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INVITED MEDICAL REVIEW

The oral microbiome in health and disease and the

potential impact on personalized dental medicine

MF Zarco, TJ Vess*, GS Ginsburg

Institute for Genome Sciences & Policy, Duke University, Durham, NC, USA

Every human body contains a personalized microbiome

that is essential to maintaining health but capable of

eliciting disease. The oral microbiome is particularlyimperative to health because it can cause both oral and

systemic disease. The oral microbiome rests within bio-

films throughout the oral cavity, forming an ecosystem

that maintains health when in equilibrium. However,

certain ecological shifts in the microbiome allow patho-

gens to manifest and cause disease. Severe forms of oral

disease may result in systemic disease at different body

sites. Microbiomics and metagenomics are two fields of

research that have emerged to identify the presence of

specific microbes in the body and understand the nature

of the microbiome activity during both health and dis-

ease. The analysis of the microbiome and its genomes will

pave the way for more effective therapeutic and diag-

nostic techniques and, ultimately, contribute to thedevelopment of personalized medicine and personalized

dental medicine.

Oral Diseases (2012) 18, 109120

Keywords: microbiome; genomics; personalized dental medicine;

diagnostics; metagenomics; medicine

Introduction

Every human body contains a personalized set offoreign inhabitants essential to maintaining health, yet

also capable of eliciting disease. The totality of thesemicroorganisms, their genomes and ecosystems encom-passes the microbiome1 (Parahitiyawa et al, 2010). Thehuman and its microbiome together make up a supra-organism (Ling et al, 2010; Rajendhran and Gunasek-

aran, 2010). The number of microbial cells within ahuman body exceeds the total number of human cells in

the body by nearly 10 times (Turnbaugh et al, 2007;Ling et al, 2010). These microorganisms contribute theirgenome, known as the metagenome, to the human body,multiplying human genes by approximately 100 times(Turnbaugh et al, 2007; Ling et al, 2010; Rajendhranand Gunasekaran, 2010). The activity of the micro-biome and, specifically, the expression of its metage-nome provide the human with resources and traits thatdid not originally evolve with the body (Rajendhran andGunasekaran, 2010). For example, the microbiomecontains genes that allow humans to digest certain plantpolysaccharides (Rajendhran and Gunasekaran, 2010).

There are various microhabitats throughout the bodythat contribute to the overall microbiome. The mouth,

skin, gut, etc. each contains its exclusive microbiome andmetagenome (Badger et al, 2011; Sonnenburg and Fisch-bach, 2011). Each microhabitat maintains a uniqueecosystem with distinct atmospheric and nutritionalcompositions that provide a setting for symbiotic inter-actions among the various microbes within that ecosys-tem and the host. Of note, microbiomes from the samelocation on the body are more similar among differentindividuals than microbiomes from different locations onthe same individual (Sonnenburg and Fischbach, 2011).

The human microbiome can be classified into a coremicrobiome and a variable microbiome (Turnbaughet al, 2007). The core microbiome is shared among allindividuals and is comprised of the predominant speciesthat exist under healthy conditions at different sites ofthe body (Turnbaugh et al, 2007; Zaura et al, 2009;Sonnenburg and Fischbach, 2011). The variablemicrobiome is exclusive to the individual and hasevolved in response to unique lifestyle, and phenotypicand genotypic determinants (Figure 1). Although indi-viduals share microbiota at similar sites of the body,there are varying differences at the species and strainlevel of the microbiome that can be as inimitable to theindividual as is the fingerprint (Dethlefsen et al, 2007).

As the correlation between the human microbiome andhealth becomes more evident, microbiome research isbecoming central to the advancement of disease diagnos-

Correspondence: Prof. Geoffrey S. Ginsburg, Center for GenomicMedicine, Institute for Genome Sciences & Policy, Duke UniversitySchool of Medicine, 101 Science Drive, DUMC Box 3382, Durham,NC 27708, USA. Tel: 919668 6210, Fax: 919668 6202, E-mail:geoffrey.ginsburg@duke.eduReceived 30 July 2011; accepted 1 August 2011*Current address: Office of Undergraduate Research, Virginia Tech,Blacksburg, VA, USA.1The terms microbiome, microflora, and microbiota can be usedinterchangeably.

Oral Diseases (2012) 18, 109120 doi:10.1111/j.1601-0825.2011.01851.x 2011 John Wiley & Sons A/SAll rights reserved

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tics and therapeutics as well as the development ofpersonalized medicine (Sonnenburg and Fischbach,2011). Because each individual harbors a unique microb-iome that plays a key role in the etiology of disease withinthe body, disease may manifest and progress differentlyamong different individuals, making personalized medi-

cine imperative for optimal health care.However, microbiome research must develop a deeper

understanding of the fundamentals and specifics ofmicrobial activity within the body during health anddisease before it can contribute to personalized care.First, there should be a clear picture of which microor-ganisms exist in the body and how they affect the hostsphysiology and health condition. Then, microbial char-acteristics of specific diseases should be studied torecognize microbiome patterns that distinguish onedisease from another. Finally, proper diagnostic meth-ods and technologies should be developed to enableprofessionals to identify individual microbial profilesand treat specific microbes responsible for disease.

The emerging field of research that targets the microb-iome for therapeutic purposes is known as microbiomics.Microbiomics aims to understand how microorganismsinterplay with its hosts physiology and health by analyz-ing their distinct functions and interrelationships (Raj-endhran and Gunasekaran, 2010). Bacteria as adetermining factor of disease was a concept that emerged

in 1882 when physicist Robert Koch wrote an articlediscussing bacteria as the etiological agents of tuberculo-sis (Socransky and Haffajee, 1992). Kochs article trig-gered years of microbiology and disease etiology researchthat eventually led to microbiomics. Figure 2 summarizesseveral groundbreaking discoveries that were steps to thestudy of the microbiome today (Socransky and Haffajee,1992). Today, the Human Microbiome Project (HMP)takes a leading role in human microbiomics. It exploresthe role of the human microbiome in physiology, health,and disease through metagenomic research, which ana-lyzes the genomes of specific microorganisms (Rajendh-ran and Gunasekaran, 2009).

