Introduction to probiotic therapy

The oral cavity is a natural habitat for more than 700 bacterial species. Most of these bacterial species are commensals, that is, they are associated with health. Remaining bacterial species are pathogenic and are responsible for causing various oral health problems including periodontal diseases. These bacterial species are not randomly distributed in the niches in the oral cavity and they have a specific place in the oral cavity. As discussed in “Dental plaque”, the plaque biofilm is a very well-organized structure with each bacteria in it having a well-defined place and role. These bacteria compete with each other to occupy various niches in the oral cavity and this is the basis for ‘probiotic’ therapy. Probiotic therapy aims at modulating the composition of microflora by administering living micro-organisms that confer a health benefit to the host when administered in a sufficient amount.

Definition

The term ‘probiotics’ was derived from the Greek word, meaning “for life” 1. It may also be defined as “live micro-organisms”, which, when administered in adequate amounts confers a health benefit on the host 2. Other definitions of probiotics and their respective authors have been listed in the following table. Various bacterial genera most commonly used in probiotic preparations are Lactobacillus, Bifidobacterium, Escherichia, Enterococcus, Bacillus, and Streptococcus. Along with these bacterial species, some fungal strains belonging to Saccharomyces have also been used in probiotic preparations 3-5.

Various definition of probiotics

Author and yearDefinition
Lilly & Stillwell (1965) Substances produced by microorganisms that promote the growth of other microorganisms.
Parker (1974) Organisms and substances that contribute to intestinal microbial balance.
Fuller (1989) A live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance.
Havenaar and Huis (1992) A viable monoculture or mixed-culture of microorganisms that, when applied to animal or human, beneficially affects the host by improving the properties of the indigenous microflora.
Schaafsma (1996) Living microorganisms that, upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition.
Naidu et al. (1999) A microbial dietary adjuvant that beneficially affects the host physiology by modulating mucosal and systemic immunity, as well as by improving nutritional and microbial balance in the intestinal tract.
FAO/WHO report (2001) Live microorganisms that, when administered in adequate amounts, confer a health benefit to the host.

Historical aspect

The benefit of consuming fermented food for the treatment of gastrointestinal ailments is centuries old, dating back as far as 5400 BC, whilst recommendations for gastrointestinal illness date back to 76 AD 12. In modern times, the history of the use of probiotics goes back to 1908 when a biologist and Nobel laureate Elie Metchnikoff described the beneficial effects of the use of Lactobacillus species from yogurt 13. Working at Pasteur Institute in Paris, he discovered Lactobacillus bulgaricus strain which he later introduced into commercial production of sour-milk products in France and throughout Europe. He claimed that cholera could be controlled by the presence of antagonistic organisms in the intestine. The term “probiotic” was coined in …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book…..

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Probiotic strains in the oral cavity

As already stated, probiotic microorganisms include various bacterial species as well as certain molds, or yeast, however, the majority of them are bacteria. The most common probiotic strains belong to the genera Lactobacillus and Bifidobacterium. Culture-based studies suggest that bifidobacteria are among the first anaerobes in the oral cavity 15. Various bacterial species and strains that have probiotic properties have been listed in the following table. Usually, a consortium of these bacterial strains is given to the patients in the form of powder, liquid, gel, paste or granules 16.

Various micro-organisms used as Probiotics
Various micro-organisms used as Probiotics

     An essential property of a microorganism to be ‘an oral probiotic’ is its ability to adhere and to and colonize surfaces in the mouth. However, it must be noted here that various bacterial species have different inherent habitat in the gastrointestinal tract. Out of all the bacterial species so far detected in the oral cavity, Lactobacilli genera constitute approximately 1% of the cultivable oral microflora 17. Using polymerase chain reaction (PCR), Teanpaisan and Dahlen (2006) 18 demonstrated that various strains of Lactobacillus species isolated from saliva include L. fermentum, L. rhamnosus, L. salivarius, L. casei, L. acidophilus, and L. plantarum.

