutans were identified by their morphology with a microscope with 10× magnification and compared with the morphology of a standard S. mutans strain (MTCC 890).
An average of colonies that were multiplied by the dilution number (dilution factor) represents the viable count per ml.[20],[21]
CFU/ml = Number of colonies × Dilution factor.
Statistical analysis
The records got processed with the Statistical Package for
Statistical Package for Social Sciences (SPSS) version 14.0 (SPSS Inc., Chicago, IL, USA).
The data obtained got analyzed by the Wilcoxon Signed-Ranks test to compare the bacterial scores before and after the intervention of curd within the groups. The testing is done to compare the 14 days, 21 days, and 28 days scores with that of baseline. A P < 0.05 got considered a statistically significant one.
We used the Chi-square (χ2) test to analyze the distribution of baseline and the transition of mutans streptococci scores and inter-relation between the groups. The number and percentages of subjects with the changes in mutans streptococci scores also got compared. A P < 0.05 got considered a statistically significant one.
O is the Observed Frequency in each category.
E is the Expected Frequency in the corresponding category.
All the study groups with probiotic curd intervention were combined. Both tests applied to test the relationship and the significance between and within the whole probiotic groups compared with control.
Results All the subjects selected were cooperative, and the compliance was excellent. None of the subjects reported any change in their general health or reported any side effects throughout the study.
There was one dropout in each of the groups P and S; 1 subject was not considered from group S because of the antibiotic administration during the study. Three subjects in group R and one in group S were unable to report at the time of saliva sample collection on the 28th day.
All the codes were revealed only after the completed data were obtained after 21 days.
No significant alteration in salivary mutans streptococci scores was observed in the control group between baseline and after 14 days, between baseline and after 21 days.
No significant alteration in salivary mutans streptococci scores was observed in the group administered with curd containing Bifidobacterium bifidum between baseline and after 14 days, between baseline and after 21 days.
A significant increase in salivary mutans streptococci scores was observed in the group administered with curd containing a blend of Bifidobacterium bifidum and Lactobacillus reuteri between baseline and after 14 days. This result was in opposition to the anticipated reduction in salivary mutans streptococci. No significant alteration observed between baseline and after 21 days, between baseline and after 28 days.
A significant decrease in salivary mutans streptococci scores was observed in groups administered with curd containing Lactobacillus reuteri between baseline and after 14 days and between baseline and after 21 days. No significant alterations were observed in this group between baseline and after 28 days. It explains that the effect of probiotic organism Lactobacillus reuteri on salivary mutans streptococci was sustained till 21–28 days [Table 1],[Table 2],[Table 3],[Table 4].
Table 1: Transition of mutans streptococci scores in P, Q, R and S groups from Baseline to 14 days,
Table 2: Transition of mutans streptococci scores in P, Q, R and S groups from Baseline to 21 days,
Table 3: Transition of mutans streptococci scores in R and S groups from Baseline to 28 days,
Table 4: Distribution of salivary mutans streptococci score Baseline, 14, 21 and 28 days in P, Q, R and S groupsChi-square (χ2) test results had revealed a statistically significant association between the groups at 14 days and 21 days. The number of subjects with unchanged increased and decreased scores of salivary mutans streptococci was tabulated.
Discussion Caries can be defined as a chemical dissolution of the dental hard tissues by acidic bacterial products from the degradation of low molecular weight sugars. Dental caries is a multifactorial infectious disease process attributed to factors such as the child's dietary and oral hygiene habits, various pathogenic microorganisms in the oral cavity, family income, parental education, parent's dental knowledge, attitude and behavior, and place of residence.[22]
The correlation between poverty, quality of life and oral health are more pronounced in developing countries like India, where 72.2% of the population lives in rural areas. The rural areas have limited access to oral healthcare due to a shortage of dental workforce, financial constraints besides other factors like lack of perceived need for dental care.[22]
Loesche stated that dental caries is one of the most widespread diseases of humankind. S. mutans is the primary etiologic agent of dental caries. There is a strong association between the level of colonization with dental caries, although other organisms like Lactobacilli have also been implicated in this disease.[23],[24]
The existing treatment modality for dental caries is to take out the infected part and replace the missing piece of the tooth with some lifeless restorative material. Although the damaged portion was treated, the actual cause for the infection is still persistent, which could involve damage to the other teeth.[25]
The considerable caries reduction could be attained if colonization of S. mutans could be prevented or reduced at the eruption of both deciduous and permanent teeth.