Specifically, studies have shown the oral cavitys

microbiome to be a key source in the etiology of manyoral and systemic diseases (Scannapieco, 1998; Garciaet al, 2001). Because the oral microbiome is vital to abodys overall health, it has become an essential focus ofmicrobiomics. It is crucial to unravel the complexities ofthe oral microbiome to learn the mechanisms by which itmaintains health or causes disease.

Microbiomics and metagenomics

To truly understand the activity of the microbiome fortherapeutic purposes, microbiomics must use metage-nomics to sequence and analyze bacterial genomes. The

HMP aims to use metagenomic techniques to sequence3000 microbial reference genomes isolated from varioussites around the human body. In doing so, it hopes tocontribute to our understanding of how the microbiomecorrelates with human health by identifying the differ-ences between bacterial genomes that encode formetabolic functions vs disease processes (Rajendhranand Gunasekaran, 2009).

Genome sequencing has greatly enhanced the analysisof pathogenic microorganisms, most of which areuncultivable (Horz and Conrads, 2007). Most meta-genomic methods are feasible because they are ableto analyze extremely small sample sizes of bacterialgenomes. The technique has led to the discovery of new

Figure 1 Overall human microbiome consists of a core microbiome,common to all individuals, and a variable microbiome, unique toindividuals depending on lifestyle and physiological differences.Reprinted by permission from Macmillan Publishers Ltd: Nature(Turnbaugh et al, 2007)

Figure 2 Summary of historical events that lead to the emergence of microbiomics

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genes, enzymes, and natural products, which may alsobe used for the development of novel chemicals andpharmaceuticals in the future (Wong, 2010).

There are several ways to sequence a metagenome orsegments of a metagenome, each technique serving aunique purpose. The most common techniques include16S ribosomal RNA sequencing, pyrosequencing, and

shotgun sequencing. Table 1 summarizes some methodsused to identify microbes, how they are performed, theirpurpose, and their positive and negative aspects. Ascertain metagenomes have already been synthesized andthe species identified, researchers can also use moreconventional techniques to determine the presence of amicroorganism. These techniques include culture-basedidentification, microscopy, enzyme analysis, and immu-nological assays. Although less advanced, these proce-dures complement the sequencing processes byproviding information on the physical characteristicsand additional behavioral and metabolic properties ofmicrobes (Horz and Conrads, 2007; Filoche et al, 2010).It may be beneficial or even necessary for the future

dentist to be familiar with sequencing and the other

identification techniques in case these laboratory proce-dures arrive in the clinical setting for an on-the-spotmicrobial identification and personalized care.

Microbiomics requires collaboration of metagenomicsand clinical research to gather accurate and in depthinformation about the correlation between the microb-iome and human health. Unfortunately, there are many

existing factors that inhibit rapid progress of micro-biomics, beginning with complications in experimentaldesign. First of all, it is challenging to gather populationsizes that are large enough for unbiased test results(Badger et al, 2011). Second, there are factors insequencing that can contribute to bias, like defining aoperational taxonomic unit that may over or under-estimate biodiversity in a sample. Researchers mustdecide whether to sequence full-length genomes orgenome segments to determine diversity, or to undergomore expensive procedures such as pyrosequencing.Also, to fully measure the microbial diversity in the oralcavity, there must be a more concrete definition ofspecies, which includes not only genotypic character-

istics, but phenotypic ones as well (Avila et al, 2009). In

Table 1 Methodologies for the detection of microbes and microbiomes

Technique Procedure Purpose Pros Cons

Metagenomics 16S rRNAsequencing

Amplification andcloning sequencingof DNA segmentsusing PCR

DNA sequences analyzedto identify species

1. Rapid 1. PCR subject to bias2. PCR sensitive to

contamination2. Accurate3. Detects viral DNA

Pyrosequencing Sequencing of smallDNA segments.Pyrophosphates areemitted as nucleotidescome together andsynthesis DNA

DNA sequences analyzedto identify species andalso biodiversity

1. Successfully determinesbiodiversity

1. Expensive2. Does not produce

full-length 16S rDNAsequences necessary fortaxonomic studies

2. Subject to less biasbecause does notrequire cloning

3. Produces manysequences

ShotgunSequencing

Long DNA israndomly fragmentedand sequenced.Several rounds ofthis are performedfor multipleoverlapping readsfor the target DNA.Computer programsuse the overlappingends of different readsto assemble them intoa continuous sequence

DNA sequences analyzedto identify whichorganisms are presentand also to suggest themetabolic processesthey are responsiblefor in the microbialcommunity

1. Less expensive2. Rapid3. Forms long stands of

continuous DNA

1. High error rate inconstructing thecontinuous set ofoverlapping sequences

4. Less need for humanintervention

5. Suggests informationon metabolic activity

2. Computationallyintensive

Conventionalmicrobiology

Cultureanalysis

Growth of bacteria onspecific medium

1. Physical, behavioral,and chemical properties

2. Metabolic requirements

1. Highly accurate2. Resistance testing is

possible

1. Time-consuming2. Many periodontal

pathogens anaerobicandor fastidious, manyspecies are uncultivable

Microscopy Observance of bacteriathrough microscope

Insight about microbialcommunities throughvisual observance

Provides informationon ecosystembehavior

1. Offers limiting information2. Requires special equipment3. Time-consuming

Enzyme assay Laboratory technologymeasures enzymeactivities

Identifies pathogenby detecting presenceor absence of certainenzyme activities

Rapid Less precise

Immunoassay Tray inoculated withantibody to detectantigen activity ofmicrobe

Identifies presence ofmicrobes by antimicrobialresistance or susceptibility

Rapid 1. Low sensitivity2. Specific antigenic

molecules for markerpathogens are yet tobe identified

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terms of the oral microbiome, time and compositionface significant challenges attributable to the highlydynamic characteristic of the oral cavity and its anaer-obic inhabitants (Badger et al, 2011).

