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Probiotics sourced from the oral cavity: A powerful modality for good oral health

The World Health Organization (WHO) Global Oral Health Report Towards universal health coverage for oral health by 2030 states: “No other disease group affects humanity across the life cycle and across all countries in the way that oral disease do”.1 Indeed, oral diseases are more prevalent globally than mental health disorders, cardiovascular diseases, diabetes mellitus, chronic respiratory diseases, and cancers combined. Worldwide, approximately $710 B is spent on treatment of oral diseases with a resulting loss of $323 B in productivity.2 In Canada, available statistics showed that oral diseases account for productivity losses of over $1 billion per year.3 Because of global population growth and increased tooth retention throughout the age span, these numbers are expected to grow substantially, with high global economic impact. To address the major oral health problem globally, the abovementioned WHO report advocates a paradigm shift in oral health policy planning “…from a conventional model of restorative dentistry towards a promotive and preventive model”.1 Several preventive oral health therapeutics have been developed and tested over the years.4-6 Many of these approaches suffer from lack of high-quality laboratory evidence or rigorous clinical trials.

The oral microbiome and its modulation offer a powerful target for next-generation preventive oral care. Known as “the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space”,7 the oral microbiome contains a community of microorganisms, including bacteria, fungi, viruses, archaea, and protozoa, and plays a major role in oral diseases8 (Fig. 1). In health, a complex equilibrium exists between the resident species in the oral cavity to maintain a healthy, or symbiotic state. Several factors, however, including diet, poor oral hygiene, tobacco smoking, and certain medications, among others, can disrupt this weak homeostatic balance to cause a dysbiotic state with potential implications on both oral and systemic health.9 Oral diseases have been consistently linked to dysbiosis of the oral microbiome, with a single or few species predominating and an associated increased risk of disease that drives inflammation and tissue damage10 (Fig. 1). For instance, increased colonization by mutans streptococci (MS), mainly of the species Streptococcus mutans and Streptococcus sobrinus, with significant acidogenic and aciduric properties, and oftentimes in synergy with other bacteria and fungi, e.g., Candida albicans,11 is primarily responsible for caries formation.12,13 The localized metabolic production of acids of these microorganisms decays the tooth through the demineralization of calcium and phosphorous from the enamel and dentin over several months, resulting in caries lesions.14

Fig. 1

Microbial dysbiosis leads to oral diseases such as dental caries and periodontal diseases, heart diseases and cancers. Created with BioRender.com.
Microbial dysbiosis leads to oral diseases such as dental caries and periodontal diseases, heart diseases and cancers. Created with BioRender.com.

Addressing the microbiome imbalance in the oral cavity requires targeted approaches, one that can actively recalibrate the microbiome toward a health-associated state. Pharmabiotics—defined as bacterial cells of human origin or their products with a proven pharmacological role in health and disease15—represent one such promising strategy. A key class of pharmabiotics is probiotics, defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”.16 By reinforcing epithelial barriers, competing with pathogens, inhibiting harmful bacteria, and modulating the host immune response, probiotics provide mechanisms that directly counter microbiome-driven pathologies.17 Depending on the strain, delivery, and host context, these effects can be local within the oral cavity or systemic. A diverse range of bacteria—including acidogenic Lactobacilli, Bifidobacteria, and Streptococci—have demonstrated the capacity to rebalance dysbiotic communities and mitigate oral disease.18-22