Properties of an ideal probiotic agent

A probiotic agent has viable microorganisms, hence, it is essential that they should not harm the host. The ideal properties of a probiotic agent include the following,

  • It should be a strain, which is capable of exerting a beneficial effect on the host animal. It should enhance host immunity against pathogens.
  • It should be non-virulent and non-pathogenic.
  • Preferred to be present as viable cells in large numbers.
  • It should be capable of surviving and metabolizing in the gut environment (e.g., endurance to low pH and carbon-based acids), and should be able to maintain
  • genetic stability in oral microflora.
  • It should be able to influence local metabolic activity.
  • It should be stable and, on storage, a large number of viable bacteria must be able to survive.

Mechanism of action of probiotics in the oral cavity

There are various mechanisms by which oral bacteria can prevent and treat oral diseases. These include,

Direct mechanism

One of the most important properties of probiotic bacteria is their ability to adhere to various surfaces in the oral cavity. These bacteria colonize the biofilm and prevent the formation of the pathogenic microbial community. These also interact with the host cells and participate in the generation of the immune response. Furthermore, probiotic bacterial strains produce various antimicrobial substances (lactic acid, hydrogen peroxide, bacteriocins) that can kill or inhibit the growth of periodontal pathogens 20.

Competitive exclusion

These bacteria compete with various pathogenic bacterial species in colonizing various niches in the oral cavity and they can also restrict the pathogens’ adhesion capabilities by changing the environmental pH. Furthermore, the probiotics compete with pathogens for essential nutrients. Because of this competitive inhibition, the overall bacterial count of the pathogenic bacterial species is reduced in the oral cavity. Thus, probiotics may significantly alter the microbial ecosystem in the oral cavity.

Possible mechanisms of probiotic actions in oral cavity
Possible mechanisms of probiotic actions in the oral cavity



Indirect actions

The probiotics may modulate the ecosystem in the oral cavity as well as the immune response. Probiotic bacteria have been shown to modulate immunoglobulin production. It is well established that IgA plays a vital role in mucosal immunity. It acts as a barrier against pathogenic bacteria and viruses. It has been demonstrated that probiotic bacteria increase the mucosal IgA production 21. These factors may indirectly benefit the growth of …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book….

     Hence, it is clear from the above-explained mechanisms that probiotic bacteria benefit the host, firstly, by competing with the oral pathogens for adhesion site and then colonizes the oral surface. Secondly, after colonization in the oral cavity, they compete with oral pathogens for nutrients and growth factors. Thirdly, they produce antimicrobial compounds which inhibit the growth of pathogens, including organic acids, hydrogen peroxide, carbon peroxide, diacetyl, low molecular weight antimicrobial substances, bacteriocins, and adhesion inhibitors. Finally, probiotics can also activate and modulate the immune system that favor the maintenance of oral health.

Application of probiotics to improve oral health

Probiotics and dental caries

Dental caries is a multifactorial disease of bacterial origin that is characterized by acid demineralization of the tooth enamel 23. Dental plaque is the primary etiology of dental decay. There are various bacterial species that have been categorized as cariogenic. These bacterial species create an acidic environment which is responsible for the demineralization of the calcified tooth structure. The probiotic microbial species have an ability to neutralize the acidic environment and prevent dental decay. However, it must be noted here that not all strains of bacteria in probiotics have the ability to attach to the tooth surface and inhibit cariogenic bacterial species. As already stated, to be effective in the oral cavity, the microorganisms in the probiotics should be able to attach to the oral surfaces. In a study, Comelli et al. (2002) 24 reported that out of 23 bacterial strains used in the dairy industry, Streptococcus thermophilus and Lactobacillus lactis species were the only ones with the capacity to integrate into a biofilm present on the hydroxyapatite surface and to interfere with the growth of cariogenic species Streptococcus sobrinus.