Furthermore, Socransky and Haffajee, (1992) presentsthe complications researchers face when forming con-clusions about oral pathogens. It is tricky to define the

predominant etiological agent of a polymicrobial diseasecaused by the opportunistic behavior of pathogenicbacteria. Even in the case of a monomicrobial disease,there is a potential to define the wrong predominantspecies because a disease caused by a pathogen within aniche may change the environment within that niche,leading other microorganism to proliferate in numbersthat can skew test results. In addition, there is aconstantly growing number of newly identified peri-odontal species, most probably because of the uniquecomposition of every individual oral microbiome (Horzand Conrads, 2007). Advanced technologies and scien-tific methods are needed to accurately identify specificbacterial compositions characteristic to disease (Horz

and Conrads, 2007).However, the most complicated issue that arises in

microbiomics and metagenomics is the significance ofstudying microbes in isolation and outside the context oftheir natural habitat within the human body (Keller andRamos, 2008). To safely mimic and manipulate themicrobiome for therapeutic purposes, tests must ensurethat microbes operate similarly in vitro and in silico asthey do in vivo.

The oral cavity and its microbiome

To understand the role of the oral microbiome within

the oral cavity, it is important to analyze its funda-mental characteristics and dynamics. The oral cavityharbors over 700 species of bacteria that contribute tothe health and physiological status of the oral cavity.Within the oral cavity, there are two types of surfacesfor which bacteria can colonize: the hard surfaces ofteeth and the soft tissue of the oral mucosa (Zauraet al, 2009). The teeth, gingival sulcus, tongue, cheeks,hard and soft palates, and tonsils (Dewhirst et al, 2010)each provide enriching environments in which micro-bial communities can flourish. Different types ofmicroorganisms prefer distinct niches according tovarying surface structures and functions (Aas et al,2005). Each niche provides the optimal conditions and

nutrients for its populating microbes (Avila et al, 2009).In fact, research has shown the maxilla, hard palate,soft palate, and even the tongues lateral sides anddorsal side each to have a different bacterial profile(Aas et al, 2005).

Furthermore, the oral microbiome is extremelydynamic because of the oral cavitys continuum withthe external environment (Parahitiyawa et al, 2010).Thus, the oral microbiota has evolved skills to facechallenges that are not experienced by other microbiotas(Avila et al, 2009). The oral cavity has multiple essentialfunctions that affect bacterial growth and activity:eating, communicating, and defending against infection.

Also, the microbial ecosystem is disturbed by oralhygiene practices. Even oral microbial colonies that areless susceptible to agitation experience changes attrib-uted to diet, age, and health (Parahitiyawa et al, 2010),as well as constant changes in pH, redox potential,atmospheric conditions, salinity, and water activity fromsaliva (Badger et al, 2011).

Biofilms and salivaOral microorganisms adapt to changing environmentswithin protective biofilms (Avila et al, 2009). Biofilmsare the complex colonies of microorganisms thatpredominate both hard and mucosal surfaces in theoral cavity (Flemmig and Beikler, 2011). While themicrobial colonies play vital roles in maintaining oralhomeostasis, they are also significant players in oraldiseases (Flemmig and Beikler, 2011).

Dental plaque is a commonly known multispeciesbiofilm that packs as layers onto tooth surfaces (Listgar-ten, 1976; Flemmig and Beikler, 2011). Dental plaque caneither entrap and prevent an existing oral pathogen from

flourishing, or provide a refuge for a pathogen to hidefrom salivary flow and the hosts immune system (Avilaet al, 2009). Under healthy conditions, an ecologicalbalance between microbe composition and activity keepsbiofilms healthy and stable (Flemmig and Beikler, 2011).Yet, detachment of biofilms is necessary because patho-gens can manifest within and cause disease (Filoche et al,2010) Both oral hygiene practices and salivary flow areresponsible for this required detachment.

Saliva is crucial to the oral cavity because it plays akey role in maintaining homeostasis and defending fromdisease (Nieuw Amerongen and Veeman, 2002). While ithelps maintain a climate that allows biofilms to flourish,

saliva also detaches layers of plaque and containsnumerous proteins, minerals, and antimicrobial enzymesthat control biofilm build up and activity (NieuwAmerongen and Veeman, 2002). Saliva also providesnutrients that protect tooth enamel and antibodiesthat defend the oral cavity and the rest of the bodyfrom infection (Nieuw Amerongen and Veeman, 2002;Filoche et al, 2010).

Studies have shown both salivary flow and microbiomecomposition to be unique to an individuals oral cavity,also suggesting that dental plaque composition andarrangement are specific to the individual (Filoche et al,2010). Varying plaque biomass, pH, and microbialresponse may result from or explain current health and

disease conditions, and also may explain why someindividuals are more prone to oral diseases than others,despite oral hygiene habits (Filoche et al, 2010).

Oral microbiome of healthThe bacterial flora in a healthy oral cavity vs a diseasedone is distinctly different, suggesting there may be aprofile for a core oral microbiome of health (Aas et al,2005). According to various studies, identical bacterialsequences have been discovered in the oral cavitiesof unrelated healthy individuals (Zaura et al, 2009;Bik et al, 2010). Bik et al (2010) performed a studybased on the largest set of near full-length sequences per

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healthy individual to date. The analysis identified 10variables shared between 11 bacterial species. However,the same study also showed that significant interindi-vidual differences exist, supporting the concept of both acore and variable microbiome within the oral cavity.Figure 3 shows the overlapping of specific bacterialgenera that were found among the 10 samples.

