INTRODUCTION TO THE HUMAN MICROBIOME
Have you ever thought of yourself as a microbial safe-haven? According to new-age science, you should. Indeed, recent endeavors into defining the human microbiome have demonstrated that we play host to seemingly infinite number of microscopic tenants.
It is this inter-kingdom relationship that has led to a reformation of what it is to truly be human including redefining our name from our ancestral Homo erectus counterpart, to the 21st century “Super-organism.” As defined by Eberl G. and Sleator RD., a super-organism is one in which the human being is no longer seen as separate from its resident microbes.1,2 In other words, we are an amalgamation (pun intended) of eukaryotic (nucleated) and prokaryotic (anucleate) microbial cells.
Certainly, the advent of “omics” technology such as Metagenomics and multi-billion dollar transnational projects including Europe’s MetaHIT (Metagenomics of the Intestinal Tract) and US National Institute of Health’s Human Microbiome Project (HMP) have revealed that we are teeming with microbes with certain body habitats considered “hot spots” for colonization including the skin, oral cavity, gut and urogenital tract.3,4
Whether you would like to believe it or not, each and every one of us harbours microbes at a ratio of 10 parts microbial cells to one part germ line and somatic cells, which is tantamount to saying we are 10 percent human and 90 percent microbes.5 This numerical quantity translates into an approximate weight of three to five pounds or one to two percent of our body mass.6 In fact, these microbes contain upwards of 8 million microbial protein encoding genes, outnumbering our very own by more than 360 fold.7 It is these figures that have led some to suggest that we are in essence more microbial than human (at least from a cellular perspective) and as Johnathan Eisen summarized in his recent Ted Talk, “there is more mass in the microbes than mass in our brains.”8
Despite modern-day technological innovations into the human microbiome, it would not be an appropriate discussion of the microbial consortia, especially from an oral healthcare perspective, without paying homage to Antony van Leeuwenhoek. In his seminal work, he studied plaque which he described as “a little white matter, which is thick as if ‘twere batter.” He further examined the plaque microscopically, which led him to account in his very own words that microbes were contained in the biofilm. “I then most always saw, with great wonder, that in the said matter there were many very little living animalcules, very prettily a-moving.” It is these observations that have been credited as the earliest reports of living microbes from a human host.9
How do we acquire such a large and varied “microbial menagerie” or in other words, microbial jungle, as Ed Yong remarked?10 Up until now, the prevailing paradigm was that in utero we are sterile and we acquire our microbial partners at birth. However, recent studies, as early as 2008, have heralded new insights suggesting that we may not entirely be born sterile and in fact, colonization of the human landscape begins prior to birth.11,12 Irrespective of these novel findings, what has been well established is that the mode of delivery, whether vaginally or caesarean delivered, influences the initial colonizing agents of a host. The mother’s vaginal and fecal flora inhabit those that are delivered through natural birthing processes and the environment affects caesarean section delivered babies.13 As a result, inter-individual disparities in our colonizers exist right at birth.
In the realm of dentistry, we are often taught the notion that our caregivers are a major driving force on the type of bacteria that are found inhabiting the mouth. In fact, it would be a prudent (albeit controversial) prescription for one to suggest that parents choose their nannies wisely. Heck, give them an oral microbial examination if necessary. To reiterate this concept from a research standpoint, studies such as that by Parisotto TM. et al. have demonstrated that infants are at a greater risk of colonization of the oral cavity with Streptococcus mutans, one of the key players in caries formation, when the salivary content of their mothers also harbors higher levels of S. mutans.14 Similarly, a recent literature review showed that the genotype or ‘genetic stamp’ of the S. mutans species found in infants was identical to that of their maternal counterparts.15 Therefore, this once again shows the transmission and inoculation of the oral milieu with potentially pathogenic bacteria from caregiver to infant.
