Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 11  |  Issue : 1  |  Page : 49-54

Effect of Probiotics on Dental Caries and Periodontal Pathogens: An In Vitro Study


Department of Public Health Dentistry, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India

Date of Web Publication9-Aug-2019

Correspondence Address:
Dr. Mithun Pai
Department of Public Health Dentistry, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_41_19

Rights and Permissions
  Abstract 


Introduction: Probiotics are microorganisms that are administered in live forms and can improve health and well being of human beings. Literature about their antimicrobial activity against oral pathogens is limited. The present study was conceptualized to ascertain the antimicrobial capabilities of common probiotics against microorganisms involved in dental caries and periodontal diseases. The aim of this article is to ascertain the action of probiotics on dental caries pathogen, Streptococcus mutans, and periodontal microbes that included Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Prevotella intermedia. Materials and Methods: The present study was an in vitro type of study. A. actinomycetemcomitans was cultivated in tryptic soy-serum-bacitracin-vancomycin-fluoride agar, Po. gingivalis in modified Wilkins–Chalgren medium, and Pr. intermedia in brain heart infusion agar. The samples were cultivated for 48 h in Brewer-like anaerobic jars. The first plate was impregnated with strains of the probiotic Lactobacillus acidophilus, the second plate with strain of the probiotic S. salivarius, and the third plate was kept as the control. Zones of inhibition were calculated by unaided eye employing a Vernier caliper to the nearest millimeter, including the diameter of the disk in the measurement. Results: Analysis of variance test exhibited significant findings at 24 and 48 h. Post hoc Tukey test revealed results that were statistically significant for all microorganisms but for A. actinomycetemcomitans and Pr. intermedia. Conclusion: The findings showed significant effect of probiotics on common microbes implicated in dental caries and periodontal diseases. The present study has crucial public health implications for the prevention and control of common oral diseases in populations.

Keywords: Dental caries, in vitro techniques, oral health, periodontal diseases, probiotics


How to cite this article:
Routh S, Pai M, Rajesh G, Shenoy R, Sarit S. Effect of Probiotics on Dental Caries and Periodontal Pathogens: An In Vitro Study. J Orofac Sci 2019;11:49-54

How to cite this URL:
Routh S, Pai M, Rajesh G, Shenoy R, Sarit S. Effect of Probiotics on Dental Caries and Periodontal Pathogens: An In Vitro Study. J Orofac Sci [serial online] 2019 [cited 2019 Sep 15];11:49-54. Available from: http://www.jofs.in/text.asp?2019/11/1/49/264185




  Introduction Top


Probiotics are live microbes that on administration in appropriate quantities lead to improvements in the health of human beings.[1] They are free from adverse health effects and also provide basic nutritive value to individuals when ingested in adequate quantities.[2]

Various mechanisms have been proposed to explain how probiotics work. Microbes secrete numerous chemicals such as hydrogen peroxide, bacteriocins, organic acids, and others, which have definitive bactericidal properties. They act at the mucosal sites of adhesion by competitive inhibition and thus prevent colonization of pathogenic microorganisms. They exert changes in pH by altering the redox potential (oxidation reduction potential), which, in turn, affects the capacity of the microorganisms to thrive in the oral environment. Finally, probiotics trigger nonspecific immunity in individuals and alter their immune mechanisms (both cellular and humoral). Lactogenic bacteria may exert their modulating influence on immune-competent cells like T cells and macrophages. This may impact the cytokine production in these cells and thus may influence the overall immune response of individuals. A combination of probiotic strains have a synergistic effect and this is used to increase those beneficial effects.[2]