This study and others offer a glimpse into the possiblemicroorganisms that predominate healthy oral cavities.The major genera with the largest representation inhealthy oral cavities include the following: Streptococ-cus, Veillonella, Granulicatella, Gamella, Actinomyces,Corynebacterium, Rothia, Fusobacterium, Porphyromon-as, Prevotella, Capnocytophaga, Nisseria, Haemophilis,Treponema, Lactobacterium, Eikenella, Leptotrichia,Peptostreptococcus, Staphylococcus, Eubacteria, andPropionibacterium (Aas et al, 2005; Jenkinson andLamont, 2005; Wilson, 2005; Zaura et al, 2009; Biket al, 2010). Developing an in depth definition of health,and understanding molecular differences between healthand disease, may give clinicians the ability to recognize

and diagnose diseases at an earlier and reversible stage(Zaura et al, 2009).

Oral symbiosis as the determining factor ofhealth and disease

The key to oral health is an ecologically balanced anddiverse microbiome that practices commensalism withinitself and mutualism with its host (Ruby and Goldner,2007; Zaura et al, 2009; Filoche et al, 2010). Commensalrelationships among microbes allow them to flourish atno expense to their co-habitants and, in turn, maintainbiodiversity within the oral cavity. Research has

demonstrated such biodiversity to be crucial to health.Analysis of plaque and saliva in healthy adults demon-strated much more diversity than originally hypothe-sized (Filoche et al, 2010). Oral microbiomes of childrensuffering from severe dental caries are much less diversethan those of children with oral health (Kanasi et al,2010). Asymptomatic lesions of infected root canals

displayed a higher level of biodiversity than did thesymptomatic ones. The need for biodiversity in healthmay suggest that every species carries out a specificfunction that is required to maintain equilibrium andhomeostasis within the oral cavity.

Furthermore, the relationship between the micro-biome and its host during health is mutually beneficialbecause the host is providing its microbial communitieswith an environment in which they can flourish and, inturn, keep their host healthy. In health, microorganismsprevent disease progression in several ways: they canprevent the adherence of pathogens onto specificsurfaces by occupying the niche preferred by a patho-gen, they can actively prevent a pathogen from occupy-

ing a site, they can hinder a pathogens abilities tomultiply, and they can degrade a pathogens virulencefactors (Socransky and Haffajee, 1992).

However, certain pathological changes within themicrobial ecosystem may occur and cause a once-beneficial microorganism to initiate disease within theoral cavity. Ecological shifts that cause pathologicalchanges are: (1) a change in the relationships betweenthe microbes and with the host; (2) an increase inrelative abundance; and (3) acquisition of virulencefactors2 (Parahitiyawa et al, 2010). In disease, microbesalter their relationship with their host from mutualisticto parasitic and with other microbes from commensal to

opportunistic (Avila et al, 2009; Parahitiyawa et al,2010). As the pathogenic bacteria flourish, the hostbecomes infected or prone to infection (Ruby andGoldner, 2007). Pathogens will grow with disregard ofits co-habiting bacteria, and any beneficial bacteria willnot be able to inhibit the diseases manifestation (NieuwAmerongen and Veeman, 2002).

Because shifts in relationships, proportion, and viru-lence properties of microbes seem to affect one another, itis not always certain which ecological shift occurred first.It is also unclear what exactlytriggers the initial ecologicalshift and, in turn, catalyzes the entire cycle (Avila et al,2009). The major factors that may be responsible forinitiating an ecological shift are poor oral hygiene,

compromised immune system, and genetics. Figure 4illustrates the cycle of the ecological shifts the oralmicrobiome may experience and the contributing factorsto these shifts, which eventually cause disease.

Poor oral hygiene is greatly responsible for theaccumulation of bacteria within biofilms. Failure todetach accumulating plaque will lead to overgrowthof bacteria that may become pathogenic, reducebiodiversity of the oral cavity, and ultimately cause

Figure 3 The inner circle presents the bacterial genera found in all 10individuals; the second circle, bacteria present in 69 individuals; thethird circle, bacteria present in 35 individuals; and the outer circle,bacteria present in 12 individuals. Adapted by permission fromMacmillan Publishers Ltd: The ISME Journal (Bik et al, 2010)

2Virulence factors are properties that bacteria acquire in becoming

pathogenic, such as bacterial toxins and surface proteins that areprotective to the pathogen but destructive to other bacteria and thehost.

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diseases such as dental caries or periodontal disease(Zaura et al, 2009). Anaerobic microflora in the cryptsof the tongue can also grow out of proportion anddevelop halitosis, or consistent bad breath (Zaura et al,2009). Proper oral hygiene practice is crucial because itis the only voluntary way to prevent oral disease.

The presence of an immune system disorder can also

cause an ecological shift in the microbiome. As theimmune system regulates interactions between themicrobiome and the host, a compromised immunesystem usually disrupts mutual or commensal relation-ships (Badger et al, 2011). Although microbial relation-ships during diseased states are parasitic, somepathogens can also facilitate the growth of otherpathogen species. In dental caries, Streptococcus mutansis responsible, in part, for creating the lactic acid richenvironment in which Veillonella species thrive (Kanasiet al, 2010). In biofilm research, Veillonella species havealso been found to enhance the growth of S. mutans(Klutymans et al, 1997). Moreover, compromised im-mune systems may inhibit the proper flow of saliva or

decrease the amount of nutrients present in saliva,allowing a buildup of dental plaque. For instance,Sjo grens syndrome is an autoimmune deficiency thatattacks the exocrine glands and inhibits the flow of anysaliva through the oral cavity, leading to dry mouth andfurther dental complications (Taubert et al, 2007).

Although usually not obvious, genetic factors can beresponsible for ecological shifts that lead to disease.First, genetic factors could contribute to oral disease inan indirect manner. An individual may have a specificgenetic makeup that encodes for a permanent immunesystem disorder, which may then affect the microbiome.

For example, a person with Crohns disease, an auto-immune disease of the gastrointestinal tract, has adecreased abundance of Bacteroidetes in the intestines(Badger et al, 2011). A similar situation in the oralcavity could result in a reduction of biodiversity andpotentially lead to disease.

