A comparative evaluation of probiotics on salivary mutans streptococci counts in Indian children.
Abstract: OBJECTIVE: Probiotics and their effect on oral health have been a focus of numerous trials in recent times. No documented trials have been reported from developing countries such as India with its focus on probiotic use, especially in the paediatric population. The aim of this study is to evaluate the effect of probiotics on salivary mutans streptococci (MS) counts of children using the two commercially and widely available preparations and to explore their anti-caries potential. STUDY DESIGN: A placebo controlled study was undertaken with 3 parallel arms comprising a total of 150 healthy children (7-14 years). The subjects were randomly divided into the groups (each comprising 50 children): group A--placebo powder, Group B--a freeze dried powdered combination of Lactobacillus rhamnosus and Bifidobacterium species, Group C--a freeze dried powdered preparation of Bacillus coagulans. The subjects were instructed to mix the preparation in 20 ml of water and to follow a swish and swallow method for 14 days. Mutans streptococci colony counts per ml of saliva were performed on Mitis-Salivarius Bacitracin agar collected on the first day and 14 days post-intervention. RESULTS: A statistically significant reduction (p<0.001) in salivary mutans streptococci counts was recorded in both groups B and C after 14 days of probiotic ingestion. CONCLUSION: A cost-effective probiotic such as Bacillus coagulans might be a subject for further research for prevention of caries in children.

Key words: Probiotics, Mutans streptococci, Bacillus coagulans, dental caries.
Article Type: Report
Subject: Children (Health aspects)
Probiotics (Health aspects)
Probiotics (Research)
Streptococcus (Health aspects)
Streptococcus (Research)
Authors: Jindal, G.
Pandey, R.K.
Agarwal, J.
Singh, M.
Pub Date: 08/01/2011
Publication: Name: European Archives of Paediatric Dentistry Publisher: European Academy of Paediatric Dentistry Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 European Academy of Paediatric Dentistry ISSN: 1818-6300
Issue: Date: August, 2011 Source Volume: 12 Source Issue: 4
Topic: Event Code: 310 Science & research
Product: Product Code: E121920 Children
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 277106772
Full Text: Introduction

WHO defined probiotics as 'Live microorganisms which when administered in adequate amounts confer a health benefit on the host' [Teughels et al., 2008]. It is also known as bacteriotherapy or replacement therapy [Hillman et al., 2007]. The basic rationale behind the tautology of probiotics was that the human body lives in a heavily contaminated environment associated with millions of bacteria and probiotics can be utilised by replacing pathogenic microorganisms with healthy ones. This concept of using beneficial bacteria has gained much popularity in the field of medical research in recent years where antibiotic resistance is an increasing global problem.

Probiotics have been found to be advantageous in combating various diseases such as gastro-intestinal infections, cancer prevention, irritable bowel syndrome, constipation, periodontal diseases, dental caries and many others [Teughels et al., 2008]. The possible mechanisms of action of probiotics in the human body are normalisation of the microbiota, modulation of the immune response and metabolic effects. As the mouth represents the first part of the gastrointestinal tract there is every reason to believe that at least some probiotic mechanisms may also play a role in that part of the system [Parvez et al., 2006].

In developing countries such as India, where the major part of the population lacks access to basic health facilities, oral health is a largely neglected area with dental caries widely prevalent across all age groups, more so in children with their fondness for confectionary. Dental caries is a cause of a great deal of continuous discomfort through impaired function and aesthetics and also the costs for operative dental treatment are significant both for individuals and society. Therefore, a need exists to identify individuals at risk for the development of caries, and to target preventive measures and active treatment for these individuals. Spreading the awareness that caries is preventable by adopting simple oral hygiene practices and the use of simple, cost-effective and easily available products is likely to be cost effective.