The interrelation between infections in the oral cavity and subsequent activities by the microorganisms is fairly well known. Individuals with adult-onset periodontitis have elevated levels of anaerobes such as Porphyromonas gingivalis and Prevotella intermedia. Individuals with local periodontitis, which is aggressive, show higher levels of Po. gingivalis, whereas patients suffering necrotizing ulcerative gingivitis exhibit high levels of Pr. intermedia. The association between occurrence of tooth decay and Streptococcus species is also well established. Streptococcus mutans has now been identified as one of the primary organisms involved in tooth decay.[3] Bacterial anchoring on the tooth surface and coadhesion of primary colonizers with other species initiate the formation of biofilm over tooth surface. Mature biofilm, also known as bacterial dental plaque, initiates a cascade of events leading to gingival inflammation. Gingivitis, either in acute or chronic form, is considered a reversible disease, as there is no loss of hard tissue surrounding the teeth. The gingival tissues mostly return to its original healthy state once the bacterial biofilm is removed.[3]

With increased use of antibiotics, clinicians have observed the advent of resistance of microorganisms to these antibiotics. In this background, probiotics offer an effective alternative to combat microbial infections in individuals. An attempt to tackle common oral diseases such as tooth decay and periodontal diseases by the use of probiotics can thus be contemplated. One of the primary reasons for the feasibility of the use of probiotics in oral health issues is their ability to thrive in the oral environment.[4]

Literature pertaining to the antimicrobial action of probiotics on dental caries and periodontal disease have been ambiguous/inconclusive, with systematic reviews unable to provide enough evidence on the same.[4],[5] The use of Lactobacilli as probiotics have been always discussed as they were one of the first bacterium to be used as a probiotic by Elie Metchnikoff, who developed the theory that the Bulgarian population had greater longevity because of consumption of fermented products containing lactic acid bacteria that improves gastrointestinal health. Montalto et al.[6] have observed that Lactobacillus may have potential to prevent tooth decay in humans. Koll-Klais et al.[7] have reported the potential role of Lactobacillus in periodontal diseases.[8] However, the use of S. salivarius as a probiotic is a new phenomenon as not much of studies are done on its potential for use as a probiotic or a prebiotic.

Therefore, the present study was conceptualized as an initial step to explore the potential antimicrobial properties of probiotics Lactobacillus acidophilus and S. salivarius against S. mutans, Actinobacillus actinomycetemcomitans, Po. Gingivalis, and Pr. intermedia.


  Materials and Methods Top


The present study employed an in vitro study design. The relevant microbiological studies were conducted in the Department of Microbiology, with Institutional Ethics Committee Protocol Reference number: 17072, of approval 19th June 2017. Pure cultures of S. mutans, A. actinomycetemcomitans, Po. gingivalis, and Pr. intermedia were procured from the Department of Microbiology, Maratha Mandal’s Nathajirao G Halgekar Institute of Dental Sciences and Research Centre.

Blood agar culture medium was used to culture S. mutans. A. actinomycetemcomitans was cultured in tryptic soy-serum-bacitracin-vancomycin-fluoride agar, Po. gingivalis was incubated using modified Wilkins–Chalgren medium, and Pr. intermedia was incubated using brain heart infusion agar and cultured in brewer-like anaerobic jars for 48 h.

Strains of probiotic microorganisms employed in the present study included L. acidophilus and S. salivarius. Commercially available preparation Neoflora (NeoFlora, Pre and Pro Biotic Capsules, Novus Life Sciences Pvt. Ltd., Mumbai, Maharashtra, India) (Carbamide Forte Probiotics) capsules containing L. acidophilus was serially diluted by employing sterile distilled water and 0.1 mL was plated on sterile De Man, Rogosa, and Sharpe (MRS) agar. Microaerophilic conditions were preserved for MRS plates that were incubated at 37°C for 48 h. Colonies that were isolated were later transferred and incubated on MRS broth at 37°C for 48 h.

Pure cultures of S. salivarius that were developed and verified and available at the Department of Microbiology, Maratha Mandal Nathajirao G Halgekar Institute of Dental Sciences and Research Centre, as no commercial available preparations, were available.

Well diffusion assay

Lawn cultures were made for S. mutans, A. actinomycetemcomitans, Po. Gingivalis, and Pr. intermedia. Three culture plates were prepared per organism, resulting in a total of 12 plates for the four pathogens. An 8-mm diameter well were cut in the media. The first plate was inoculated with L. acidophilus, the second plate with the strain of the probiotic S. salivarius, and the third plate was kept as the control. This procedure was carried out for all the four test organisms. They were then incubated for 24 and 48 h. Vernier caliper was employed to assess the zones of inhibition (ZOI) with unaided eye to the nearest millimeter, including the diameter of the well in the measurement.