     It has been demonstrated in various studies that the consumption of products containing probiotic lactobacilli or bifidobacteria could reduce the number of S. mutans streptococci in saliva 25-33. In a study, Kang et al. (2006) 34 demonstrated that W. cibaria isolates had an ability to inhibit, both in vitro and in vivo, biofilm formation by S. mutans. Furthermore, these isolates also prevented the proliferation of S. mutans. In another study, Petti et al. (2008) 35 demonstrated that yogurt containing S. thermophilus and L. bulgaricus had selective bactericidal effects on streptococci of the mutans group. Two in vitro studies have demonstrated the inhibitory effect of one strain of L. rhamnosus, and the species L. casei on two important cariogenic bacteria, S. mutans and S. sobrinus 25, 36. Nikawa et al. (2004) 27 in a study have demonstrated that consumption of yogurt containing Lactobacillus reuteri over a period of 2 weeks reduced the concentration of S. mutans in the saliva by up to 80%. In a long term study on 594 children (1-6 years of age), Näse et al. (2001)25 studied the effect of consuming milk supplemented with a strain of L. rhamnosus on incidences of dental caries. It was concluded in the study that children consuming milk containing this probiotic had significantly fewer dental caries and lower salivary counts of S. mutans than controls. The results were particularly more applicable in the age group of 3-4 years.

     As discussed above, many studies have demonstrated the beneficial effects of probiotic application on prevention of dental caries but, at present, no conclusive statement can be made about the effects of probiotic bacteria on dental caries. This is because most of the studies conducted so far to find out any relationship between dental caries and probiotics are fairly short term. However, long term studies can be designed to authenticate the beneficial effect of probiotics in the prevention of dental caries.

Probiotics and periodontal diseases

As discussed in, “Microbiology of periodontal diseases”, periodontal diseases are multifactorial diseases with microbial etiology being their primary etiology. Various bacterial species that have been implicated in the etiopathogenesis of periodontal diseases include P. gingivalis, Treponema denticola, Tannerella forsythia and Aggregatibacter actinomycetemcomitans 37. These bacteria produce various metabolic products which evade the host immune response and cause periodontal destruction. Various animal and human studies have been done to evaluate the effects of probiotics on periodontal status. Various animal studies have been summarized in the following table.

Various animal experiments done on probiotic formulations
Various animal experiments done on probiotic formulations



     It has been demonstrated that Lactobacilli can significantly alter the subgingival biofilm ecosystem. Various studies have reported that Lactobacilli can inhibit the growth of various periodontopathogens including P. gingivalis, Prevotella intermedia and A. actinomycetemcomitans 41, 42. Studies have been done to compare the prevalence of Lactobacilli in chronic periodontitis as compared to healthy patients. In one study, Koll-Klais et al. (2005) 42 demonstrated that the prevalence of Lactobacilli, particularly L gasseri and L fermentum was more in healthy individuals as compared to chronic periodontitis patients. In another study done on moderate to severe gingivitis patients, the subjects were given the probiotic incorporated into chewing gum for 14 days. The results of the study demonstrated that the oral cavity of the patients was colonized by L. reuteri and the plaque index was reduced. Although, the exact mechanism of reduction in gingivitis is not known; however, the authors suggested three possible mechanisms. Firstly, reuterin and reutericyclin are two bacteriocins secreted by L. reuteri, that inhibit the growth of a wide variety of pathogens 43, 44. Secondly, L. reuteri strongly adheres to the host tissues, thereby competing with pathogenic bacteria 45. Thirdly, L. reuteri is known to inhibit the secretion of pro-inflammatory cytokines as demonstrated on intestinal mucosa 46. This may indirectly reduce the inflammation in the gingival tissues. The anti-inflammatory effect of probiotic bacteria has also been observed with other bacteria of the Lactobacillus group. Lactobacillus brevis has also been shown to have anti-inflammatory activity. Vivekananda et al. (2010) 47 in a study on 30 adult patients evaluated the effect of …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book……

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     In a study, Shimazaki et al. (2008) 53 analyzed the epidemiological data to assess the relationship between consumption of dairy products such as cheese, milk and yogurt and periodontal health. The authors observed that individuals (particularly non-smokers), who regularly consumed yogurt or beverages containing lactic acid exhibited lower probing depths and less clinical attachment loss than individuals who consumed a few of these dairy products.