Numerous efforts such as topical or systemic use of fluorides, fissure sealants, dietary control, replacement therapy, and even caries vaccine were on to eradicate the most expensive disease; the efficacy of these methods is not enough to eliminate dental caries in humans.[26]
In developing countries, a rapid increase in caries has been observed both in children and adolescents. The low dentist to population ratio and the lack of organized dental health care limit utilizing conventional caries-preventive methods.[22] Therefore, the search for new, more effective methods to prevent dental caries was optimistic of which the resurfaced concept of using probiotics (harmless microbes) is of great value. The effort is to find alternative ways to combat infections, thus preventing their rapid growth, especially in the light of the frequent use of antibiotics with subsequent risk for developing resistant strains.[27]
With the rise of investigative interest in probiotics as prevention and the publication of positive study results, it seems that probiotics someday soon could play a significant role in the control of many common childhood conditions that have socioeconomic impacts.[28]
Lactobacillus reuteri and Bifidobacterium bifidum were selected because both are typical commensal microorganisms in the intestine with no pathogenic potential and have a long history of safe use in foods. Usually, they are known as (generally regarded as safe) probiotic organisms.[29],[30] Many recent experimental studies and results from a randomized controlled trials[5],[6],[7],[8],[9],[10] have exerted beneficial effects in the oral cavity by inhibiting cariogenic Streptococci using different vehicles.
In order to overcome the influence of risk indicators such as socioeconomic factors, social factors and oral health habits, the subjects were selected belonging to the same economic strata.
The age band of 6–14 years selected as they are cooperative to participate in the study and dental caries remains one of the most common diseases among 5–17 years old.[31] Also, 7–8-year-old age range (initially mixed dentition) represents the period in which there is the highest probability of detecting elevated mean counts of the microorganisms.[32]
All strains of genus Bifidobacteria were sensitive to penicillins: penicillin G, amoxicillin, piperacillin, ticarcillin, imipenem and macrolides, clindamycin, pristinamycin, vancomycin and teicoplanin. Probiotic organisms are more susceptible when co-administered with antibiotics, Bifidobacteria being more vulnerable. Hence, only subjects who did not administer antibiotics 2 weeks before the study were included in this study.[33]
All the subjects were caries active with no active deep lesions. Evaluating patients' risk to develop caries is the need for the earliest possible clinical identification of the beginning of caries activity. In this study, subjects selected based on the criteria that DMFS/defs ≥5 were caries active. Loesche and Straffon[34] observed a statistically significant correlation between DMFS index and salivary mutans streptococci levels. A DMFS score of 5 was used to identify individuals with low or high initial caries scores and a highly significant relationship between S. mutans and initiation in the high-caries active children.[23]
Curd, a type of yoghurt commonly used in India, was selected as a vehicle for our study because it was considered the conventional ingredient of an Indian diet. At the same time, it maintains the viability of the probiotic bacteria and increases the contact time between the oral cavity and the probiotic organisms compared to liquids.
Milk and dairy foods are rich sources of calcium because they contain significant amounts of calcium in a bioavailable form. Other dairy-related vehicles such as milk,[6] yogurt,[6],[9] cheese,[35] paneer[36] (cottage cheese), fruit yogurt,[37] yogurt drinks and ice cream.[11] Lozenges,[7] powder, liquid,[38] capsules,[38] juice,[3] gelatin, straw,[8] or tablets[8] were successfully used as vehicles. The probiotic administration in the current study was done along with food at the time of lunch to know the effect without disrupting standard consumption patterns.
The instruction not to consume any solids or liquids for 1 h after lunch was to offer some time for the organisms to colonize in oral cavity if any.
As some cases reported of bacteremia in immune-suppressed after probiotic therapy, children with immune compromise conditions[39],[40] were excluded from our study. Special children were not considered because of the compliance in cooperation during examination and sample collection.
This study was double-blind as the subjects who consumed the curd were unaware of which probiotic was given. The microbiological analysts who cultured the saliva samples were unaware of which Probiotic was administered for that particular group. The codes were revealed only after the complete data were obtained and analyzed after 21 days.