Also, because an individuals genotype contributes to

the makeup of its unique microbiome (Turnbaugh et al,2007), ones genetic makeup could directly eitherprevent the existence of certain beneficial bacteria inthe body or produce a bodily environment in whichcertain pathogenic species can reside. For example, 20%of people are long-term carriers of Staphylococcusaureus. These people are more prone to staph infections,especially if the bacteria are not controlled. In addition,certain individuals may lack genes that encode forspecific protective proteins and antibodies in saliva and,thus, be more prone to plaque accumulation or cavities.

Once a pathogen possesses virulence factors, exists inabnormal proportions, and demonstrates parasitism, allof the following conditions which are required for

disease have been satisfied: (1) The local environment isone in which the species can express its virulenceproperties; (2) the pathogen is in numbers that exceedthe threshold for that host; (3) other bacterial speciescan foster, or at least not inhibit, the diseases manifes-tation; and (4) the host is susceptible to this pathogen,i.e., currently compromised immune system or specificgenetic composition (Socransky and Haffajee, 1992).Overall, it is crucial that there be an ecological balanceamong microorganisms to prevent pathological changesand disease from occurring. A healthy microbiome canonly be maintained with good oral hygiene and a well-functioning immune system (Nieuw Amerongen and

Veeman, 2002).

The oral microbiome and the etiology of majororal diseases

Oral diseases such as dental caries and periodontaldisease are among the most prevalent diseases world-wide (Horz and Conrads, 2007; Selwitz et al, 2007),affecting nearly all ages and geographic populations.Hence, discovering the etiological factors responsible fordisease activation and progression will make wayfor advanced methods of treatment and prevention.

Dental caries

Dental caries, also recognized as tooth decay and theprimary cause of oral pain and tooth loss, is a diseasethat can begin as minor surface changes and persist untilthere are lesions in the dentin (Selwitz et al, 2007). Assupragingival biofilm matures on teeth, acid-producingmicrobial colonies accumulate in dental plaque andlower the pH of the oral cavity, creating an environmentin which they can thrive and produce more plaque(Selwitz et al, 2007; Ling et al, 2010). These opportu-nistic pathogens cause dietary carbohydrates to ferment,producing acidic byproducts that destroy eitherthe enamel of the crown or the root of the tooth. Thelow-pH environment facilitates the diffusion of calcium,

Figure 4 Cycle of ecological shifts in the oral microbiome that causedisease. Poor oral hygiene, immunological disorders, and certaingenetic compositions are major factors that contribute to the start ofthis cycle. One condition may lead microbes to either grow abnormalor acquire virulence factors, and in turn, activate the rest of the cycleand eventually disease

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phosphate, and carbonate out of teeth, which usuallyprotect the enamel from these pathogens. Although aspecific microbiome that signals dental caries is yet to befound (Ling et al, 2010), the most common bacteriaresponsible for dental caries are S. mutans, Streptococ-cus sobrinus, and Lactobacillus acidophilus (Streckfusand Bigler, 2002; Selwitz et al, 2007). Dental caries is the

most preventable and reversible childhood disease,which can be avoided with proper oral hygiene, diet,and fluoride exposure, which enables mineral resorptionback into the teeth (Streckfus and Bigler, 2002; Selwitzet al, 2007).

Periodontal diseasePeriodontal disease also results from subgingival plaqueaccumulation that causes shifts in the microflora from ahealthy state to a diseased state (Horz and Conrads,2007; Filoche et al, 2010). Periodontal disease is apolymicrobial inflammatory disorder of the periodon-tium (Pihlstrom et al, 2005). Gingivitis is the mildestform of periodontal disease (Horz and Conrads, 2007).

Microorganisms within biofilms begin to form patho-genic characteristics that aggravate and inflame thegingiva when disturbed by actions such as flossing(Pihlstrom et al, 2005). Fortunately, gingivitis is easilyreversible with good oral hygiene (Horz and Conrads,2007).

Periodontitis, on the other hand, is a severe, irrevers-ible infection that attacks all soft tissue and bone thatsupport the periodontium and teeth structures (Horzand Conrads, 2007). Like dental caries, multiple oppor-tunistic pathogens overgrow in dental plaque and theseabnormal proportions become pathogenic (Horz andConrads, 2007; Van Essche et al, 2011). Microbes

release proteolytic enzymes that break down host tissueand may result in gingival inflammation, loss of gingivalattachment, periodontal pocket formation, and alveolarbone and teeth destruction. The predominant pathogensinvolved in periodontitis are Aggregatibacter actinomy-cetemcomitans, Porphyromonas gingivalis, Prevotellaintermedia, Fusobacterium nucleatum, Tannerella for-sythia, and Eikenella corredens, and Treponema denticola(Filoche et al, 2010; Dashiff and Kadouri, 2011).

Periodontitis is extremely difficult to treat because ofthe nature of the disease, the complications of antimi-crobial therapy, and the lack of information on themicrobial interactions occurring during the disease.Once pockets form in the periodontium, periodontitis

officially becomes irreversible (Pihlstrom et al, 2005).For one, the periodontium is unable to reattach to boneonce separated. Also, the causative pathogens deepwithin the pockets become nearly impossible to targetwith antimicrobial solutions (Horz and Conrads, 2007).Furthermore, periodontal pathogens develop virulentfactors, like encapsulation, that make them resistant toantibiotics (Horz and Conrads, 2007; Van Essche et al,2011). Pathogens hiding within plaque are one thousandtimes more resistant to antimicrobials than those whichare more exposed (Van Essche et al, 2011). Even ifpathogens are successfully targeted, there are highchances of recolonization at treated sites because of

bacterial reserves in the mucous membranes that line theoral cavity (Horz and Conrads, 2007). Today, the mostadequate treatment for periodontitis is simply reducingthe number of pathogens present with antibiotics tomaintain control of the disease. As antibiotics destroyan array of communities, they cannot be distributedloosely. The oral cavity should maintain certain Gram-

positive bacteria that shield pathogens from damaginghard and soft tissues (Van Essche et al, 2011).