Statistical analysis

Statistical Package for Social Sciences (SPSS, version 16, SPSS Inc., Chicago IL) was employed for statistical analysis. The disc diffusion values obtained at all levels of concentrations were considered for statistical analysis. Descriptive statistics was used to tabulate the baseline data. One-way analysis of variance (ANOVA) was used to compare the ZOI in millimeter among the four groups of pathogens and post hoc Tukey test was used to determine the difference in means between the four groups. Level of significance was fixed at 5%.


  Results Top


Comparison of ZOI (L. acidophilus) at 24 h

One-way ANOVA test showed that the mean value of S. mutans (1.3) was highest, followed by Po. gingivalis (1.22) and A. actinomycetumcomitans (1.18), and least in Pr. intermedia. This difference was found to be statistically significant with a test value of 59.13 and P value of <0.001. Results of analysis by post hoc Tukey test shows that the difference between S. mutans, A. actinomycetumcomitans, and Po. gingivalis was statistically significant with a mean difference of 0.3 and P value of <0.001. The difference between A. actinomycetumcomitans and Pr. intermedia was not found to be statistically significant [[Table 1], Graph 1].
Table 1 Inter group Comparisons of Zone of inhibition (ZOI) of microorganisms against Lactobacillus at 24 and 48 hours using ANOVA and post hoc Tukey.

Click here to view




Comparison of ZOI (L. acidophilus) at 48 h

Comparison of ZOI after 48 h using one-way ANOVA test shows that the mean value of S. mutans (1.59) was highest, followed by A. actinomycetumcomitans (1.41) and Po. gingivalis (1.30), and least in Pr. intermedia (1.08) [Table 1], Graph 1]. This difference was found to be statistically significant with a test value of 124.57 and P value of <0.001. Results of the post hoc Tukey test revealed that the difference between all the organisms was statistically significant.

Comparison of ZOI (S. salivarius) at 24 h

One-way ANOVA revealed that the mean value of S. mutans (1.11) was highest, followed by A. actinomycetumcomitans (0.81) and Po. gingivalis (0.714), and least in Pr. intermedia (0.598). These variations were statistically significant with a test value of 184.8 and P value of <0.001. It can also be observed that the analysis by post hoc Tukey test revealed that the difference between S. mutans, A. actinomycetumcomitans, Po. Gingivalis, and Pr. intermedia was statistically significant, with a mean difference 0.512 to 0.03 and P value of <0.05 [[Table 2], Graph 2].
Table 2 Inter group Comparisons of Zone of inhibition (ZOI) of microorganisms against S. salivarius at 24 and 48 hours using ANOVA and post hoc Tukey.

Click here to view




Comparison of ZOI (S. salivarius) at 48 h

One-way ANOVA test showed that the mean value of S. mutans (1.21) was highest, followed by A. actinomycetumcomitans (0.97) and Po. gingivalis (0.91), and least in Pr. intermedia (0.82) [[Table 2], Graph 2]. These dissimilarities were statistically significant with a test value of 224.95 and P value of <0.001. Results of post hoc Tukey test showed that the difference between S. mutans, A. actinomycetumcomitans, Po. Gingivalis, and Pr. intermedia was statistically significant, with a mean difference of 0.29 to 0.38 and P value of <0.05.


  Discussion Top


Probiotic microorganisms have to be introduced within the biofilms that are formed on tooth surface for effective interactions with other oral microbes. More importantly, these probiotic microbes should be able to thrive, grow, and colonize in the oral environment.[9] Although probiotic microbes have potential to render health benefits to individuals, their systemic effects should also be considered.[10],[11],[12] The present study was conducted with the objective of comparing the effect of probiotic L. acidophius and S. salivarius strains on the growth of other microbes implicated as causative agents of dental caries and periodontal diseases.

