The Use of Probiotic Strains in Caries Prevention: A Systematic Review

This paper aims to provide a systematic review of the caries-prevention effect of probiotics in human. The hypothesis was that the administration of probiotic strains might play a role in caries lesion prevention and in the control of caries-related risk factors. The main relevant databases (Medline, Embase) were searched. Quality of the Randomized Clinical Trials (RCTs) was classified using the “Consolidated Standards of Reporting Trials” (CONSORT) checklist and the Impact Factor (IF) value of each journal was recorded. Sixty-six papers were identified, and 23 fulfilled the inclusion criteria. Only three studies had caries lesion development as outcome, all the others reported caries risk factors as interim evaluation. Using the CONSORT Score, the papers were coded as 4 excellent, 9 good and 10 poor. The mean IF value recorded was 1.438. Probiotics may play a role as antagonistic agent on mutans streptococci (MS), acidogenic/aciduric bacteria that contributes to the caries process. In two-thirds of the selected papers, probiotics have demonstrated the capacity to reduce MS counts in saliva and/or plaque in short-term. The effect of probiotics on the development of caries lesion seems encouraging, but to date, RCTs on this topic are insufficient to provide scientific clinical evidence.

Several appropriate vehicles of administration of probiotic strains have been proposed. Dairy products supplemented with probiotics are a natural means of oral administration and easily adopted in dietary regime for adults and children. However, specifically formulated devices with slow release of the microbial strain might be needed in order to oral diseases prevention and control.
Another uncertain aspect of the probiotic use is whether the probiotics species really are able to colonize the oral habitat, and how long the microbial shift was induced [37]. It is well established for probiotics in the gastrointestinal tract that they usually colonize for a short time only [38]. Therefore, a prolonged administration of the probiotics bacteria seemed is mandatory to improve the benefits of the treatment.
The hypothesis behind this systematic review was that the administration of probiotic strains might play a role in the caries lesion prevention and in the control of caries-related risk factors.

Eligibility Criteria
The studies included in the present review are Randomized Clinical Trials assessing the in vivo role of probiotics administration on caries lesion development and on caries risk factors control (cariogenic micro-flora, plaque pH, etc.). Only human studies considering subjects without any stated medical condition were considered. Only studies in English were collected, due to the virtual absence of research published in other languages as a result of preliminary electronic database searches. All in vitro studies, all studies not focusing on probiotics administration for caries prevention and studies where probiotics were administered for other reasons were excluded.

Search Strategy
The main important electronic databases were searched: Medline from 01 January 1966 to 15 May 2013 and Embase from 1973 to 15 May 2013. Two preliminary searches were conducted in March 2013 in order to obtain an overall idea of findings and to polish search terms (MeSH words) and limits. The MeSH Browser was accessed to identify entry terms and compose the final Boolean searches [39].
The first step was the association of MeSH terms Dental Caries and Probiotic(s); after that, a combination of key words derived from the two previous MeSH terms were searched for a total of 18 inquiries. The key words used were: Caries, Probiotic Bacteria, Probiotic Lactobacilli, Bacteriotherapy, Dental Disease, Oral Health, Oral Streptococci, Cariogenic bacteria, Plaque pH and Dental Caries Susceptibility.
A comparison of the 18 different searches was carried out to delete the repeated studies. Then, two authors (M.G.C. and S.M.) examined independently all abstracts of the selected papers. All studies, which appeared to meet the inclusion criteria, were obtained in the full text format. The two authors assessed the papers independently, to establish whether or not the studies met the inclusion criteria.
Disagreements were resolved by discussion. If not possible, other authors were consulted. All studies meeting the inclusion criteria then went to a validity assessment. Studies rejected at this or subsequent stages are reported in the Table 1 of excluded studies with the reasons for exclusion . For each trial, the following information was recorded: citation details; participants: including demographic characteristics and criteria for inclusion; intervention: including type and duration of intervention, duration of follow-up and method of administration.

Quality Assessment
The quality of the trials was assessed through the -Consolidated Standards of Reporting Trials‖ (CONSORT) guidelines [83], using the CONSORT 2010 checklist. The 25-items checklist is focused on how the trial was designed, analyzed and interpreted. The quality was classified in three categories according to CONSORT score: excellent (≥20 items), good (between 13 and 19 items) and poor (≤12 items) [84].
The Impact Factor, for each journal where the RCTs were published, was determined from ISI Journal Citation Report, 2011 JCR Science Edition [85].

Results
Sixty-six (66) papers were identified and assessed, and of these, 23 fulfilled the inclusion criteria and they are reported in Tables 2-4 .
No differences were observed between the two main databases used. Selected papers were divided between those performed on children/adolescents and those on adults. All studies utilized parallel arms with intervention and a placebo/control or a crossover design. The sample sizes were generally small or medium, and the majority of them (80%) were short-term interventions (between 10 and 42 days). Different vehicles for the administration and different dosage of probiotics were used. The quality of published papers recorded using the Consort Score was: 4 excellent, 9 good and 10 poor. All papers, except two [19,23], were published on Journals with positive IF with a mean value of 1.438.