Although traditionally viewed as pathogenic, especially from a dental care perspective, we humans can derive several health benefits from our microbial partners, which include but are not limited to: regulation of nutrient and vitamin production, conferring resistance to colonization of pathogenic microbes, and host immunomodulation.16,17,18 Indeed, it is these beneficial effects that have led investigators to study the potential to manipulate the host bacteria in health and disease in order to create a more favorable environment for colonization of beneficial microbes. This has been an impetus in spawning the current multi-billion dollar probiotic industry.
WHY SHOULD ONE CARE?
By now you may be wondering — what is the utility of this knowledge and how does it affect me as a dental professional? The simple answer is that we need to investigate the microbiome to understand what is out there, both in health and disease. In fact, acquiring this knowledge is fundamental to the research on how to manipulate the microbiome towards a healthier subtype, particularly in dental conditions. This point will be further elucidated later in the article.
To address the question of what is out there in health and disease, one must first understand the atmosphere in which microbes are tested at diverse body sites. Although oversimplified in the context of this article, cataloging the microbes at various body sites is a daunting task that requires extensive transnational efforts with participants including Europe’s MetaHit (Metagenomics of the Intestinal Tract 2008) and United States’ HMP (Human Microbiome Project 2007-current), which over years of research have investigated both the gut microbiome and that of several body sites respectively.
How are these researchers creating a library of knowledge of the bacterial species found in humans? The technology being employed can be thought of as akin to the process of identifying humans at a crime scene through DNA profiling (i.e. genetic fingerprinting). The only difference is that we are identifying bacteria using bacterial DNA detection.
In essence, bacterial DNA is isolated from a sample obtained at a particular body cavity using commercial kits or techniques that do not isolate eukaryotic DNA. Hence, no human DNA is found in the sample.
In turn, out of the entire bacterial DNA sample obtained, a particular gene called 16S rRNA (ribosomal RNA) acts like a fingerprint that can be used to identify the particular bacterial subtypes in the entire sample. This gene is unique in that it contains conserved sites (pertaining to all bacteria) and variable sites (unique to particular bacterial genera or species) that can be targeted through molecular tags to see which bacteria are present in the clinical sample (Fig. 1).
FIGURE 1. 16S rRNA sequencing for microbial community analysis./201412041446570ryfxua45fxxuea55450rkjez.jpg)
Using primers, which are the molecular tags, the bacterial DNA sample is exposed and then bound by their specific tags. After the binding between the primers and bacterial 16S rRNA target occurs, the 16S rRNA gene is amplified (i.e. many copies are made of the 16S rRNA gene). Following amplification, sequencing of specific variable regions of the 16S rRNA gene is completed to enable for resolution of the particular bacterial cohorts present in the polymicrobial sample.
Analysis is then performed to compare the sequenced samples of the unknown microbial identities to a database of known sequences that correspond to particular microbial cohorts. Knowledge of the particular microbe’s presence is obtained when the sequence matches that of the database as the bacteria’s specific “fingerprint” is matched.
This “omics” technology should not be underestimated. In fact, through utilization of 16S rRNA sequencing technique, our knowledge of the repertoire of the human microbiome and the oral microbial consortium has dramatically progressed. This is especially important considering that traditional techniques, such as culturing, have yielded low returns in the microbial arena, with less than half of the microbes in the oral cavity having the capability of being cultivated.19
ORAL MICROBIOME IN HEALTH & DISEASE
Transitioning to a greater understanding of the oral microbiome, it is important to note that recent research reveals that the oral cavity itself is home to upwards of 700 diverse bacterial species and of these, 49 percent have been named, 17 percent remain unnamed but cultivated, and 34 percent are known only as uncultivated species.20 Alternatively, other estimates suggest that currently upwards of 50 percent of the oral microbiome remains uncultivable.19 Considering that the oral cavity contains a varied landscape, it serves to reason that the bacteria present throughout the oral environment are vast and varied according to the surfaces on which they reside.