Oral cancerA third oral disease that deserves substantial attention isoral cancer. Oral cancer is the sixth most prevalentcancer, affecting over 300 000 people each year aroundthe world (Gill, 2011). There may be a correlationbetween the structure and function of the oral microb-iome and oral cancer (Meurman, 2010; Gill, 2011). TheGill Lab at the University of Rochester Medical Centerhas shown that bacterial cells of oral tumors can impactsignal pathways that initiate and advance oral cancer(Gill, 2011); however, the sources of activation of

cancerous and precancerous oral lesions have yet to beidentified (Mehrotra and Yadav, 2006). Researchersshould take full advantage of the ability to easily accessthe oral cavity and examine its microbiome and otherprecancerous characteristics for the purpose of earlydiagnosis, effective treatments and better chances ofsurvival (Mehrotra and Yadav, 2006).

Most patients infected with oral cancer practice poororal hygiene (Meurman, 2010). In general, numerousstudies conducted around the world have shown poororal health and tooth loss to increase the risk ofgastric, pancreatic, and other cancers. Inflammation isusually the first symptom of compromised oral health

and it gets worse as health regresses. Approximately1520% of human tumors contain pathogenic agentsderived from inflammatory infections. Proper oralhygiene will maintain control of such inflammatoryagents that may contribute to oral cancer (Meurman,2010).

Conversely, cancer can lead to poor oral health.Carcinogens can introduce toxic agents into salivaryfluid that damage DNA, cause mutations, and damagethe integrity of oral cavity (Meurman, 2010). The oralcavity reacts to the toxins with an inflammatoryresponse, which then produces the pathogenic agentsthat contribute to tumor development, thus maintaininga vicious carcinogenic cycle.

Despite the limited research available about therelationship between oral cancer and the oral microb-iome, it is well known that the two most important riskfactors for oral cancer are tobacco and alcohol(Johnson, 2001). Certain pathogenic strains of oralmicroorganisms tend to increase carcinogenic acetalde-hyde concentrations in saliva when metabolizing ethanoland tobacco smoke (Meurman, 2010; Yang et al, 2011).However, not all who drink alcohol or smoke are subjectto oral cancer. Those individuals are at higher risk. Also,each microbiome differs in the rate at which it metab-olizes the ethanol and tobacco compounds (Meurman,2010).

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Relationships among oral disease, the oralmicrobiome, and systemic diseases

The oral cavity is the primary gateway to the humanbody; therefore, microorganisms that inhabit that areaare very capable of spreading to different body sites(Dewhirst et al, 2010). Pathogens that originate in the

oral cavity can be frequently detected in blood cultures asthey destroy and pass through oral mucous membranesand periodontal pockets (Horz and Conrads, 2007). Thissuggests a mechanism by which pathogens derived froma periodontal inflammatory response make their way totumors in the gut or pancreas. Pathogens may enter theblood stream, alter proper immune responses, or pro-duce excessive and deregulated amounts of inflammatorymediators, and in turn, cause disease at different bodysites (Williams et al, 2008). Figure 5 illustrates a sum-mary of the pathways that periodontal pathogens maytake to a systemic target organ. Persistent inflammationand frequent bacterial attacks not only lead to bacter-emia, but also organ abscesses and severe systemic

diseases, such as diabetes and cardiovascular disease(Horz and Conrads, 2007; Williams et al, 2008; Meur-man, 2010). This correlation further supports theimportance of the oral microbiome to overall health.

DiabetesDiabetes and periodontal disease hold a strong bidirec-tional relationship (Pihlstrom et al, 2005; Kuo et al,2008; Williams et al, 2008). In one direction, the bacteriainvolved in periodontal disease jeopardize the bodyscontrol of glycemic levels (Kuo et al, 2008). Porphyro-monas gingivalis, a chief agent in periodontal disease,produces a lipopolysaccharide (LPS) that is toxic to

certain cytokine proteins that regulate insulin activityunder normal conditions. Other bacterial infections canalso decrease the ability of skeletal muscles to uptakeinsulin-mediated glucose. This can produce whole bodyinsulin resistance (Kuo et al, 2008). Fortunately, peri-odontal treatments can benefit patients with diabetes by

inhibiting pathogen secretions of LPS and improving thebodys glycemic control (Pihlstrom et al, 2005).

Poorly controlled diabetes increases the risk ofperiodontal disease activation and severity, and the rateof periodontal bone loss (Pihlstrom et al, 2005; Preshaw,2009; Filoche et al, 2010) A hyperglycemic conditioncan lead to a chronic inflammatory-immune response

that produces an excessive and deregulated amount ofinflammatory mediators, such as cytokines and otherenzymes (Pihlstrom et al, 2005). The excess inflamma-tory mediators make their way into the periodontium,casing periodontal detachment, pocket formation, andeven alveolar bone destruction (Preshaw, 2009). Diabe-tes can also result in other oral complications such asburning mouth syndrome, fungal infections, dentalcaries, and salivary functional disorders (Kuo et al,2008).

Cardiovascular diseasePeriodontal pathogens signal excessive amounts ofantigens, endotoxins, cytokines, and C-reactive proteins

that also contribute to cardiovascular complicationssuch as lipid deposition, smooth muscle proliferation,and platelet aggregation (Kuo et al, 2008). Pathogenslike P. gingivalis and Streptococcus sanguis have abilitiesto induce platelet aggregation and accumulate as arterialplaque (Williams et al, 2008). Aggregatibacter actino-mycetemcomitans in the periodontal pockets has alsobeen discovered in atherosclerotic plaque (Bahekar et al,2007). The organism accesses the circulatory systemthrough oral tissue and makes its way to the arterieswhere it secretes LPS and inflammatory-response medi-ators, resulting in atherothrombogenesis. As in diabetes,periodontal treatments may also alleviate cardiovascular

diseases (Tonetti et al, 2007; Kuo et al, 2008). The exactpathway from cardiovascular disease to periodontaldisease has yet to be established.

