1 INTRODUCTION

Periodontal diseases, which include a range of conditions from gingivitis to periodonitits, are some of the most prevalent oral health diseases.1-3 Specifically, periodontitis—a disease characterized not only by gingival inflammation but also by loss of connective tissue attachment, leading to resorption of alveolar bone and subsequent tooth loss4-6—affects approximately half of adults over 30 years of age in its mildest form and over 60% of individuals over 65 years of age.7, 8 Microbial biofilms are the primary etiology in chronic periodontitis; however, the condition can be modulated by many other local and systemic factors.9, 10

In treating chronic periodontitis, the main goal is addressing the primary etiology.11 Successful treatment of chronic periodontitis results in a reduction in periodontal pocket depth with gains in clinical attachment levels.12 Traditionally, the main treatment modality for chronic periodontitis is nonsurgical periodontal therapy, including oral hygiene instructions and scaling and root planing.13 Scaling and root planing therapy does not always result in improvements, especially for sites with deep probing depths and when patients suffer from comorbidities (diabetes mellitus, obesity, cardiovascular disease).14-16 Adjunctive therapies, such as antibiotics or probiotics, may prove useful in these multifactorial conditions where scaling and root planing therapy alone is not successful.

In the past, systemic antibiotic therapy has been used to reinforce mechanical therapy and support the host defense by killing the subgingival pathogens remaining after nonsurgical therapy.17 Systemic antibiotics may offer additional benefits in conjunction with scaling and root planing, but they are not innocuous drugs.18 Antibiotics have side effects, and there is the potential for emergence of antibiotic-resistant bacterial strains.18 As a result of these side effects, efforts have commenced to explore the use of probiotics as a method to modulate the composition of pathogenic biofilms in conjunction with scaling and root planing.19

The World Health Organization (2014)20 defines probiotics as live cultures of microorganisms that confer a health benefit on the host when administered in adequate amounts. Research has highlighted success in many areas of medicine with the eight known classes of probiotics.21 These successes include the treatment of diseases related to the gastrointestinal tract and oropharyngeal infections.19, 22 However, the role of probiotics in modulating periodontal diseases is not fully understood. The main focus of this literature review is to examine the in vitro and in vivo/preclinical, microbiological, and immunological effects of probiotics in the context of periodontal disease.

2 IN VITRO MOLECULAR FINDINGS

In order for a probiotic candidate to be used orally in periodontal-disease–related conditions, it must first be tested using in vitro experimentation (Table 1). Similar to probiotics used in the gastrointestinal tract, probiotics for periodontal disease applications should demonstrate the absence of pathogenic gene sequences and the presence of antibacterial activity against periodontopathic bacteria, colonizing ability on oral epithelial cells, and the ability to modulate the immune response in the presence of periodontal pathogens.23

TABLE 1. Summary of probiotic effects from in vitro studies Author Type of study Oral biofilm model Type of prebiotic/probiotic Groups (n) Test method Probiotics effects Species composition Antimicrobial activity Immunology 2005 Kõll-Klais et al26 Screening of oral lactobacilli in chronic periodontitis and healthy patients and antimicrobial activity against Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans P. gingivalis, P. intermedia, S. mutans, and A. actinomycetemcomitans grow in suspension Several lactobacilli strains Two groups: healthy (n = 15) and chronic periodontitis (n = 20) Collection of saliva and subgingival samples, plated onto Man–Rogosa–Sharpe agar plates and incubated for 72 h at 37°C and 10% carbon dioxide. The isolated lactobacilli colonies were further analyzed by 16S ribosomal ribonucleic acid (RNA) gene sequencing Lactobacillus gasseri and Lactobacillus fermentum were most prevalent lactobacilli identified in healthy patients, whereas Lactobacillus plantarum was the most prevalent strain in periodontal disease patients. Healthy patients exhibited higher counts of homofermentative strains than periodontitis patients did The majority of probiotic strains significantly suppressed P. gingivalis, P. intermedia, S. mutans, and A. actinomycetemcomitans growth — 2010 Zhu et al29 Screening of bacteria isolated from yogurt for antimicrobial activity against pathogens P. gingivalis, Fusobacterium nucleatum, A. actinomycetemcomitans, P. intermedia, Prevotella nigrescens, Peptostreptococcus anaerobius, Porphyromonas circumdentaria, Bacteroides fragilis, and Streptococcus sanguinis grown on agar plates Aliquots of yogurt containing Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium strains — Antimicrobial effects based on inhibition zone diameter (in millimeters) on agar plates —

