microbiology past, present & future

Oral Microbiology: Past, Present and Future

Xuesong He1, Xuedong Zhou2, and Wenyuan Shi1,*1UCLA School of Dentistry, Los Angeles, California 90095, USA2West China College of Stomatology Sichuan University, 14 South Road of Renmin 610041,Chengdu City, Peoples Republic of China

AbstractSince the initial observations of oral bacteria within dental plaque by van Leeuwenhoek using hisprimitive microscopes in 1680, an event that is generally recognized as the advent of oralmicrobiological investigation, oral microbiology has gone through phases of reductionism andholism. From the small beginnings of the Miller and Black period, in which microbiologistsfollowed Kochs postulates, took the reductionist approach to try to study the complex oral microbialcommunity by analyzing individual species; to the modern era when oral researchers embraceholism or system thinking, adopt new concepts such as interspecies interaction, microbialcommunity, biofilms, poly-microbial diseases, oral microbiological knowledge has burgeoned andour ability to identify the resident organisms in dental plaque and decipher the interactions betweenkey components has rapidly increased, such knowledge has greatly changed our view of the oralmicrobial flora, provided invaluable insight into the etiology of dental and periodontal diseases,opened the door to new approaches and techniques for developing new therapeutic and preventivetools for combating oral poly-microbial diseases.

IntroductionLike many other biological sciences, the study of microbiology has gone through phases ofreductionism and holism. For a long time, microbiologists took the reductionist approachto study complex microbial communities by analyzing individual bacterial species. Thestrategy was to understand the whole by examining smaller components, and it has been thehallmark of much of the industrial and scientific revolutions for the past 150 years. Whilereductionism has greatly advanced microbiology, it was recognized that assembly of smallerpieces cannot explain the whole! Modern microbiologists are learning system thinking andholism. From global gene regulation to metagenomics to biofilms, microbiology isentering an exciting new era with emphasis on revealing and decoding the interactions ofdifferent elements within a microbial community. The knowledge obtained from systemthinking is changing our understanding of microbial physiology and our ability to diagnose/treat microbial infections, and will have great impact on oral microbiology as well.

Commonly known as dental plaque, oral microbial communities is one of the most complexbacterial floras associated with human body. So far, more than 700 different bacterial specieshave been identified from human oral cavity, and the majority of them are associated withdental plaque (1,64,65). Extensive animal and clinical studies have indicated that the oralmicrobial flora is responsible for two major human oral diseases: dental caries (tooth decay)and periodontitis (gum disease) (13,48,52,62,74). For a long time, oral microbiologistsfollowed guideline of Koch's postulates, and used reductionism approach to try to identify the

*To whom correspondence should be addressed. [email protected]; Tel: 310-825-8356.

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Published in final edited form as:Int J Oral Sci. 2009 June ; 1(2): 4758.

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key pathogens responsible for oral microbial pathogenesis (16,26,72). However the limitationof reductionism forced microbiologists to embrace new concepts such as interspeciesinteraction, microbial community, biofilms, poly-microbial diseases, etc. These new researchdirections have revealed many new physiological functions critical for the pathogenesis, whichresult from interactions between different components within oral microbial flora, and mightnot be observed with individual organisms. The new system thinking, empowered with themodern techniques for analyzing complex microbial community serves as a new foundationfor studying oral microbial community, providing invaluable insight into the etiology of dentaland periodontal diseases, developing new therapeutic and preventive tools for combating oralpoly-microbial diseases.

Oral Microbiology at Early StageThe initial discovery of oral microbes

Although minute and primordial, bacteria are incredibly versatile and diversified. Judging bytheir numbers and biomass, they are arguably the most successful living organisms on earth.They can tolerate environmental extremes and colonize almost every habitat on earth, includinghuman oral cavity. However, due to their minuteness, their existence was not revealed untilfairly recent times, around 1680, when Antony van Leeuwenhoek (16321723) (22), a Dutchdry goods merchant, observed and described first microorganisms in the tartar from his teethwith his primitive microscope. In his notebook, he recorded I didnt clean my teeth for threedays and then took the material that has lodged in small amounts on the gums above my frontteeth I found a few living animalcules. The microbes sketched in his notebook are nowknown as some of the most abundant bacteria resided within oral cavity, including cocci,spirochetes, and fusiform bacteria. These fascinating observations at the birth of microbiologyhad already signaled the complexity of the oral microbial community.

Revealing the Infectious Nature of Dental Caries and PeriodontitisLike many other sub-disciplines of microbiology, the early stage of development in oralmicrobiology was largely driven by medical needs. As two of the most prevalent human oraldiseases, dental caries and periodontitis have always been the focus of oral microbiologyresearch.

Dental caries is a human oral disease with a long history. Archaeological evidence shows thattooth decay is an ancient disease dating far back into prehistory (79). Interestingly, studies byMoore and Corbett of the dentition of Britons in various periods demonstrated that, until about1850, caries occurred relatively infrequently; after 1850, coinciding with the increasingavailability of cane sugar and refined flour, there has been an explosive increase in dental carieslesions (61). Even from ancient time, long before the visual observation of microorganism,people had suspected the possible causative link between certain form of living organism--described as tooth worm in a Sumerian text about 5000 BC, and dental caries (79). However,the revelation of the true identity of tooth worms had to wait for another 6000 years.

W. D. Miller was arguably one of the most important individuals who greatly advanced oralmicrobiology. As a practicing dentist with a thorough training in natural sciences, he spent hisevenings and weekends at Robert Kochs laboratory for the purpose of identifying the germsthat were responsible for tooth decay. In his 1890 book titled Microorganisms of the HumanMouth (55), he proposed a chemoparasitic theory which suggested that, in susceptible hostswho frequently consumed fermentable carbohydrates, oral microorganisms would convertthese carbohydrates into acid, which would result in the demineralization of teeth. Miller'schemoparasitic theory, together with the description of "gelatinous microbic plaques" now

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commonly known as dental plaque, by G. V. Black (2) and J. L. Williams (84), provided thekey elements for our modern concept of the etiology of dental caries.