     It is now well established that the reduction in periodontal pathogenic microflora is the cornerstone for periodontal health. Various means such as application of mechanical plaque control measures and chemical plaque control measures have been applied to reduce the bacterial load in the oral cavity. Use of antimicrobials is an effective method to reduce the bacterial load in the oral cavity, but this effect is not a permanent process due to recolonization in due course of time and there are problems associated with emerging resistant bacteria. Hence, the application of probiotics seems to be an effective and beneficial strategy to reduce the pathogenic bacteria in the oral cavity.

Halitosis and probiotics

Halitosis is a very common oral health problem in the general population. It is primarily caused by various anaerobic bacteria present in various niches in the oral cavity. Various periodontopathogens have been found to be associated with the production of various compounds that are associated with the development of oral malodor. Cultivable oral cavity bacteria associated with halitosis include mainly Porphyromonas gingivitis, Treponema denticola and Tenerella forsythia 54-56. Halitosis is caused by the production of volatile sulfur compounds due to the degradation of S-containing amino acids by bacteria in the oral cavity. One effective way of controlling halitosis is reducing the number of anaerobic bacterial species in the oral cavity by a reduction in their total counts by occupying their inches with probiotic bacterial strains.

     Only a few studies have evaluated the effect of using probiotics on halitosis. In a study, Kang et al. (2006) 57 have reported that various strains of W. cibaria could inhibit the production of various volatile sulfur compounds (VSCs) produced by F. nucleatum. The reason suggested for this inhibition was the production of hydrogen peroxide by W. cibaria, which inhibited the proliferation of F. nucleatum. In another study, Kazor et al. (2003) 58 compared the microbial species isolated from the tongue of halitosis patients to that isolated from healthy patients. The authors reported that most commonly associated bacterial species with halitosis patient included Atopobium parvulum, Eubacterium sulci, and Fusobacterium periodonticum whereas Streptococcus salivarius was …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book……

Probiotics and oral Candida

Diverse numbers of fungal genera have been identified in the oral cavity, including Candida, Saccharomyces, Penicillium, Cladosporium, Malassezia, and Fusarium with varying densities. Out of all these genera, Candida is the most dominant commensal in the oral cavity and is present in about 25%-75% of the microbiota of healthy individuals 61. Imbalance in the resident microbiota may promote the growth of opportunistic microorganisms with Candida being the commonest of them, causing candidiasis 62. The majority of cases of candidiasis are associated with Candida albicans isolates 63. Other Candida species isolated from the oral fungal infections include C. krusei, C. tropicalis, C. glabrata, C. parapsilosis and C. dubliniensis 61, 64-66. Oral fungal infections are more common in elderly people and people who are immunocompromised. With the use of anti-fungal drugs, the chances for the development of resistant fungal strains has increased. Various studies have been done, especially on elderly patients, to evaluate the effect of probiotics on the growth of oral fungal species. In a study, the test group of elderly people was asked to consume cheese containing L. rhamnosus strains GG and LC705 and Propionibacterium freudenreichii species shermanii JS for 16 weeks, followed by estimation of total yeast count. The results of the study demonstrated that the total yeast counts decreased but no changes were observed in mucosal lesions 67.

     In another study, Kraft-Bodi et al. (2015) 68 investigated the effect of a daily intake of probiotic Lactobacilli on the prevalence and counts of oral Candida in frail elderly patients living in nursing homes. The subjects were given one lozenge containing 2 strains of the probiotic bacterium Lactobacillus reuteri (DSM 17938 and ATCC PTA 5289) or placebo twice daily (morning and evening). The intervention period was 12 weeks, and saliva and plaque samples were collected at baseline and follow-up. The results of the study demonstrated a statistically significant reduction in the prevalence of high Candida counts in the probiotic group but not in the placebo group. However, no significant differences between the groups were noted concerning the levels of supragingival plaque or bleeding on probing. The authors concluded by saying that the daily use of probiotic lozenges may reduce the prevalence of high oral Candida counts in elderly patients.