The most commonly used probiotic bacterial strains belong to group lactobacilli [Koll et al., 2008] or to the genus Bifidobacterium [Saxelin et al., 2005]. Clinical studies have confirmed that probiotic lactobacilli can reduce the counts of salivary mutans streptococci (MS) after ingestion of L. rhamnosus-GG [Nase et al., 2001; Ahola et al., 2002], L. reuteri [Caglar et al., 2005; Nikawa et al., 2004] and a lactobacilli mix [Montalto et al., 2004]. Most of these studies used either a dairy vehicle for probiotics or a specially formulated mode of administration as lozenges [Caglar et al., 2006] or chewing gums [Caglar et al., 2007]. In India, none of these formulations are readily available for oral health. So there exists a need to explore an easily available and cost-effective alternative.

The present study aimed to explore the effect of oral probiotics on salivary MS count and to evaluate the relative efficacy of two commercially available probiotic preparations, thus assessing oral probiotic as a preventive tool against the development of dental caries.

Materials and methods

The present study was carried out in the Department of Paedodontics and Preventive Dentistry in association with Department of Microbiology, CSM Medical University, Lucknow, India. The research protocol was reviewed and approved by the Institutional Ethics Committee, CSM Medical University, Lucknow. Consent forms were signed by parents or legal guardians, prior to patient enrolment in the study. The prospective investigation had a randomised, double-blind placebo controlled study design with an experimental period of 14 days.

Subjects These were 150 healthy children in the 7-14 years age group who attended the paediatric dental clinic at FODS, CSM Medical University Lucknow, and were included in the study. Children without a history of antibiotic intake or topical fluoride therapy for the preceding one month were included. None of the subjects exhibited untreated active carious lesions as acute foci of infection in the oral cavity. Those children with signs of acute infections, congenital abnormalities, mental disorders, systemic disorders and chronic debilitating diseases were excluded.

The socio-economic status as recorded by Agarwal's modified scale [Agarwal, 2008] was in conformity with all the subjects of the present study. Caries status was recorded using the World Health Organisation index.

Study group and vehicles

The subjects were randomly assigned to one of the three groups. Group A (n=50) were given packet A containing a placebo powder. Group B (n=50) were given packet B containing Darolac (Aristo pharmaceuticals, India) containing 1gm powder of 1.25 billion freeze dried bacterial combination. It comprised of a mixture of, Lactobacillus rhamnosus, Bifidobacterium longum, and Saccharomyces cereviasae. Group C (n=50) were given packet C containing Sporolac (Uni-Sankyo Ltd., India) containing 1gm powder with 150 million spores of Bacillus coagulans. The subjects and samples were assigned alphanumeric codes.

Saliva samples were collected at baseline and a day after the 14 days of intervention. During the intervention period, the subjects were encouraged to maintain normal oral hygiene habits and to continue to brush their teeth twice daily.

Each subject was given one of the product packets with a given code according to the assigned group. On the first day, the subject to performed mouth rinsing in the clinic in front of the operator and guardians were instructed to maintain strict compliance. One sachet powder was mixed in 20 ml of water in a measuring cup. Each subject was instructed to swish the mixture in the mouth for one minute before swallowing it. The procedure was to be repeated one hour after lunch for the next 14 days. After 14 days, saliva was again collected for microbial assay. To ensure positive compliance during the intervention period telephone reminders were made on the 7th day.

Saliva samples and microbiological assessment

Saliva sampling was performed between 9-10 a.m. in the morning to minimise the effect of diurnal variation. Paraffin stimulated whole saliva was collected for 5 mins in a saliva collection cup. Collected saliva was transferred to the microbiology laboratory within three hours. Saliva samples were vortexed and serially diluted in 10-fold steps in 0.05M phosphate buffer.