Inoculation of L. acidophilus led to a ZOI, which was compared with that caused by probiotic S. salivarius. Investigators have established the fact that L. acidophilus and S. salivarius are common commensals found in individuals with adequate oral hygiene.[13],[14] It has also been established that S. mutans is the crucial microbe for bacteria to adhere on to the tooth structures, thereby leading to the formation of oral biofilms.[15],[16] S. mutans is hence central to the colonization of other microbes in the oral cavity.[9] Colonization of probiotic microbes in the oral environment is essential for them to exert their action on other pathogenic oral flora. The current study revealed that colonization of probiotic microbes might influence the growth of indigenous oral flora. The growth of Po. gingivalis, Pr. intermedia, A. actinomycetumcomitans, and S. mutans was reduced significantly in vitro.

This could be due to the acidogenic environment produced by L. acidophilus and also due to increased competition for nutrition. L. acidophilus might also reduce vitamin K, which acts as a common growth factor for many microbes.[17] This might lead to inhibition in the growth of Po. gingivalis, Pr. intermedia, and A. actinomycetumcomitans. Zhu et al.[17] have reported that probiotic Bifidobacterium inhibited the growth of Po. gingivialis if it were inoculated first, whereas there was no inhibition if both (which is the other microorganism) the microbes were inoculated simultaneously the other microbe being S. sanguinis. Probiotic bacteria might produce substances that inhibit the growth of Po. gingivialis and it might compete with it for nutrition.[13] S. mutans can survive in low pH conditions and can also nullify the effect of acids. It is thus more potent in causing dental caries.[18]Greater ZOI observed with L. acidophilus on Po. gingivalis could be due to the reduced levels of Fusobacterium nucleatum.[18] Results of the present study also indicate that L. acidophilus had significant ZOI on S. mutans. This is in contrast to the inhibition of growth of probiotic Bifidobacteria by S. mutans.[19] Few clinical studies had shown that probiotic Bifidobacteria decreased the number of S. mutans in saliva.[20],[21] Therefore, attempts to control caries by using probiotic Bifidobacteria may not prove to be very effective.


  Conclusion Top


The findings of the present study showed significant effect of probiotics L. acidophilus and S. salivarius on common microbes associated with dental caries and periodontal diseases. L. acidophilus and S. salivarius might affect periodontal microbes in the oral microflora, but their ability to inhibit microbes involved in dental caries seems to be rather constricted. The present study paves the way for further clinical studies that explore the possibility of using probiotics for the prevention and control of common oral diseases. As oral health problems are widely prevalent in all communities, the present study has crucial public health implications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Guidelines for the evaluation of probiotics in food. Report of a joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. London; Ontario, Canada. April 30 and May 1, 2002. Available at: http://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf. [Accessed May 16, 2017].  Back to cited text no. 1
    
2.
Jasberg H, Soderling E, Endo A, Beighton D, Haukioja A. Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model. Eur J Oral Sci 2016;124:251-8.  Back to cited text no. 2
    
3.
Gruner D, Paris S, Schwendicke F. Probiotics for managing caries and periodontitis: systematic review and meta-analysis. J Dentistry 2016;48:16-25.  Back to cited text no. 3
    
4.
Seminario-Amez M, López-López J, Estrugo-Devesa A, Ayuso-Montero R, Jané-Salas E. Probiotics and oral health: a systematic review. Med Oral Patol Oral Cir Bucal 2017;22:e282-8.  Back to cited text no. 4
    
5.
Anusha RL, Umar D, Basheer B, Baroudi K. The magic of magic bugs in oral cavity: probiotics. J Adv Pharm Technol Res 2015;6:43-7.  Back to cited text no. 5
[PUBMED]  [Full text]  
6.
Montalto M, Vastola M, Marigo L, Covino M, Graziosetto R, Curigliano V et al. Probiotic treatment increases salivary counts of lactobacilli: a double-blind, randomized, controlled study. Digestion 2004;69:53-6.  Back to cited text no. 6
    