Aliquots of fresh yogurt significantly inhibited all the periodontal pathogens, whereas heat-treated yogurt inhibited all the periodontal pathogens except F. nucleatum and P. gingivalis

Fresh yogurt but not heat-treated yogurt also inhibited the commensal bacteria S. sanguinis

Probiotic antimicrobial activity varied depending on the sequence of inoculation (probiotics inoculated first, co-inoculated, or last)

— 2012 Zhao et al53 Evaluation of the effect of L. acidophilus on secretion of interleukins by gingival epithelial cells exposed to P. gingivalis Gingival epithelial cells + P. gingivalis L. acidophilus Four groups: control (gingival cells); gingival cells + P. gingivalis; gingival cells + L. acidophilus; gingival cells + P. gingivalis + L. acidophilus RNA and protein levels for interleukin (IL)-1β, IL-6, and IL-8 at 2, 6, and 24 h after bacterial inoculation measured by reverse transcription polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay — — Probiotic significantly decreased IL-1β, IL-6, and IL-8 levels in a dose-dependent manner at all time points Bosch et al85 Screening of acid lactic bacterial strains from healthy children (up to 10 y old) for probiotic candidates P. gingivalis, F. nucleatum, Treponema denticola, Prevotella denticola and S. mutans grown on agar plates and tested for response to probiotics Lactic acid bacteria — Antimicrobial effects based on inhibition zone diameter (in millimeters) on agar plates —

From 46 lactic acid bacteria initially screened, 32 inhibited P. denticola, 29 inhibited S. mutans, 24 inhibited F. nucleatum, 24 inhibited T. denticola, and only seven inhibited P. gingivalis

From 46 lactic acid bacteria, none were able to inhibit all five pathogenic strains, and 11 strains were able to inhibit four out of the five pathogenic strains

— Chen et al86 Inhibition of periodontal pathogens by both L. fermentum and Lactobacillus salivarius and by their conditioned broth S. mutans, S. sanguinis, and P. gingivalis grown on agar plates L. fermentum and L. salivarius — Antimicrobial effects based on inhibition zone diameter (in millimeters) on agar plates and the broth was evaluated by counting the number of bacterial colonies on agar plates after broth treatment — Both bacterial strains and their broth exhibited significant inhibition of periodontal pathogens; however, the broth presented a lower inhibition effect than the bacterial strains did — 2013 Saha et al87 Development and characterization of carboxymethyl cellulose oral thin films for probiotic delivery — L. fermentum — — — L. fermentum remained viable in the oral thin film for 142 d and its antioxidant activity was maintained for 150 d, peaking at 48 h — 2015 Terai et al26 Screening of acid-producing bacterial strains isolated from healthy patients for probiotic candidates P. gingivalis, P. intermedia, A. actinomycetemcomitans, S. mutans, and Streptococcus sobrinus grown on agar plates 14 lactobacilli and 36 streptococci strains isolated from 32 healthy patients — 16S ribosomal DNA sequencing was used for bacterial identification; antimicrobial effects based on inhibition zone diameter (in millimeters) on agar plates; cariogenic potential was measured by demineralization of bovine tooth enamel; experimental endocarditis infection was assessed in rats by in vivo heart infection with the pathogenic bacteria — Isolated Lactobacillus crispatus YIT 12319, L. fermentum YIT 12320, L. gasseri YIT 12321, and Streptococcus mitis YIT 12322 showed no cariogenic potential Isolated L. crispatus YIT 12319, L. fermentum YIT 12320, L. gasseri YIT 12321, and S. mitis YIT 12322 showed low endocarditis potential 2016 Amižic et al88 Screening of probiotic toothpaste and mouthrinse for antimicrobial effects Candida albicans, C tropicalis, E faecalis, S. salivarius, and S aureus grown on agar plates L paracasei or L acidophillus 12 groups: saline; control toothpaste (no probiotic); L paracasei toothpaste; L acidophilus toothpaste; mouthrinse 1; mouthrinse 2; and the combinations of the three toothpastes with the two mouthrinses Antimicrobial effects based on inhibition zone diameter (in millimeters) on agar plates —

Probiotic toothpaste significantly inhibited C. albicans and S. salivarius compared to the toothpaste without probiotics and all the mouthrinses

With some exceptions, the combinations of probiotic toothpaste and mouth rinses were not significantly different from probiotic toothpaste alone