The oral cavity contains both shedding and non-shedding surfaces alike. The dentition represents a significant non-shedding surface onto which both supragingival and subgingival plaque can be found, and these consist of diverse bacteria depending on the site.21,22 Shedding mucosal surfaces, such as the tongue and buccal mucosa, represent a diverse niche in which a varied population can be derived. Even salivary samples have been suggested to represent a snapshot of the bacterial populations found in the mouth with populations varying in health and disease, including in dental caries23 and periodontitis.24
Certainly, disruptions of the bacterial populations found throughout the oral cavity have been demonstrated in particular oral conditions with the cohorts differing between those in a healthy state, and those in particular oral diseases. With regard to dental caries, a preponderance of bacterial populations of the Streptococcus, Lactobacillus, Propionibacterium, Actinomyces and Veillonella genera have been demonstrated in higher relative abundance in the plaque of adults exhibiting active caries compared to those without caries.22, 25
Etiologically, periodontal disease is multifactorial (Fig. 2), and it is well accepted that proliferation of specific Gram negative, anaerobic microorganisms in subgingival pockets contribute to disease progression including Socransky’s keystone Red Complex pathogens: Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, previously Bacteriodes forsythus.26 Recently, however, research has also shown (through the employment of deep sequencing methods) that not only do subgingival pockets harbour these proposed periodontal pathogens, but both supragingival and tongue dorsum plaque contain Socransky’s known periodontal pathogens, as well as potentially novel bacterial biomarkers associated with Chronic Periodontitis (CP). These areas may provide alternative sampling sites for investigation of potential pathogens associated with CP.27
Endodontically, multi-species biofilms have been implicated in the initiation and progression of periapical conditions.28
From an oral malodour standpoint, studies have suggested that the tongue dorsum of individuals with halitosis often harbour bacteria that are capable of breaking down protein products resulting in the production of the offending fatty acids and sulphur by-products, known as volatile sulphur compounds.29,30
By no means however is this meant to be an exhaustive review of the literature as the implications of the bacterial consortia in oral conditions has been reviewed elsewhere.31
It is important to consider that it is not sufficient to merely state that the presence of a perceived pathogen is the cause of a disease condition but rather, the relative abundances of certain disease-associated pathogens and the multi-factorial nature of these conditions must also be taken into account (Fig. 2).
FIGURE 2. Multifactorial nature of oral health conditions. Etiology of oral health conditions involves a tripartite relationship between intrinsic host factors, extrinsic environmental factors and the oral microbial consortia./201412080957580plpcxzeyrounhpbrfne0eyez.jpg)
The data provided through assessment of the disease-associated microbial consortia, although not causative, has led scientists to speculate on the potential for using bacteria therapeutically, including probiotics in dentistry as an adjunct to one’s clinical armamentarium.
PROBIOTICS IN DENTISTRY
Probiotics as defined by FAO/WHO (Food and Agriculture Organization and World Health Organization) are “live microorganisms that when administered in adequate amounts confer a health benefit on the host” (2001). In other words, these are specific bacteria that are intentionally placed in formulations ranging from freeze-dried capsules, to sprays, topical lotions and dairy products, in order to provide the host with a measurable benefit. Criteria that grant probiotic status include but are not limited to the fact that the bacteria used are “generally recognized as safe”, owing in part to the fact that they may be derived from a healthy human host. In addition, they must be living at the time of administration but may die inside the host and either the microbe, or its components, must elicit a response at the site of action.32 Hence, the term probiotics refers to the microbes themselves that are added to commercial products.
Prebiotics, on the other hand, are selectively fermented ingredients that promote specific changes in the composition, and/or activity of the gastrointestinal microbiota that confer benefits to the host.33 Prebiotics can be likened to “food” for beneficial microbes in the form of non-digestible carbohydrates. When consumed, prebiotics provide a selective advantage for potentially beneficial microbes indigenous to the host to survive and/or grow.
Synbiotics represent products that contain a combination of probiotics and prebiotics34 and as a result, not only provide the supposedly beneficial microbes to the host, but by providing the microbes their “food” also help them to survive and thrive in vivo.