Aliquots of 100 pl of the appropriate dilutions were cultured into selective media for MS, which has mitis salivarius bacitracin (MSB) agar (Hi Media Company, India) [Gold et al., 1973]. All plates were cultivated at 37[degrees]C in a micro-aerophilic environment in 5% CO2 for 48 hours. The colonies were identified on the basis of their morphology and counted using a digital colony counter. Confirmation of MS was performed under light microscopy after staining a heat fixed smear slide. Microbial counts were expressed as colony-forming units (cfu) per ml of stimulated saliva. The laboratory staff and clinicians evaluating the culture plates were blinded to the subject's group assignment.

Statistical methods

The statistical analysis was performed using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical Analysis Software. The data was analysed using non-parametric tests. The change in microbial counts before and after intervention was assessed using Wilcoxon signed rank test (non-parametric equivalent to paired 't' test) whereas the difference between two groups was assessed using Mann-Whitney 'U' test (non-parametric equivalent of Student's 't' test). The confidence level of the study was kept at 95%, hence a 'p' value less than 0.05 indicated a statistically significant difference. Colony forming units were expressed in terms of 10n units. Mean values in each group and each strata are calculated.

Results

Demographic parameters of the subjects such as age, sex, socioeconomic status along with Oral Hygiene Index are given simplified and decayed, missing and filled teeth in Table 1. Of the subjects in the study 76% belonged to middle and lower socio-economic strata which represent the major part of the population in India. The data from the subjects was segregated according to socio-economic status and DMFT scores were evaluated. The mean DMFT status of lower, middle and upper socio-economic status was 7, 5 and 4 respectively indicating a high prevalence of caries in lower socio-economic strata. All subjects had a stimulated saliva secretion rate within the normal range of 1.0-2.5 ml/min.

The comparison of MS counts between control group (A) and study groups (B and C) before and after intervention is shown in Table 2. All subjects had detectable levels of salivary MS at baseline. The groups (A versus B and C) at baseline were matched and had no statistically significant difference (p=0.875). Post-intervention comparison of the groups revealed a statistically significant difference (p=0.019). A comparison of change in MS counts in the control group revealed no significant change in microbial counts post-intervention (p=1) whereas a statistically significant post-intervention change was observed in the study groups (p<0.001). A significant reduction in MS counts was observed after administration of probiotics for 14 days.

Comparison of MS counts between the two study groups (groups B and C) before and after intervention is recorded in Table 3. A statistically significant (p<0.001) reduction of MS count was observed in both the groups after 14 days of probiotic administration. On comparing group B with group C at baseline both the study groups were matched and had no statistically significant differences (p=0.880) and also post-intervention MS counts were not found to be statistically significant (p=0.917). Thus the change produced in both the study groups was similar.

Group B had 43 subjects with decreased MS counts, five of which had unchanged counts and two had increased counts. In Group C 44 subjects had decreased counts, six that had unchanged counts and none exhibited increased counts.

Discussion

In India, the prevalence of dental caries in children has been reported

to be 87%. In low socio-economic strata the prevalence of caries was reported to be higher (96.2%) than high socio-economic strata (77.6%) [Sudha et al., 2005]. A major part of the population in India resides in rural areas (70%) where there is little awareness about oral health, especially for children who have unhealthy eating habits with large quantities of cariogenic products in their diet. The oral health care systems in developing countries mainly focus on curative care whereas community-based prevention and oral health promotion has not been systemically implemented. For research to be successful in India it has to be directed towards cost-effectiveness and easy availability of products for health. The present study was conducted to evaluate the effect of two commercially available probiotics preparations on the oral health of children in India as a possible way of providing low-cost prevention.

Earlier studies have demonstrated a beneficial effect of probiotic lozenges and chewing gums on oral health. [Caglar et al., 2006; Montalto et al., 2004] However, none of these formulations are available in India. Though the cost of these probiotic preparations may be within reach for a population man in developed countries it is beyond the reach of an average Indian. The cost of the probiotic lozenges is about $1 (US dollars) but the preparations used in the study cost were just 3-9 Indian rupees (INR) (1US dollar~45 INR). Thus in this study one of the aims had been to find a cost-effective alternative to probiotic lozenges and chewing gums. The preparations used in the present study are easily accessible, cost-effective, and commonly prescribed for general body health but their possible role in oral health had not yet been evaluated.