- 2018 Slomka et al47 Screening of prebiotic substrates to modulate pathogens/beneficial bacteria in oral biofilm Multispecies biofilm containing A. actinomycetemcomitans, F. nucleatum, P. gingivalis, P. intermedia, S. mutans, S. sobrinus as representative pathogens and Actinomyces naeslundii, Streptococcus gordonii, Actinomyces viscosus, S. mitis, Streptococcus oralis, S. salivarius, S. sanguinis, and Veillonella parvula as beneficial bacterial species β-Methyl-d-galactoside; N-acetyl-d-mannosamine; l-aspartic acid; succinic acid; lactitol, turanose, methionine-proline, phenylalanine-glutamic acid — Quantitative PCR used to measure bacterial levels at 3, 5, and 10 d — N-Acetyl-d-mannosamine, succinic acid, and methionine-proline prebiotics reduced pathogenic bacteria to <5% in the biofilm; N-acetyl-d-mannosamine resulted in a dose-dependent increase in the beneficial bacteria, reducing the pathogenic bacteria to 3% at the highest concentrations — Yamada et al89 Effect of probiotic metabolite on P. gingivalis infection P. gingivalis infection of gingival epithelial cells (Epi-4); P. gingivalis inoculation in mouse model Metabolite generated by probiotic bacteria (10-hydroxy-cis-12-octadecenoic acid [HYA]) Four groups: control, P. gingivalis, HYA, P. gingivalis + HYA Barrier disruption was measured by quantification of desmosomes visualized by transmission electron microscopy — HYA significantly inhibited gingival epithelial barrier disruption induced by P. gingivalis HYA significantly suppressed P. gingivalis E-cadherin degradation and proinflammatory cytokines IL-1β, tmor necrosis factor-α, and IL-6 in mouse gingival tissue Zupancic et al23 Probiotic incorporation into chitosan-poly(ethylene-oxide) nanofibers A. actinomycetemcomitans grown on agar plates Bacillus sp isolated from a healthy patient Two groups: A. actinomycetemcomitans; A. actinomycetemcomitans + nanofibers Antimicrobial effects based on inhibition zone diameter (in millimeters) on agar plates — Probiotic significantly inhibited pathogen growth — 2019 Cornacchione et al30 Identifying the different strains of L. delbrueckii, that inhibit and do not inhibit the growth of P. gingivalis P. gingivalis grown on agar plates L. delbrueckii isolated from commercial yogurt products — Inhibitory effects based on the inhibitory zone diameter (in millimeters) on agar plates — The STYM1 strain of L. delbrueckii inhibited the growth of P. gingivalis by producing inhibitory amounts of hydrogen peroxide — 2.1 Absence of pathogenic gene sequences

A healthy oral cavity consists of more than 700 bacterial species, predominantly colonizing the tooth and the surrounding structures, buccal epithelium, hard and soft palate, and tongue dorsum.24 Given this bacterial richness, it is important to screen potential bacterial probiotic strains to ensure that they do not contain pathogenic gene sequences that may be harmful to the oral bacterial community, and thereby to oral health. Dental plaque, tongue coatings, saliva, and subgingival plaque have all been used to evaluate the different probiotic strains and their by-products that could be used in the context of oral diseases.25, 26 The potential pathogenicity of probiotic strains that were isolated from the oral cavity was identified using standard gene sequencing analysis. This analysis was used to generate a list of probiotic microorganisms that are safe for human use and that could be added to food or feed.27 This list included a number of lactobacilli and streptococcal strains.27

2.2 Antibacterial activity against periodontopathic bacteria

Many lactobacilli and streptococcal strains isolated from healthy oral cavities show antibacterial activity against Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans,25, 26, 28 and Fusobacterium nucleatum.29 Studies also show that different lactobacilli strains can inhibit the ability of multiple microbial species. For example, Lactobacillus fermentum is active against microaerophiles, and Lactobacillus gaseri is active against anaerobes.26 Other Lactobacillus strains, including Lactobacillus paracasei and Lactobacillus acidophilus have inhibitory effects on the growth of Staphylococcus aureus,30 a pathogen found in aggressive periodontitis.31 Streptococci inhibit anaerobes, such as P. gingivalis and P. intermedia through the production of lactic acid and other organic acids.32 The growth of P. gingivalis is also inhibited by the production of hydrogen peroxide from some probiotic strains isolated from commercial yogurt products. One study33 showed that the STYM1 strain of Lactobacillus delbrueckii releases intracellular proteins that can produce inhibitory amounts of hydrogen peroxide.33 Hydrogen peroxide can be produced and generated by several different enzymes in many Lactobacillus strains, including pyruvate oxidase,34 lactate oxidase, NADH oxidase 35 and NADH flavin–dependent reductase.36 In this study,33 the growth of P. gingivalis in agar plates treated with hydrogen oxidase from L. delbrueckii was inhibited significantly compared with the untreated plates.