     In an in vitro study, Hasslöf et al. (2010) 69 tested eight probiotic Lactobacilli strains for growth inhibition on three reference strains and two clinical isolates of Mutans streptococci and two reference strains and three clinical isolates of Candida albicans with an agar overlay method. The authors observed that selected probiotic strains showed a significant but somewhat varying ability to inhibit the growth of oral Mutans streptococci and Candida albicans. In a study done on elderly denture wearers, Ishikawa et al. (2015) 70 evaluated the …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book……

Commercial oral preparations available

Gum PerioBalance

It is the first probiotic specifically formulated to fight periodontal diseases. It is a chewing gum containing two strains of L. reuteri (strains DSM 17938 and ATCC PTA 5289.). These two strains have been specifically selected for their synergetic properties in fighting cariogenic bacteria and periodontopathogens. Each dose of lozenge contains at least 2 × 108 living cells of the bacteria. The recommended use is to take one lozenge every day either after a meal or in the evening after brushing the teeth, to allow the probiotics to spread throughout the oral cavity and attach to the various dental surfaces.

PerioBiotic

It is a toothpaste containing Lactobacillus paracasei strain (LP-33)). PerioBiotic™ toothpaste also contains xylitol and calcium glycerophosphate which are also its active ingredients. The ingredients not found in this toothpaste, which are found in most other toothpastes include fluoride, sodium lauryl sulfate (SLS), and saccharin.

Bifidumbacterin

It is a commercial preparation that contains the lyophilic dried mass of probiotic bacteria of the strain Bifidobacterium bifidum, not less than 108 of microorganisms in one dose and it is available in vials of 5 and 10 doses. It is claimed to improve both clinical and microbiologic parameters in gingivitis and mild periodontitis patients. After routine mechanical debridement, 2 tablets to be dissolved in the mouth, three times a day for 20-30 days for improved outcome.

Wakamate D

This probiotic tablet contains 6.5 x 108 colony forming units (CFU) per tablet of Lactobacillus salivarius WB21 and xylitol (280 mg/ tablet). This preparation was originally prepared to contribute to the intestinal microbial balance by providing acid tolerant L. salivarius WB21. However, it was observed that …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book…..

Acilact

Acilact, a bacterial biopreparation representing a complex of live lyophilized acidophilic lactobacteria. This preparation was first clinically tested by Pozharitskaia et al. in 1994 71 and they found improved clinical parameters in periodontitis patients and shifts in local microflora towards gram-positive cocci and lactobacilli. In a subsequent study, Grudianov et al. (2002) 72 obtained a probiotic mix in the tablet forms, viz Acilact and Bifidumbacterin, and found normalization of microflora and reduction of signs of gingivitis and periodontitis.

ProDentis

ProDentis contains a natural lactic acid bacteria, Lactobacillus reuteri (L. reuteri DSM 17938 and L. reuteri ATCC PTA 5289) developed by Swedish company BioGaia®. Because L. reuteri naturally colonizes in the oral cavity, it has been observed to exert beneficial effects on periodontal health 47, 49, 73, 74. It is available as a lozenge and chewing gum in mint flavor. The lozenge needs to be dissolved slowly in the mouth. When using the gum it must be chewed for at least five minutes.

     All the above discussed commercial probiotic products have been claimed to have beneficial effects on the maintenance of periodontal health; however, additional studies are required to evaluate the long-term effects of using these commercially available products.

Safety associated with probiotics

Presently, probiotics are extensively used and most of them have a history of safe use. In healthy humans, lactobacilli are normally present in the oral cavity (103-104 CFU/g), the ileum (103-107 CFU/g), and the colon (104-108 CFU/g), and they are the dominant microorganism in the vagina 75. Infections due to the use of probiotics, specifically due to Lactobacilli and Bifidobacteria are extremely rare and are estimated to represent 0.05%-0.4% of cases of infective endocarditis and bacteremia 76, 77. Most of the rare cases of infection with Lactobacilli occur in patients with underlying conditions that are predominantly of a severe nature. Lactobacillemia is considered as indicative of serious underlying illness 78. The list of organisms that have been associated with …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book…..