Probiotics and Caries Prevention in Children/Adolescents
Eleven studies were evaluated [14][15][16][17][21][22][23][26][27][28]36]. Only one study was performed to verify the effect of the early administration of probiotics (Bifidobacterium animalis subsp. lactis BB-12) on the oral colonization of mutans streptococci (MS) in 106 infants from a low-caries population [17]. Subjects received probiotic bacteria, xylitol or sorbitol (polyol 100-300 mg) from the age of 1-2 months to the age of 2 years, twice a day. The MS concentration in plaque of the mothers at the start of the study was high and similar in all subjects, without significant differences. At the end of the study, children showed a rather low MS colonization percentage, with a statistically significant difference among groups. At the age of 4 years, the same children were re-evaluated to assess the MS level in plaque and the occurrence of dental caries in deciduous teeth [14]. No differences were observed for both parameters among the three groups.
The effect of milk containing L. rhamnosus on MS counts was evaluated in four papers (two short and two long-term studies). In the short-term studies [16,26], the effect of milk containing Lactobacillus rhamnosus (hct 70 or LB21) for few weeks was registered in small groups of adolescents. The difference in post treatment regarding MS count between test and control group was not statistically significant, while the difference in follow-up was highly significant [16]. No statistically significant differences in SM were recorded in subjects who received milk with probiotic compared to subjects using milk without probiotic [26]. In the long-term studies [28,36], L. rhamnosus was administered for several months (7 and 21 months respectively). Statistically significant reductions were recorded with Lactobacillus rhamnosus GG, ATCC use [36], while no statistically significant changes were observed in SM counts in subjects receiving Lactobacillus rhamnosus LB21 [28].
Two studies were performed with yogurt as probiotics vehicle [22,27]. The effect of the administration of yogurt containing Lactobacillus rhamnosus GG for three weeks in 105 children was evaluated with a significant decrease in SM count immediately after probiotics use alone, but recolonization was described during the five consecutive weeks [22]. Pre-treatment with chlorhexidine produced a statistically significant reduction in salivary SM counts that enhances during the five consecutive weeks. A double-blind, crossover study was carried out on 24 healthy adolescents, undergoing orthodontic treatment, with the aim to assess the effect of yogurt containing Bifidobacterium animalis subsp. lactis DN-173010 administered once daily [27]. Statistically significant reduction of MS was recorded after probiotic yogurt consumption.
One study used ice-cream as probiotic vehicle [21]; a combination of Bifidobacterium lactis Bb-12 and Lactobacillus acidophilus La-5 was evaluated in 40 adolescents. Significant reduction in salivary MS scores was reported after consumption of the probiotic compared to baseline.
One study was performed using lozenges as probiotic vehicle. The effect of lozenges containing Lactobacillus brevis CD2 administered for six weeks was evaluated in 191 high caries risk children [15]. A statistically significant reduction of the cariogenic microorganism was recorded.
One study used two powders as probiotic vehicle in 150 children aged 7-14 years, containing the first Lactobacillus rhamnosus, Bifidobacterium longum and Saccharomyces cereviasae and the second Bacillus coagulans, and compared them to a placebo powder [23]. Powders dissolved in 20 mL of water were used as a mouth rinse for one minute for 14 consecutive days. Data analysis showed a statistically significant reduction in MS counts in both probiotics groups.
Five studies of the ten reported above, investigated the effect of the probiotics strain on Lb level also [17,21,[26][27][28]. In all studies, a statistically significant change in Lb counts in saliva and/or plaque was not observed. Moreover, one study evaluated the effect of the probiotic on oral yeasts, failing to prove any statistically effect [17]. The effect of probiotics on plaque pH modification after a rinse with a 10% sugared solution was investigated and plaque acidogenicity resulted significantly lower in subjects that have used probiotic lozenges [15]. Two studies evaluated the probiotic effect on caries lesion development [14,28]. A statistically significant difference in caries increment was recorded only in one paper in subjects who received probiotic and fluoride compared to subjects who received placebo milk [28].
No statistically significant differences in MS counts were recorded immediately after consumption of cheese containing Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LC 705, but a significant reduction was reported three weeks after the experimental period [35].
Conversely, the other six of the twelve studies did not reveal an effect of probiotics administration on MS counts [18][19][20]24,25,34]. Four short-term studies were performed using tablets containing Lactobacillus rhamnosus or Lactobacllus reuteri; MS counts remained stable after the administration of both probiotics twice a day for two weeks in 13 adults [18]. No significant differences were also observed after the use for two weeks of Lactobacillus reuteri on MS counts in 18 adults [19], and using the same strains after full mouth disinfection with chlorhexidine on 62 adults on regrow of MS [20]. Tablets containing Lactobacillus paracasei GMNL-33 were unsuccessfully administered to 80 young adults [25]. One long-term study evaluated the effect of Lactobacillus rhamnosus LB21 delivered in milk on MS count in saliva and supra-gingival plaque in 160 older adults for 15 months [24]. No statistically significant reduction in MS count was registered. Results from a study utilizing several strains of Lactobacillus spp. in liquid and capsules form in 35 adults revealed no significantly MS count reduction [34].
Two studies evaluated also the effect of probiotics on plaque acidogenicity, but no significant changes were found [18,19]. Two studies investigated the effect on buffer capacity failing to demonstrate a statistically significant difference on it [25,35]. One of these did not demonstrate an effect on oral yeast yet [35].