7.
Koll-Klais P, Mandar R, Leibur E, Marcooe H, Hammarstrom L, Mikelsaar M. Oral lactobacilli in chronic periodontitis and periodontal health: species composition and antimicrobial activity. Oral Microbiol Immunol 2005;20:354-61.  Back to cited text no. 7
    
8.
Terai T, Okumura T, Imai S, Nakao M, Yamaji K, Ito M et al. Screening of probiotic candidates in human oral bacteria for the prevention of dental disease. PLoS One 2015;10:e0128657.  Back to cited text no. 8
    
9.
Toiviainen A, Jalasvuori H, Lathi E, Gursoy U, Salminen S, Fonatana M et al. Impact of orally administered lozenges with Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 on the number of salivary mutans streptococci, amount of plaque, gingival inflammation and the oral microbiome in healthy adults. Clin Oral Investig 2015;19:77-83.  Back to cited text no. 9
    
10.
Stamatova I, Meurman JH. Probiotics and periodontal disease. Periodontol 2000 2009;51:141-51.  Back to cited text no. 10
    
11.
Devine DA, Marsh PD, Meade J. Modulation of host responses by oral commensal bacteria. J Oral Microbiol 2015;7:26941.  Back to cited text no. 11
    
12.
Ericson D, Hamburg K, Bratthall G, Sinkiewic GE, Ljunggren L. Salivary IgA response to probiotic bacteria and mutans streptococci after the use of chewing gum containing Lactobacillus reuteri. Pathog Dis 2013;68:82-7.  Back to cited text no. 12
    
13.
Zhu Y, Xiao L, Shen D, Hao Y. Competition between yogurt probiotics and periodontal pathogens in vitro. Acta Odontol Scand 2010;68:261-8.  Back to cited text no. 13
    
14.
Bik EM, Long CD, Armitage GC, Loomer P, Emerson J, Mongodin EF et al. Bacterial diversity in the oral cavity of 10 healthy individuals. ISME J 2010;4:962-74.  Back to cited text no. 14
    
15.
Haukioja A, Yli-knuuttila H, Loimaranta V, Kari K, Ouwehand AC, Meurman JH et al. Oral adhesion and survival of probiotic and other lactobacilli and bifidobacteria in vitro. Oral Microbiol Immunol 2006;21:326-32.  Back to cited text no. 15
    
16.
Nagaoka S, Hojo K, Murata S, Mori T, Ohshima T, Maeda N. Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays. FEMS Microbiol Lett 2008;281:183-9.  Back to cited text no. 16
    
17.
Hojo K, Nagaoka S, Murata S, Taketomo N, Ohshima T, Maeda N. Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis. J Appl Microbiol 2007;103:1969-74.  Back to cited text no. 17
    
18.
Takahashi N, Saito K, Schachtele CF, Yamada T. Acid tolerance and acid-neutralizing activity of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. Oral Microbiol Immunol 1997;12:323-8.  Back to cited text no. 18
    
19.
Taipale T, Pienihakkinen K, Alanen P, Jokela J, Soderling E. Administration of Bifidobacterium animalis subsp. lactis BB-12 in early childhood: a post-trial effect on caries occurrence at four years of age. Caries Res 2013;47:364-72.  Back to cited text no. 19
    
20.
Çaglar E, Kuscu OO, Selvi Kuvvetli S, Kavaloglu Cildir S, Sandalli N, Twetman S. Short-term effect of ice-cream containing Bifidobacterium lactis Bb-12 on the number of salivary mutans streptococci and lactobacilli. Acta Odontol Scand 2008;66:154-8.  Back to cited text no. 20
    
21.
Cildir SK, Germec D, Sandalli N, Ozdemir FI, Arun T, Twetman S et al. Reduction of salivary mutans streptococci in orthodontic patients during daily consumption of yoghurt containing probiotic bacteria. Eur J Orthod 2009;31:407-11.  Back to cited text no. 21
    



 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Tables

 Article Access Statistics
    Viewed68    
    Printed0    
    Emailed0    
    PDF Downloaded18    
    Comments [Add]    

Recommend this journal