     Infections by probiotic organisms may occur in immunocompromised patients. However, there is no published evidence that the consumption of probiotics that contain lactobacilli or bifidobacteria increases the risk of opportunistic infection among such individuals. Two studies have been done on patients with HIV infection to find out the safety of probiotics and results from both the studies support the safety of probiotics consumed by these patients 82, 83.



Safety of probiotics in infants and young children

The use of probiotics containing Lactobacilli and Bifidobacteria in infants and children has been found to be safe. Many studies have reported that the administration of probiotics containing Lactobacilli and Bifidobacteria are well tolerated in this age group 84-86. Presently, there are only a few case reports of Lactobacillus GG-associated bacteremia in children. One case report is a child with short gut syndrome, 2 cases of bacteremia in children who have central venous catheters, 1 case of endocarditis, and 1 case of a liver abscess 87-91. A few cases of bacteremia associated with Bacillus subtlis have also been reported 92. However, there is scanty literature regarding the safety of use of other probiotic bacterial species in infants and children and more studies are required to authenticate their safe use in these age groups.

Contraindications and precautions

Most important precaution associated with manufacturing the probiotics is to prevent contamination. Caution is also warranted in patients with central venous catheters. In literature, there are several case reports regarding fungemia associated with a Saccharomyces cerevisiae (Saccharomyces boulardii) probiotic. It was found that the infection was due to the contamination of indwelling catheters 93. Lactobacillus preparations are contraindicated in persons with hypersensitivity to lactose or milk. S. boulardii is contraindicated in patients with a yeast allergy. Probiotics should be used with caution in patients with immunosuppression (including a debilitated state, HIV infection or malignancy) and prematurity in infants. Other minor risks associated include the presence of a central venous catheter, impairment of the intestinal epithelial barrier (such as with diarrheal illness), cardiac valvular disease (Lactobacillus probiotics only), concurrent administration with broad-spectrum antibiotics to which the probiotic is resistant. Probiotics should also be used with caution in patients where probiotics are administered via a jejunostomy tube. In this case, because the probiotics bypass all acid contents of the stomach so the total viable count of bacteria is more.

     As far as the use of probiotics to prevent or treat periodontal diseases is concerned, the most important concerned factor is the …….. Contents available in the book………… Contents available in the book……. Contents available in the book……. Contents available in the book………

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Future perspective

As discussed above, except for certain systemic conditions, probiotics can be safely given to the patients. These can be used as passive local immunization agents against dental caries. High immunoglobulin titer against cariogenic bacteria may help to prevent dental caries. Furthermore, genetic engineering can be employed on probiotic acidogenic bacteria to further improve their caries preventing capability. Mutations can be induced in specific bacteria to achieve increased bacteriocin production. These strains can inhibit the growth of pathogenic bacteria in the oral cavity. However, these mutant strains should not cause any unwanted effects in the oral cavity. To confirm that, various in vitro and in vivo studies should be performed. Dairy products which have probiotic lactobacilli in combination with prebiotics are also being currently developed. Many studies have been done to evaluate the effects of probiotics in the gastrointestinal tract and many of them have been used to treat oral infections, specifically periodontitis. However, the research on the effect of probiotics on periodontitis is still in a preliminary stage. More long term studies are required to authenticate their application in the oral cavity. In the future, various techniques such as molecular biology, nano-techniques, biochemical, microbiological and Immunological techniques, and tissue engineering sciences are expected to come together in bringing up an ideal or near-ideal probiotic preparation, specifically for the treatment of periodontitis.

Conclusion

In the above discussion, we discussed various aspects of probiotics and their application for the treatment of oral diseases. Because this is a recent treatment strategy in periodontics, we need more research in this field. The use of various antibiotics and antimicrobial for the treatment of periodontal diseases is associated with the development of resistance and various side-effects associated with these drugs. Probiotics are a very good alternative to substitute these drugs. In the near future, we are likely to see various probiotic preparations and their application for the treatment of various oral diseases specifically periodontitis.




References

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