Our research team comprising a dental clinician/scientist and an oral microbiologist at the University of Toronto Faculty of Dentistry collaboratively undertook a mission: To discover new probiotic strains specifically from the oral cavity to improve oral health. We focused on identifying strains of the oral commensal Streptococcus salivarius (Fig. 2) for a more sustainable and longer-term probiotic benefit compared to species from external sources (such as dairy products). S. salivarius is a species known to offer significant value to oral health. A pioneer colonizer of the oral cavity and a predominant member of the native microbiota that persists throughout the human life-span,23,24 S. salivarius are well adapted to growth in the mouth and oral biofilms. Importantly, they are known to produce anti-microbial substances against other bacteria.25 Additionally, many S. salivarius
strains are regarded as having “generally recognized as safe” (GRAS) status in the USA25 and as natural health products by Health Canada.26 This means that they can be used in food or health products for human consumption. We focused specifically on identifying S. salivarius strains from the dental plaque of caries-free children, with the hypothesis that the highly structured plaque biofilm of caries-free children would harbor a high proportion of S. salivarius strains with strong inhibitory activities against cariogenic bacteria. As the inhibitory activities against MS are observed in only ~1% of S. salivarius strains,27,28 we followed a systematic screening approach to identify these potential candidates (outlined in Fig. 3). Each step of the experimental screen was designed, and validated, to eliminate less suitable options and refine the pool of strains to those showing the strongest inhibitory activities. Presumptive S. salivarius strains were first identified from the harvested plaque samples by cultivation in culture broth specific for their growth. Any positive clones were then “fingerprinted” to verify that they contained S. salivarius gene sequences and produced general inhibitory substances. Positive clones were further tested and ranked by their ability to show the strongest inhibitory activities against the major cariogenic group of bacteria, the MS. Those that met the pre-set criteria were selected for a more thorough testing via in vitro means, such as their ability to inhibit the growth of biofilms from monocultures of the major cariogenic S. mutans12 and dual cultures containing both S. mutans and C. albicans. Additionally, we also tested the ability of the select group of candidates for their ability to bind salivary-coated hydroxyapatite (enamel substitute) and dissected rodent incisors.

Fig. 2

Scanning electron microscopic images of Streptococcus salivarius, at low (left) and high (right) magnification showing the spherical or oval-shaped cocci arranged in chains.
Scanning electron microscopic images of Streptococcus salivarius, at low (left) and high (right) magnification showing the spherical or oval-shaped cocci arranged in chains.

Fig. 3

Screening strategy to identify novel S. salivarius probiotic strains. Supragingival plaque samples were collected from the buccal surfaces of teeth of caries-free children and streaked on S. salivarius selective agar plate. Individual bacterial colonies were screened to ensure that they contained S. salivarius sequences via PCR amplification. S. salivarius candidates were subjected to microbiological testing for their inhibitory activity against cariogenic bacteria. Next generation sequencing was performed on one specific strain, LAB813. Pre-clinical experiments using the rodent model of caries can be performed to test the safety and efficacy of specific probiotic strains with the goal of translating these discoveries for use as a preventive oral health therapeutic for human use. Created with BioRender.com
Screening strategy to identify novel S. salivarius probiotic strains. Supragingival plaque samples were collected from the buccal surfaces of teeth of caries-free children and streaked on S. salivarius selective agar plate. Individual bacterial colonies were screened to ensure that they contained S. salivarius sequences via PCR amplification. S. salivarius candidates were subjected to microbiological testing for their inhibitory activity against cariogenic bacteria. Next generation sequencing was performed on one specific strain, LAB813. Pre-clinical experiments using the rodent model of caries can be performed to test the safety and efficacy of specific probiotic strains with the goal of translating these discoveries for use as a preventive oral health therapeutic for human use. Created with BioRender.com

We screened approximately 600 S. salivarius strains from the supragingival plaque samples harvested from the buccal surfaces of teeth of 60 healthy children. Several potential candidates were identified and characterized. In vitro experiments showed that the killing efficiency of at least five probiotic strains against S. mutans biofilms approached 90% (as measured by their ability to inhibit the growth of S. mutans; Fig. 4). One candidate, LAB813, was especially efficient, with its inhibitory activities against the biofilms approaching 99%. Significantly, when grown on materials used in orthodontic treatment (such as metal and ceramic brackets and plastic aligner materials made of thermoplastic polyurethane/copolyester), the killing efficiency of LAB813 of S. mutans biofilms approached 99%29 (Fig. 4; orange bars). Indeed, when LAB813 was pre-formed on these materials, the killing efficiency against S. mutans increased further, approaching 99.9% (Fig. 4; blue bars). Furthermore, and of great clinical significance, is our finding that it only takes 2 hours for LAB813 to kill 99% of S. mutans biofilm cells, as shown in a time killing kinetic study. The inhibitory abilities of LAB813 are likely to be mediated via the production of anti-inhibitory molecules such as peptides and other compounds, a hypothesis that was strengthened after a thorough analysis of the sequenced LAB813 revealed several potential anti-bacterial peptide sequences.30