2.3 Antibacterial effects mediated by bacteriocins

Studies have found that bacteriocins contribute greatly to the antibacterial activities of many probiotics, including lactobacilli. Bacteriocins are antimicrobial proteins or peptides that are secreted by bacteria as a defense mechanism. Bacteriocins are classified into three main classes depending on their molecular size and function.37, 38 The bacteriocins relevant to the oral cavity include salivaricin from Lactobacillus salivarius and Streptococcus salivarius,39 reuterin from Lactobacillus reuteri,40 plantaricin from Lactobacillus plantarum,41 and nisin from Lactobacillus lactis.42 The bacteriocin plantaricin contributes to the improvement of systemic health.40 In the context of oral health, plantaricin shows its antimicrobial activities by preventing the growth of P. gingivalis.28 Salivaricin is known to suppress oral bacteria related to malodor,39 whereas reuterin has potential applications for periodontal disease as it has antibacterial effects against Tannerella forsythia ATCC 43037.40

Similarly, nisin has antibacterial effects against multiple periodontal pathogens43 in addition to its effects on gastrointestinal-associated gram-positive bacteria.44 Nisin exhibits significant antibiofilm and antimicrobial effects against many gram-negative periodontal pathogens, including P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, and Treponema denticola.45 Importantly, nisin also does not exhibit cytotoxic effects against various human cells that line the oral cavity or which are relevant to the periodontium45. Other drug-resistant oral bacteria, such as S. aureus can also be actively inhibited by the application of bioengineered and other forms of nisin produced by L. lactis.46

2.4 Growth modulators and prebiotics to enhance antibacterial activities

The antibacterial activities of lactobacilli and streptococci are modulated by several factors, including growth media conditions. L. salivarius, L. plantarum, Lactobacillus rhamnosus, L. fermentum, and S. salivarius express better antimicrobial activity when cultured on media with additional glucose (1%-2%).26 In contrast, anaerobic bacteria, including P. intermedia, P. gingivalis, and F. nucleatum, are inhibited in the presence of high glucose, compared with standard low-glucose–containing media (0.2%).32 In addition, a nutritionally rich environment and use of prebiotics (compounds found in food that promote the growth and activity of beneficial microorganisms) promote antimicrobial activities of probiotics. This has been elucidated by studying probiotic strains co-cultured with pathogenic bacteria using different prebiotics, growth media, and conditions.47, 48 The probiotic strains tested included Streptococcus gordonii ATCC 49818, Streptococcus mitis ATCC 49456, Streptococcus oralis, S. salivarius TOVE-R, and Streptococcus sanguinis LMG 14657. The pathogenic bacteria tested included A. actinomycetemcomitans ATCC 43718, F. nucleatum ATCC 10953, P. gingivalis ATCC 33277, P. intermedia ATCC 25611, and Streptococcus mutans ATCC 25175. The results from these studies showed that treating biofilms with different prebiotics altered the proportion of beneficial bacterial species as well as that of pathogenic bacteria. Thus, prebiotics, specifically N-acetyl-d-mannosamine, showed a significant beneficial effect in shifting the bacterial communities toward a more beneficial composition while reducing the number of pathogens.48 The rationale for choosing this compound was based on its close relationship to metabolic enzymes derived from salivary mucin: N-acetylneuramine and N-acetylmannosamine. These enzymes are digested by and promote the growth of several Streptococcus strains, including S. mitis, S. oralis, S. sanguinis, and S. gordonii. Further, most probiotic strains are lactic acid bacteria, meaning that they produce lactic acid as the major metabolic end product of carbohydrate fermentation, and this acidic environment inhibits the growth of pathogenic bacteria. This acidification and the production of proteinaceous bacteriocins by several lactic acid bacteria support their growth and inhibit that of pathogenic microorganisms.