Discussion
The role of the administration of probiotic strains in caries prevention was the aim of this systematic review. Results described by various research groups were encouraging [15][16][17][21][22][23][24]27,[29][30][31][32][33]35,36], but the scientific evidence is still unclear and often not very high. The main goal for the use of probiotics in caries prevention is to replace and displace cariogenic bacteria, mainly mutans streptococci, with noncariogenic bacteria [13].
Most clinical trials reviewed had a small sample size and reported caries risk factors as intermediate or surrogate endpoints, which limited the conclusions about the real efficacy of probiotics administration in caries lesion prevention. From the analysis of the RCTs selected, it reasonable to affirm that probiotic strains may play a role as antagonistic agent on cariogenic bacteria. In the two-thirds of the selected papers, probiotics have demonstrated the capacity to reduce MS counts in saliva and/or plaque regardless of the product or strain used. However, this effect is variable and probably short-lasting. In addition, MS are no longer considered the main cariogenic bacteria involved in the caries progress, since the important role of non-mutans acidogenic and aciduric bacteria was clarified [5]. Different results are reported on the effect of probiotics on lactobacilli counts. From the fourteen studies that evaluated the changes of this interim outcome, just two reported a positive result [19,35]. The other interim outcomes considered (yeasts and plaque acidogenicity) were investigated in few studies and the results are unclear. Only three selected papers [14,24,28], two performed on children and another one on adults/elderly samples, had caries lesion development as outcome; two studies reported a statistically significant difference in caries increment after 15/21 months of probiotics use [24,28].
Several mechanisms of action for probiotic are described in literature, same of them still not fully understood. Several local and systemic effects are describing, including adhesion, co-aggregation, competitive inhibition, production of organic acids and bacteriocin-like compounds and immune-modulation [86]. However, probiotic bacteria are not able to colonize oral cavity permanently [29], so a continuous regular, almost daily intake is required. This may be a compliance aspect to be considered.
One study evaluated the combined effect of probiotics and fluoride on cariogenic bacteria and caries lesion increment. No statistically significant differences were recorded between the group using probiotics alone and those using probiotic and fluoride together [24]. Another paper studied the combined effect of probiotics and low dosage of xylitol on cariogenic microorganisms [31], but no statistically significant differences were noted compared to probiotics alone. Finally only one study has investigated the effect of probiotic on MS counts after chlorhexidine mouthwash disinfection [22]. Pre-treatment with chlorhexidine produced a long-lasting reduction in salivary SM compared to probiotics alone.
It is interesting to note that up to day none products have successfully approved by the European Food Safety Authority (EFSA) [88,89].
A theoretical risk of the probiotic assumption is the increase of caries risk due to the capacity of probiotic strains to form biofilm and produce acids, but this aspect was not taken into consideration by any papers.
Two approaches have been used to assess the quality of RCTs in the present review: the CONSORT checklist and the journal Impact Factor. A significant association between the CONSORT score and the impact factor was reported [90].
The CONSORT checklist takes into account 25 important methodological items, providing an accurate evaluation of the methodological correctness with which the study was planned and carried out. From the analysis of the checklists of the selected papers, the main deficiencies observed were the lack of information on methods to define the hypothesis, the sample size calculation, the absence of data on the results of estimated effects size and their precision. These methodological weaknesses reduce the validity of studies and the interpretation of the results may lead to biased findings. Moreover, few studies reported correctly the results of the RCTs not taking into account other sources of bias. In general, the quality of reporting of RCTs was quite low, with half of all studies scoring as poor with the exception of three studies that were scored as excellent. These results are similar to those reported of other systematic review [13], but it is possible to observe a progressive improvement in the scientific evidence of the effect of probiotic on caries prevention.
The journal impact factor has been used widely as a quality measure of the published papers [91]. All selected studies except two were published in journals with impact factor and all except one in dental journal [34]. The mean value of impact factors of the selected studies (1.438) might seem low when compared with IF of journals from other areas of medicine. However, the mean impact factor value of the 81 impacted dental journals is quite low (1.455-range 0.037-3.961), with the journals with the highest IF values dedicated to other topic of dentistry, different from caries prevention. Therefore, the mean IF value recorded in the present review has to be considered a quite good score.

Conclusions
The use of probiotic strains for caries prevention showed promising results even if only few studies have demonstrated clear clinical outcomes. Therefore, the scientific evidence is still poor. A continuous regular almost daily intake is probably required; this maybe a compliance aspect to be considered. However, for all products effective in caries prevention (i.e., fluoride and chlorhexidine) a frequent intake is required, so a possible way of administration could be to insert probiotic in other daily preventive products like toothpaste.