The subjects selected in this study were in the 7-14 years age group, which was important for the present study in two ways. First, this is the period during which permanent teeth are erupting and new surfaces would be colonised by pathogenic bacteria. Any measure(s) directed towards prevention against early colonisation might be beneficial in the long term for the prevention of dental disease. The second reason for selecting this age group was the intellectual ability of the child. In accordance with Jean Piaget at the age of seven years a child largely corresponds to an increase in cognitive development whereby the child develops a sense of semi-logical reasoning to infer physical cause-effect relationships. Thus, in this age group a positive compliance could be expected from a child. To our knowledge, no documented study has been reported in the literature for use of oral probiotic in a paediatric age group of 7-14 years.

The results of the present study showed that the daily consumption of both the probiotics for two weeks reduced the salivary levels of MS colony counts. The combination of probiotic strains used in Group B were similar to those used by Ahola et al. [2002] in their study. The results of the present study are in agreement with previous findings [Ahola et al., 2002; Caglar et al., 2006 and 2007]. However, according to Montalto et al. [2004], oral administration of probiotics increased the salivary lactobacilli counts but MS counts remained unchanged.

In Group C subjects the probiotics containing Bacillus coagulans (also called Lactobacillus sporogenes) was administered for 14 days which resulted in a significant reduction of salivary MS colony counts. It is an established probiotic for intestinal health [Cremonini et al., 2002] but no study untill now had reported its relation to oral health. Bacillus coagulans is a spore forming microorganism. Spores are known to be more resistant than vegetative cells to harsh environmental conditions. This characteristic allows spores to survive industrial manufacturing and ensures a long-term viability than more labile bacteria [Sanders, 2003]. This feature offers advantages such as low cost production processes, ease of preparation, resistance to production process and an extended shelf-life over a wide range of temperatures [La Rosa et al., 2003].

The effects of probiotics on the microbiota can be due to the competition for binding sites or secretion of antimicrobial components [Silva et al., 1987; and Morita et al., 2001] and lactic acid bacteria can produce different antimicrobial components [Silva et al., 1987; and Haukioja et al., 2006]. Adhesion of probiotics in biofilms and co-aggregation with other bacteria has been demonstrated by in vitro studies [Stamatova et al., 2009]. Protection against pathogenic microbes and an enhanced maturation of the immune system can also be due to strengthening of the mucosal barrier [Caballero-Franco et al., 2007], or effects on cytokine or chemokine production [Latvala et al.,2008]. This in turn affects the cell-mediated and humoral immune responses, and partly explains the systemic effect produced by probiotics.

There have been concerns expressed in some quarters about the cariogenic potential of lactic acid bacteria. [Teughels et al., 2008]. However, Fitzgerald et al., [1980] showed that only three of 50 lactobacillus strains isolated from the dental plaque of schoolchildren induced significant caries activity in conventional hamsters. Additionally, in an artificial caries model, Lactobacilli produced significantly shallower caries lesions than S. mutans and Actinomyces israelii, although a synergistic effect on the growth of S. mutans and A. israelii was observed in mixed cultures where L. acidophilus was present [Shen et al., 2004]. Therefore, it can be concluded that in contrast to MS, lactobacilli are implicated in caries progression rather than in the initiation of a carious lesion [Maltz et al., 2002]. This was the reasoning for the exclusion of subjects with open and untreated dental carious lesions in the present study. Strains of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus salivarius, and Lactobacillus rhamnosus have been suggested as potential probiotics for use in the oral cavity [Koll et al., 2008].