2.5 Beneficial colonizing properties

The processing time of probiotics in the oral cavity is shorter than in other areas of the digestive tract.49 Therefore, it is important that oral probiotics show good colonizing potential, including the ability to adhere to oral epithelial cells. Unlike the intestine, oral epithelial tissue is comprised of a thin mucin-covered mucosa, which allows microbes to attach directly to the oral epithelium.49 Pili from some streptococcal strains, including S. sanguinis and S. salivarius, help the bacteria adhere to mucin, a major component of saliva, thus increasing the colonizing properties of these probiotic strains.25 Within minutes after being consumed, several streptococcal strains, such as S. sanguinis, S. gordonii, S. oralis, and S. mitis, attach quickly to the pellicle on the tooth surface by connecting with the colonizing receptors on the pellicle.25, 50 However, compared with streptococci, lactobacilli generally have lower adherence ability when it comes to salivary-coated hydroxyapatite (human tooth simulation assay).25 The surface of many lactobacilli exhibit traits that promote colonization, including high hydrophobicity, auto-aggregation, and high adhesive capacity to intestinal epithelial cell lines.51 In the oral environment, lactobacilli and bifidobacteria compete with the pathogen P. gingivalis for adhesion to epithelial cells. These probiotics alter the interaction of epithelial cells with P. gingivalis by modulating the pathogen's ability to adhere to and invade these cells.51

2.6 Immune modulation

The antimicrobial activity of probiotics also involves altering host-microbial signaling and the subsequent host immune response. Several studies found that lactobacilli modulate the inflammatory response to periodontal pathogens.52, 53 Specifically, when gingival epithelial cells were infected with P. gingivalis W83 and ATCC 33277 then treated with Lactobacillus and Bifidobacterium strains, different host immunological responses were noted.52 While the levels of inflammatory cytokines, such as interleukin (IL)-1β,52, 53 IL-6,53 and tumor necrosis factor-α,52 increased in the presence of P. gingivalis, their levels were reduced following treatment with lactobacilli. Although evidence regarding the change in levels of IL-1β, IL-6, and tumor necrosis factor-α following treatment with probiotics is generally consistent, data regarding the levels of IL-8 expression in a similar context are still controversial. Some studies reported an increased expression of IL-8 in gingival epithelial cells, which in turn attracts leukocytes and activates neutrophils, thereby mediating an inflammatory response to P. gingivalis.54 However, in the presence of Lactobacillus, this increased expression was inhibited with increasing concentrations of the probiotic; showing Lactobacillus’ ability to reduce the inflammatory response to the pathogen, P. gingivalis.53 In contrast, other studies found that the IL-8 protein might be degraded by P gingivalis protease genes, especially in a mixed bacterial environment like the oral cavity, where there are multiple pathogens.55 At the same time, lactobacilli can activate CXCL8, an IL-8–encoding gene, which increases the expression of IL-8 and counteracts its degradation by P. gingivalis, thus promoting an anti-inflammatory response against this pathogen.52

2.7 Preclinical Studies

Whereas in vitro studies analyzed the molecular contributions of probiotics to periodontal-related conditions, preclinical studies focused on the effects of specific types of bacteria on experimental periodontal diseases, including gingivitis and periodontitis (Table 2). Most preclinical studies examined the antibacterial effects of lactobacilli, bifidobacteria, and streptococci.56-61 Other probiotic strains that have been examined included the yeast Saccharomyces cerevisiae.62 Most studies evaluated alveolar bone changes following probiotic treatment.56, 59, 61, 62 However, there has also been increasing interest in the immunomodulatory effects mediated by probiotics.57-60, 62 Most studies were performed using murine models,56-62 which lend themselves to the study of both alveolar bone changes and immunomodulatory effects, although one study utilized the canine model to evaluate radiographic changes in alveolar bone levels.63 In these animal models, periodontitis was often induced by placing ligatures around the first molar teeth59, 61 to mediate alveolar bone loss.

TABLE 2. Summary of probiotic effects in animal studies Author Type of study Defect site/size Type of probiotic Animal model (n) Groups Test method Probiotics effects Alveolar bone Microbiology Immunology 2008 Nackaerts et al63 Split-mouth double-blind randomized trial Moderate periodontitis; bilaterally in the lower jaw Streptococcus sanguinis, Streptococcus salivarius, Streptococcus mitis Dog (n = 8) Two groups with different treatments Radiography Scaling and root planing (SRP) followed by local irrigation with probiotics significantly decreased bone loss and increased bone density — — 2013 Messora et al61 Randomized preclinical trial Ligature in same position for 14 d on the mandibular right first molar (experimental periodontitis, EP) Bacillus subtilis Rat (n = 32) Four groups: control, EP, probiotics, EP + probiotics Histomorphometric analysis Probiotics-only significantly reduced bone loss —