From an economic standpoint, as of 2010, the probiotic business has been shown to represent a multi-billion dollar industry with a forecasted increase in global market projected to reach upwards of 30 billion dollars in 2015.35
Given th
e large array of products available on the market, sifting through the literature may be difficult. However, from an oral healthcare perspective, although several probiotics have been tested from a research standpoint, very few have yet been commercialized for the purpose of dental treatment.
From a cariogenic perspective, studies have focused on the use of probiotics from both the Bifidobacteria and Lactobacillus genera. This includes Lactobacillus paracasei ssp. paracasei, Lactobacillus rhamnosus36 and Lactobacillus reuteri37 alongside bifidobacteria species.38 These bacteria have all collectively been demonstrated to affect colonization of the potential cariogenic pathogen S. mutans. One of the non-traditionally sought out probiotics, Weissella cibaria has been shown to be effective not only in curtailing caries development through the prevention of biofilm formation of cariogenic S. mutans,39 but has also been shown to affect Fusobacterium nucleatum colonization, a microbial pathogen implicated in both periodontitis and oral malodour.40
Furthermore, in regards to periodontitis, L. reuteri ATCC 55730 containing chewing gum41 and Lactobacillus brevis42 have been demonstrated to decrease gingival crevicular fluid levels of cytokines and Matrix Metalloproteinases in saliva respectively. These substances have been shown to be key players in the biology of periodontal disease. From a periodontal pathogen inhibitory standpoint, lactobacilli have once again been shown to have an inhibitory effect on the growth of known periopathogenic bacteria, including P. gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans.43 Even in the case of gingivitis, Krasse P. et al. demonstrated the benefit of utilising L. reuteri formulations in decreasing plaque and gingivitis scores in individuals exhibiting moderate to severe varieties of the disease.44
A summary of the proposed dental probiotics can be found in Table 1. Once again, the aforementioned studies are by no means exhaustive and more in-depth reviews on potential probiotics for dental conditions can be found elsewhere.45-47
With regards to marketed probiotics in the realm of dentistry there is a general lack of products specifically marketed for dental health. Two of the most widely recognized products in the area of oral healthcare include SUNSTAR’s lozenge, GUM® PerioBalance® (Fig. 3) and Oragenics EvoraPlus® chewable tablet containing ProBiora3® (Fig. 4). ProBiora3® is also readily available as an additive for interested companies, enabling the incorporation of this proprietary probiotic cocktail into many different types of delivery vehicles including mouthwashes, chewing gums, lozenges and fast-dissolve tablets.
FIGURE 3. G•U•M® PerioBalance® by SUNSTAR. Each lozenge contains the probiotic L. reuteri Prodentis./201412041446582ryfxua45fxxuea55450rkjez.jpg)
FIGURE 4. EvoraPlus® by ORAGENICS. Each tablet contains ProBiora3™, a probiotic cocktail of Streptococcus oralis KJ3®, Streptococcus uberis KJ2®, and Streptococcus rattus JH145®./201412041446581ryfxua45fxxuea55450rkjez.jpg)
GUM® PerioBalance® has been suggested to promote healthier gums by reducing plaque formation and gingivitis when used in conjunction with good oral hygiene. The active probiotic contained in the product is L. reuteri Prodentis. It is generally recommended that one lozenge be used after brushing, flossing and the use of mouth rinses and should be dissolved in the mouth for a period of 10 minutes. The product must be consumed for a period of 14 to 28 days; in conjunction with good oral hygiene in order have appreciable effects.48
The EvoraPlus® tablet contains ProBiora3®, a cocktail of Streptococcus oralis KJ3™, Streptococcus uberis KJ2™, and Streptococcus rattus JH145™. This product has been suggested to promote fresher breath and whiter teeth, and protect the health of teeth and gums in between dental visits.49 More specifically, the company proposes through the production of hydrogen peroxide both S. oralis KJ3™ and S. uberis KJ2™ inhibit the growth of potential periodontal pathogens. In vitro, they have been shown to decrease stains on ceramic disks, an effect attributed to their hydrogen peroxide production potential. As well, Streptococcus rattus JH145™ decreases S. mutans levels by competitive inhibition; that is by occupying the same niche and preventing S. mutans colonization without being cariogenic itself.49
Despite ongoing research into the field of probiotics in the realm of dentistry, there are several things a prudent health professional should deliberate when discussing or considering probiotic usage for their patients, including understanding how they are regulated.