In the present study only the effect of short term administration of probiotics was assessed. As this also resulted in significant reduction of cariogenic bacterial counts it seems plausible that prolonged administration of probiotic preparations may have a preventive role against caries development. Yli-Knuuttila et al. [2006] reported that one of their subjects who had received LGG milk at the age of 10 years for a period of one year was found to be salivary LGG positive even after withdrawal of probiotics, and speculated that permanent colonisation could take place in childhood. Caglar et al., [2009] also advocated that constant uptake of probiotics were necessary for its permanent colonisation. Therefore, was further long term studies are advised for evaluation of its permanent colonisation.

In the present study, Bacillus coagulans produced a reduction in cariogenic microorganisms. This effect was similar in efficacy as the combination of Lactobacillus rhamnosus and Bifidobacterium. When comparing the cost of the products used in this study, Darolac was three times more expensive than Sporolac. Hence, the present study endeavoured to search for an economically viable bacterial product such as Bacillus coagulans for maintaining oral health.

Conclusion

The administration of probiotics in a group of Indian children resulted in a significant reduction of a cariogenic microorganism, mutans streptococci. With an increasing global problem of antibiotic resistance, contributing to preventing the effective treatment of microbial diseases, probiotics by virtue of their natural therapy appear to be a novel approach for caries prevention. Dietary supplementation of probiotics might be used as a suitable, easy and safe method for anti-caries prevention. Further long term research is needed for determining the minimum duration of probiotic administration required to produce an optimal effect and to establish the role of Bacillus coagulans in oral health as a cost-effective alternative.

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G. Jindal *, R.K. Pandey *, J. Agarwal **, M. Singh **

* Department of Paedodontics, C.S.M. Medical University, Lucknow, India, ** Department of Microbiology, C.S.M. Medical University, Lucknow, India.

Postal address: Dr G.Jindal, Room No - 205, R.H., KGMC, CSMMU, LUCKNOW--22600, India.

Email: dr.gjindal@gmail.com
Table 1. Demographic profile of the three groups
of Indian children in a study of probiotics effect
on salivary MS.

                              Group a   Group b   Group c
                              (n=50)    (n=50)    (n=50)

Gender          Male            26        22        27
                Female          24        28        23

Age             7-10 Years      30        27        30
                10-14 Years     20        23        20

Socioeconomic   Upper           12        10        14
status          Middle          32        32        28
                Lower            6         8         8

OHI-s (mean values)            3.24      2.89      3.07

DMFT (mean values)             3.72      3.70      3.92

Table 2. Comparison of mutans streptococci counts
between control and study groups before and after
intervention with probiotics.

S.No.                Colony      Control    Study     'z'     'p'
                    Forming       Group     Group
                     Units       (n=50)    (n=100)

Before intervention

1.                <[10.sup.3]       4         5      0.157   0.875
2.                 [10.sup.4]      10        22
3.                 [10.sup.5]      22        45
4.                >[10.sup.5]      14        28

After intervention

1.                <[10.sup.3]       4         7      2.350   0.019
2.                 [10.sup.4]      10        38
3.                 [10.sup.5]      22        43
4.                >[10.sup.5]      14        12

Before                'z'           0       6.00
vs After              'p'           1      <0.001
intervention *

* Wilcoxon signed rank test

Table 3. Comparison of mutans streptococci counts between two
study groups before and after intervention with probiotics.

S.No.               Colony        Study       Study      'z'     'p'
                   Forming       Group B      Group
                    Units        (n=50)     C (n=50)

Before intervention

1.               <[10.sup.3]        2           3       0.151   0.880
2.                [10.sup.4]       10          12
3.                [10.sup.5]       25          20
4.               <[10.sup.5]       13          15

After intervention

1.               <[10.sup.3]        2           5       0.104   0.917
2.                [10.sup.4]       21          17
3.                [10.sup.5]       22          21
4.               <[10.sup.5]        5           7

Before               'z'          4.359       4.123
vs After             'p'         <0.001      <0.001
intervention *                   (1.31x       (3.7x
                                [10.sup.    [10.sup.
                                  -7])        -5])

* Wilcoxon signed rank test
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