Disease manifestations in the oral cavityAnalysis of the oral cavity and its microbiome maybecome a means to diagnose systemic diseases that tendto manifest in the periodontium (Pihlstrom et al, 2005).Herpetic infections, leukemia, tuberculosis, and evendermatological diseases are some examples of diseasesthat present major gingival swelling, oral lesions, andgingival discoloration because of cellular infiltration(Pihlstrom et al, 2005). These diseases may also invis-ibly manifest in the oral cavity before any symptoms

become apparent in the body. For instance, respiratorypathogens may colonize the mouths of individuals witha high risk of pneumonia even when respiratorysymptoms are absent (Scannapieco, 1998). The oralbiofilms serve as a reservoir for these pathogens andcontribute to the diseases progression. In fact, there isalso an altered oral microflora in individuals with HIV,as well as individuals who are pregnant, lactating, ortaking antibiotics. Although not all of these conditionsare diseases, this evidence suggests that homeostaticalterations in the body manifest in the oral microbiome.Detecting bacteria related to oral or systemic disease atearly or asymptomatic stages may increase the chances

Figure 5 Periodonto pathogen pathways from the oral cavity tosystemic organs. Adapted from (Scannapieco, 2004)

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of rapid disease reversal and alert the patient to practicepreventative measures.

Treatment and prevention methods

To maintain oral and systematic health, it is vital toprotect the periodontium from pathogens that cause

inflammatory infections. The foundation of periodontaltherapy is anti-infective, non-surgical treatments aimedto control biofilms and the proliferation of pathogenicbacteria within the oral cavity (Pihlstrom et al, 2005).First and foremost, practicing good oral hygiene is theprincipal preventative measure of oral diseases. Dentalprofessionals may perform scaling and root planning toremove plaque on tooth surfaces or infected tissue of theperiodontal pockets. These procedures, combined withpersistent oral hygiene practices, can reduce tissueinflammation, pocket depths, and improve periodontalattachment (Pihlstrom et al, 2005).

Antibiotics

When manual treatments are supplemented with localand systemic antibiotics, the oral cavity experiences achange in composition and abundance of variousbacteria. (Pihlstrom et al, 2005). Local antibiotics killor freeze an array of species at diseased sites in the oralcavity, as well as heal oral lesions and halt plaqueaccumulation (Horz and Conrads, 2007). Systemicdrugs target pathogens at sites around the body inaddition to the oral cavity but are limited to the speciesthey target. They can also reduce any bleeding in theperiodontium. However, systemic drugs are convention-ally used as a last resort in the treatment of periodontaldiseases for cost-effective purposes (Flemmig and Bei-

kler, 2011).There can be variation in the pathogen and hostresponse to different drugs. For example, growth ofA. actinomycetemcomitans is inhibited by tetracyclinesbut unaffected by clindamycin (Horz and Conrads,2007). Moreover, an individuals intestinal microbiotaaffects the metabolism of drugs and toxins. As thehuman microbiome is unique to the individual, oralvaccines maybe processed differently in each body,depending on both oral and gut microbial communities(Ferreira et al, 2010).

Effective use of antibiotics in the future requiresgenomic analysis of the patients oral microbiome torecognize the microbes that are present and to determine

whether they will respond to specific treatments. There-fore, the oral microbiome will likely play a central rolein the development and advancement of personalizedmedicine.

Probiotics and prebioticsWhile antibiotics are synthetic drugs that harm themicroflora, probiotics are live microbes that are part ofthe natural microflora. The utilization of antibioticsimplies that disease is already in progress. Instead offighting to cure disease, medicine today should focus onhow to maintain health and prevent disease. Probiotictherapy or bacteriotherapy (Rajendhran and Gunasek-

aran, 2010) has the potential to naturally cure andprevent disease at its early stages by incorporatingbeneficial bacteria that can reestablish an ecologicalbalance or enhance the biodiversity of a microflora. Forexample, research showed that individuals with highamounts of Capnocytophaga ochracea had loweramounts of P. gingivalis and displayed no periodontal

disease progression. Individuals with low C. ochraceadid exhibit disease progression. In probiotic treatment, apatient found to have elevated levels of P. gingivaliscould be given C. ochracea probiotics to reestablish ahealthy equilibrium before any periodontal diseasegenerates. Prebiotics may also be used for similarpurposes. Prebiotics are oligosaccharides, or complexsugars, that aim to stimulate the growth of beneficialbacteria in the host (Badger et al, 2011). Both probioticsand prebiotics would strengthen the beneficial microfl-ora so the body can naturally fight off disease-causingagents.

However, a clear definition of health, including thecomposition and interrelationships of the healthy

microbiome, is necessary before any probiotics can bedeveloped or used properly. In addition to the ambigu-ity of health, the probiotic industry faces challengesthat have prevented their market appearance (Kleinet al, 2010). First, the effects of probiotic organisms onthe host and the mechanism by which they exert theseeffects are still uncertain (Sonnenburg and Fischbach,2011). Blind delivery of probiotics is dangerous becausethere is yet to be any true evidence of how they influencein vivo physiology and functionality. Second, theprobiotic industry bears production parameters it mustovercome to be able to create safe and reliable probioticsubstances (Klein et al, 2010). The production process

would have to consider factors such as the technologyused, temperature, and fermentations conditions, oxy-gen content, organic ingredients used, etc. Therapeuticantibiotics and probiotics face limitations because of alack of knowledge of the oral microbiome and itsnumerous, complex constituents.