Fig. 4

Ability of LAB813 to inhibit growth of S. mutans. LAB813 was capable of killing about 99% of 24-hour old S. mutans biofilms grown on metal and ceramic orthodontic brackets or on aligner materials (orange bars). When these orthodontic materials were first coated with LAB813 followed by addition of S. mutans, the inhibitory activities of LAB813 against S. mutans went up to 99.5 - 99.9% (blue bars).
Ability of LAB813 to inhibit growth of S. mutans. LAB813 was capable of killing about 99% of 24-hour old S. mutans biofilms grown on metal and ceramic orthodontic brackets or on aligner materials (orange bars). When these orthodontic materials were first coated with LAB813 followed by addition of S. mutans, the inhibitory activities of LAB813 against S. mutans went up to 99.5 – 99.9% (blue bars).

Our in vitro data gives substantial support for commercializing these probiotic products as preventive oral health products for use in humans. Currently, we are actively engaged in planning and/or conducting activities that will move our products into the market. For example, we are planning a series of pre-clinical studies on mice, a powerful way to test the efficacy and safety of therapeutics before translating to humans. Fortunately, in dental research, there exists a well-established rodent model of caries where the formation of caries is induced in rodents by exposing the animals to a high sugar diet combined with inoculation of S. mutans.31 We plan to take advantage of this model and combine our studies with the powerful and highly sensitive micro-computed tomographic technique to generate ultrahigh-resolution images of rodent teeth to determine the impact of our probiotic administration on tooth cavitation (Fig. 5). Another immediate goal of our research is to develop innovative probiotic products that can be readily incorporated into current oral healthcare and treatment modalities for use in the oral cavity. For example, we are actively working with bioengineers and industrial partners to develop delivery formats including powder forms, tablets, and microcapsules (Fig. 6). We are also actively involved in conducting manufacturing, stability and efficacy testing of our finished products, conducting clinical trials to validate claims and safety, obtaining federal regulatory approval for human use, etc.

Fig. 5

Mouse model of dental caries. A) Inoculation of any probiotic strain to the oral cavity of mice. B) Jaws of mice inoculated with the probiotic can be processed, and subject to micro-computed tomographic scanning (C, E- slice through the scans). D) Teeth can be hemi-sectioned to visualize presence of enamel and dentin breakdown, i.e. cavities.
Mouse model of dental caries. A) Inoculation of any probiotic strain to the oral cavity of mice. B) Jaws of mice inoculated with the probiotic can be processed, and subject to micro-computed tomographic scanning (C, E- slice through the scans). D) Teeth can be hemi-sectioned to visualize presence of enamel and dentin breakdown, i.e. cavities.

Fig. 6

Different delivery formats of LAB813 in the form of A) powder, B) capsules, and C) gels.
Different delivery formats of LAB813 in the form of A) powder, B) capsules, and C) gels.

We are excited and optimistic about the potential of our probiotic products to transform preventive oral health strategies for individuals across all age groups and for oral healthcare providers worldwide. Importantly, they hold promise for making a meaningful difference in the lives of those most at risk of dental caries, including Indigenous individuals, communities, and peoples—helping close critical gaps in oral health. 

Oral Health welcomes this original article.

Acknowledgement: We would like to acknowledge the significant help and contribution of numerous students (undergraduate, dental and graduate students, dental specialty students), postdoctoral fellows and research associates, who have worked tirelessly in our research labs at the Faculty of Dentistry, University of Toronto over the years. Credit also goes to dental students from Tokyo Medical and Dental University (now Institute of Science Tokyo) who participated in research in our labs as part of the summer research program.

We also extend our thanks to the federal, private and institutional agencies that fund our work – Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada, Canada Foundation for Innovation, Align Technology Research Award Program, the Connaught Innovation Award Program and the University of Toronto Innovations and Partnerships Office.

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About the author

Dr. Gong is a scientist, educator, and dental clinician (orthodontist). Through her affiliation with the University of Toronto, she has contributed to studies in oral biology, orthodontic tooth movement and the development of probiotics for oral health. 

Dr. Céline Lévesque is a professor and microbiologist at the University of Toronto. Her research focuses on oral bacteria, biofilm formation, and developing new approaches to prevent dental caries. She leads collaborative projects and mentors students in microbiome science, combining lab work with global partnerships to improve dental health.