PROBIOTIC REGULATIONS
Firstly, one should consider the US Food and Drug Administration guidelines when contemplating the use of probiotics. This regulatory authority, which oversees probiotic products in the US, currently regulates probiotics, which are sold over-the-counter, as dietary supplements and not pharmacological agents. Hence, they are not subjected to the same rigorous controls and inspections that pharmaceutical companies must adhere to, thereby rendering probiotics more lax from a regulatory standpoint.50 The only exception to the less stringent regulations is if the company manufacturing the probiotic product intends to use their product in accordance with benefits paralleling those of pharmaceutical agents.50
Health Canada has provided little assistance with regard to probiotic regulation and since 2009, has permitted a limited number of claims or statements about the inherent nature of probiotic microorganisms. These claims predominately involve suggesting that the probiotics either form a part of, or contribute to, healthy gut flora.51 Collectively, these results imply that due to a lack of stringent regulation from officials, one should question the dose, content and strains in a given probiotic product; the lack of regulation may result in these levels being subpar and in fact, not contain what is suggested on the package or in the quantities necessary to elicit a host response.
Moreover, despite the number of positive results shown in the aforementioned clinical studies, there are others that have demonstrated limited effects with the same probiotics on clinical parameters such as those measured in periodontal conditions52 and their proposed anti-cariogenic nature.53 Similarly, studies on probiotics and oral healthcare examining the length of treatment, dose response, time-course of action and the best vehicle of probiotic delivery (including gums, yogurts, tablets or mouth rinses) are lacking. Even the proposed bacteria that are suggested to aid in alleviating dental conditions, such as those of Lactobacillus and Bifidobacteria genera, need to be examined as they are acidogenic in n
ature. As a result they, themselves, can mediate detrimental changes in the oral cavity.54 Similarly, cost affiliations, risk-benefit analysis, duration of effect, and mechanisms of action are also lacking from a research standpoint in the probiotic arena.
Ultimately, although promising preliminary results exist regarding probiotic benefits in oral healthcare, one should not be over-zealous in the recommendation of probiotics, as further research is needed to determine clinical efficacy.
CONCLUSIONS
All in all, despite the potential for probiotics to be of benefit in the realm of dentistry, the field is still very much in its infancy and to date, does not offer a panacea for dental health issues. The prudent dentist should, however, consider enhancing their knowledge in this field as the research is ongoing with promising results. Given that patient populations are consuming more and more probiotics (as evidenced by the rise in the multi-billion dollar oral probiotic industry) and that patients may seek advice from their dental professionals as to the use of probiotics for oral health, probiotic discussions are a necessary addendum to 21st century dental education.
Considering we live in an era where bacterial fecal therapy has been accepted by patients, it does not seem far-fetched for the public to consider probiotics for their oral health, as it is part of the alimentary tract. What is more, in view of an increase in antibiotic resistance, (and antibiotics are a treatment modality which dentists use extensively), patients may elect to consume probiotics in conjunction with prescribed antibiotics. Therefore, the onus remains on the health professional to provide the appropriate guidance as to the use of probiotics in order to facilitate the best oral health and overall health outcomes. OH
Natasha Singh Hons. BSc, MSc, DDS Candidate 2016; is a third year DDS student at the Faculty of Dentistry, University of Toronto. She has previously completed a Masters Degree specializing in Molecular and Microbiology at the Department of Nutritional Sciences, University of Toronto, St. George, and currently remains active in the field of Dental Research. Natasha can be contacted at natasha.singh@mail.utoronto.ca.
Oral Health welcomes this original article.
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