Pathogen predatorsAside from mainstream antibiotics and emerging pro-biotic approaches, an alternative treatment strategyinvolving biological antimicrobial agents may establisha groundbreaking approach to curing and preventingdisease. Research has discovered bacterial lineages ofBdellovibrio, Bacteriovorax, and Peredibacter, called

Bdellovibrio-and-like organisms or BALOs, that serveas predators to kill anaerobic, Gram-negative bacteria(Dashiff and Kadouri, 2011; Van Essche et al, 2011).Because most periodontal pathogens are anaerobic,Gram-negative bacteria, these BALOs may play asignificant role in the treatment of periodontal infections(Horz and Conrads, 2007; Van Essche et al, 2011).These highly motile species are abundant in aquaticenvironments but cannot be isolated in large amounts(Van Essche et al, 2011). As BALOs do not prey onGram-positive bacteria, and most beneficial bacteria inthe oral cavity are Gram-positive, BALOs may even bepreferred over antibiotics when curing oral diseases.

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While antibiotics are non-specific and destroy an arrayof microbial communities, BALOs will only kill thebad microbiome and sustain the good microbiome(Dashiff and Kadouri, 2011; Van Essche et al, 2011).Furthermore, antibiotics cannot target pathogens deepwithin layers of biofilm. BALOs, on the other hand, caninfiltrate and attack surface-attached bacteria (Dashiff

and Kadouri, 2011). They can also assist antibiotics bydetaching biofilm surfaces and exposing the presentpathogens (Dashiff and Kadouri, 2011). Mixed micro-biota environments do not inhibit BALOs predationefficiencies (Van Essche et al, 2011) and neither doessaliva nor high temperatures (Dashiff and Kadouri,2011); however, predator-prey interactions are highlyBALO strain specific, which underscores the importanceof identifying which pathogens are present for effectivetherapy (Van Essche et al, 2011).

The utilization of BALOs seems to be a groundbreak-ing approach to oral disease therapy, as these biologicalantimicrobial agents possess the benefits of both anti-biotics and probiotics. Unfortunately, several parame-

ters exist that prevent the use of BALOs in clinicalsettings. First, the tests that have been performed toanalyze BALOs behavior have only taken place inexperimental settings. The use of BALOs as therapeuticsrequires a better understanding of how these predatorsfunction in the physiological setting (Van Essche et al,2011). Second, high inoculum concentrations of BALOsare more efficient in killing a substantial amount ofpathogens (Van Essche et al, 2011); yet, BALOs are notextracted from the environment in plentiful amounts.Finally, BALOs are unable to function under anaerobicconditions. This limitation means that BALOs wouldnot be able to prey on the anaerobic bacteria that reside

deep within periodontal pockets formed in periodontitis(Dashiff and Kadouri, 2011).

Personalized dental medicine

Because the microbiome is the biomarker of diseaseactivity, further research and advancements in microbi-omics and metagenomics are essential to understandingthe microbiology and etiology of oral diseases. Genomescollected through metagenomic techniques will not only

be used for the analysis of microorganisms, but also inthe engineering of therapeutic agents needed to manip-ulate the microbiome according to personal needs (Parkand Kim, 2008). Understanding changes in the oralmicrobiome at the early stages of chronic oral diseaseswould allow clinicians to diagnose and treat anunhealthy oral cavity before the appearance of any

dental lesions or periodontal pockets (Zaura et al,2009). Additionally, the use of probiotics or otherbiological antimicrobial agents at early stages of diseasecould naturally restore microbial equilibrium and, thus,minimize the need for antibiotics.

The diagram in Figure 6 represents the course ofchronic oral disease over time, the clinical tools thatshould be used to track the disease burden, and thesuggested use of probiotics and antibiotics to slow orstop the disease process. For example, if specificpathogens are recognized following screening methods,probiotics could be administered locally, according tothe amount and type of those pathogens present.However, the role that each microorganism plays in

disease progression or regression must be accurate andwell understood for safe and effective manipulation ofa microbiome (Zaura et al, 2009). In addition, usingsuch clinical detection methods would require thedevelopment of novel technologies, especially forperforming on-the-spot tests. Personalized dental med-icine that focuses on the oral microbiome will haveextensive effects in health care, considering the oralmicrobiomes importance to both oral and systemichealth.

Conclusion

Although invisible to the naked eye, the microbiomeshould not be underestimated as a key determinant ofhealth and disease. The oral microbial ecosystem isparticularly vital to maintaining both oral and overallhealth in the body. Salivary flow and biofilms on theteeth and soft tissue maintain microbial equilibriumwithin the oral cavity and protect pathogens frommanifesting. Disturbing the homeostasis of the oralcavity can stir pathogen activity and lead to oral disease.Because the oral cavity is the primary gateway to the

Figure 6 Diagram of the course of chronicdisease over time (depicted by the red line)and the clinical points of intervention thatmight reduce disease burden, along with thepotential uses of probiotics (blue arrows).Targeted antibiotic therapy (red arrow) mightreduce disease burden and activity (dashedred line). Adapted from (Ginsburg andWillard, 2009)

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body, severe cases of oral disease may result in thespread of infection to other body sites, producingsystemic diseases such as cardiovascular disease orexacerbating an already compromised immune system,as in diabetes. Practicing good oral hygiene and main-taining stable oral biofilms is essential to keeping a bodyhealthy and also preventing rapid spread of disease to

other individuals.Microbiomics and metagenomics must collaborate tofully elucidate the nature of the microbiome during bothhealth and disease, which will, subsequently, pave theway for more effective therapeutic and diagnostictechniques. Ultimately, the analysis of the humanmicrobiome will significantly contribute to the develop-ment of personalized medicine and personalized dentalmedicine.

Acknowledgements

We want to thank Marla VacekBroadfoot for editorialassistance in preparation of the manuscript. This work was

supported by the Center for Genomic Medicine in the DukeInstitute for Genome Sciences and Policy.

Author contributions

Zarco researched the topic, wrote the initial draft, and editedthe final draft. Vess assisted Zarco in the research, and editedearly and late drafts. Ginsburg initiated the idea, guided Zarcoand Vess in their research and writing, and edited andapproved the final manuscript.

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