The collection of saliva samples were terminated after 21 days in groups P and Q as there remained no significant change that could be observed in mutans streptococci scores and after 28 days in groups R and S as there was a significant change in mutans streptococci scores in these two groups.
The results obtained in the group administered with Lactobacillus reuteri in curd were in line with the previous studies. It reiterates the effect of Lactobacillus reuteri on mutans streptococci, in contrast to control and other groups. The reduction in mutans streptococci scores was more pronounced in the postintervention period at 21 days rather than immediately after the 14-day intervention. It suggests a sustained action of the probiotic organism even after discontinuation, and the reason behind this was not apparent. More clinical trials are required to observe this effect.
Studies conducted by Katz et al.,[41] have demonstrated that intranasal immunization of rats with an antigen-induced a protective salivary immune response, which was associated with a reduction in Streptococci mutans colonization and S. mutans induced caries. Kleanthous et al.,[42] conducted experiments through rectal immunization with nonoral bacteria antigens such as Helicobacter pylori resulting in secretory immunoglobulin A antibodies in distant salivary sites. These studies suggest the role played by the host immune system to antigens is independent of the area of antigen exposure and immune-modulation at a remote site. The reaction could be the same in the case of probiotic therapy showing its action without colonization.
Our study could not reproduce the effect of Bifidobacteriumbifidum on mutans streptococci as shown by previous studies[9],[11] on Bifidobacteria. There were no significant variations of mutans streptococci scores observed in this group. The reason for this behavior of the organism could be the environment of the oral cavity, but the same basis has to be scrutinized.
The unique feature in this study is the use of multispecies probiotics in the oral cavity. The effect of the multistrain and multispecies probiotic in GIT was considered to be synergistic.[43] The data of our study showed a significant increase in the mutans streptococci levels immediately after the intervention. The result was opposite to the anticipated decrease in mutans streptococci scores.
The cause for the differing effect from the GIT could be attributed to the individual variations in diet, salivary rate, and composition besides the local environmental influence, immune mechanisms involved in the induction and regulation of host immune systems operative in the oral cavity. Or the inhibitory effect of these organisms on each other in the oral cavity has to be investigated.
The reduction in mutans streptococci counts seems to be ended between 21 and 28 days. As many researchers had previously mentioned, probiotic bacteria must be taken in sufficient amounts daily for a prolonged duration to attain sustainability and permanent colonization in the oral cavity. Its time to decide the amount and the period required for permanent colonization.
The exact mechanism by which a probiotic act in the mouth is difficult to evaluate because of the various local influences in the oral cavity with a mixture of microorganisms, wide range of pH, shedding and nonshedding surfaces and the host defense effects of saliva and crevicular fluids. The underlining host-dependent factors play a role in the adhesion of probiotics and their action. Even the mechanical cleansing of the oral cavity by tooth brushing or frequent rinsing can change the habitat. The probiotic organisms may compete for the nutrients or adhesion sites, produce antimicrobial substances, immunostimulation act by nonimmunologic defense mechanisms or by local and systemic immunomodulation.[44],[45]
This study's results and findings must be interpreted with caution because of the limited sample size, unavailability of previous data about the synergistic effect of probiotic bacteria in the oral cavity, and the nonestablishment of the optimal daily dose.
A thorough analysis of the cost-effectiveness, risk weighed with benefits of a probiotic against dental caries in various societies and subgroups has to be performed.
Summary and Conclusions The following conclusions drew from the present study:
The curd containing probiotic organism Lactobacillus reuteri had significantly reduced the mutans streptococci scores and the effect was sustained 8–14 days after the intervention period.
The curd containing probiotic organism Bifidobacterium bifidum had no significant change in the mutans streptococci scores.
The curd containing a multispecies probiotic with Lactobacillus reuteri and Bifidobacterium bifidum significantly increased the mutans streptococci scores immediately after the intervention period.
Although the sustained probiotic effect on mutans streptococci is still under investigation, our study results showed that the probiotic Lactobacillus reuteri effectively reduces the mutans streptococci scores for 8–14 days after discontinuation of the probiotic curd in children.
However, further investigations on these probiotic treatments with larger samples, different vehicles, and various combinations of organisms and for more extended periods are necessary to explore the exact mechanisms of probiotic action, their synergy and sustainability. Control of various influencing factors will also play a key role in probiotic research.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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