Due to the limited bacterial isolation and cultivation technique available in 19th century, W.D. Miller was unable to identify the causative agent(s) of dental caries. In 1924, J. K. Clarkefirst isolated a bacterial species from human dental caries site, it was named Streptococcusmutans, and was shown to be capable of fermenting several sugars and producing a pH of 4.2in glucose broth (7). But unfortunately, Clarke was unable to demonstrate that this organismactually caused caries.

Since 1950, there has been an increasing interest in using experimental animal to betterunderstand the nature and etiology of oral disease, including dental caries. Many relativelysophisticated animal models had been developed which has greatly facilitated oralmicrobiology studies (31,59). In 1960, two important paper were published which wereconsidered the cornerstone of dental caries research. In the first paper, using hamster as animalmodel, P. H. Keyes revealed the infectious and transmissible nature of dental caries (32). Thenin another elegant paper (20), R. J. Fitzgerald and P.H. Keyes successfully demonstrated cariesinduction in an animal harboring a conventional microflora, by a single type ofstreptococcus--the same bacterial species that had been isolated by J. K. Clarke more than 30years ago.

Other than dental caries, another major human oral disease is periodontitis, it is widely regardedas the second most common disease worldwide. The involvement of bacteria in thedevelopment of periodontal disease was suggested by the fact that administration of penicillininhibited periodontitis in hamster (56); while the infectious nature of this disease wassubstantiated by demonstration of its transmissibility (33). Early experimental animal studyalso identified several oral isolates, such as Actinomyces species, which could potentially playan important role in eliciting the disease (30).

Establishing Dental Plaque as the Cause of Dental Caries and PeriodontitisOne of the early achievements in oral microbiology is to link dental plaques to dental andperiodontal diseases. As mentioned earlier, dental plaque was one of the first substancesAntony van Leeuwenhoek examined under his microscope (22). His recording of the livingmicro-organisms within the plaque gave the first hint to the complex nature of dental plaquein term of its diversified microbial inhabitants. Later on, both Erdi and Ficinus described thepresence of microorganisms within the membrane on teeth (79). However, the fullimplications of dental plaque were not realized until the publication of Blacks 1898 paper, inwhich he referred dental plaque as gelatinous microbic plaques, a gelatin-like substance thatcarried microorganisms (2). Based on his clinical experience and experimentation, he believedthat the cause of dental caries was due to the attack from acids generated by bacteria withinthese plaques. Blacks work, together with Millers chemoparasitic theory established theimportant role of dental plaque in the etiology of dental caries and has become one of theessential paradigms of oral biology. And the progress in dental plaque study has become amajor indicator of the development of oral microbiology.

Oral microbiology---PresentGenetic Studies of Bacterial Isolates from Dental Plaque

Since the establishment of microbes within dental plaque as the cause of dental and periodontaldiseases, oral microbiologists followed the guideline of Kochs postulates and tried to isolatespecific microorganisms that could be the causative agents and responsible for these disease.However, like in other microbiology fields, one of the challenges faced by early researchersin oral microbiology was the isolation and cultivation of pure bacterial culture. By 1960s,

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anaerobic bacteria had been recognized as the predominant microbial component of the gastro-intestinal tract (including oral cavity) associated flora of human and mammals (67). Duringthe process of bacterial cultivation, researchers realized that the majority of these anaerobicbacteria cannot be cultivated aerobically, or with the conventional anaerobic cultivationtechnique, such as, anaerobic culture tube introduced by Laidlaw (44), the vacuum jar designedby Noguchi (63), and the anaerobic containerthe McIntosh bomb developed by McIntoshand Fildes (53). The introduction of anaerobic glove boxes--a primitive version of now widelyused anaerobic chamber, by Socransky (76) and Rosebury et al (68) in 1960s greatly facilitatedthe isolation and cultivation of anaerobes, particularly those obligate (strict) anaerobicmicroorganisms, from human oral cavity.

With improved anaerobic cultivation technique and availability of newly developed complexmedia for bacterial cultivation, pure cultures of more than 300 different oral bacteria specieshave been isolated from oral cavity in the past 40 years, including bacterial species harboredin both supra-gingival and sub-gingival dental plaque samples taken from healthy and diseasedsites (37). The isolation and cultivation allowed detailed phenotypic and genetic study on thoseclinically important bacteria, such as Streptococcus, Actinobacillus, Actinomyces,Porphyromonas and Treponema species (37). The combination of classic genetic approaches,such as isolation and characterization of mutants, with molecular genetic techniques, includingrecombinant DNA methodology developed in the late 1970s and genetic mapping (8), has beenused to genetically manipulate and characterize those microorganisms important in dentalcaries and periodontal diseases. The genetic studies, together with the additional bio-informaticinformation obtained from whole genomic sequences of many oral bacteria (88) allowmicrobiologists to dissect the development and function of oral microbial community at themolecular level. Progress has been made in determining the molecular genetic basis of biofilmformation of oral isolates using data from in vitro studies on either single, dual species or multi-species systems. Studies have shown that in certain virulent organisms many different typesof genes encoding a variety of functions are required for biofilm development and successfulestablishment within the dental plaque, including genes encoding virulence factors, generalstress response pathways, quorum-sensing systems, two-component systems sensingenvironmental cues, and those encoding surface adhesions involved in cellcell or cell-to-genesencoding surface interactions (14,36,37,43).

Getting a Closer Look at Dental PlaqueThe dental plaque has long been linked to the etiology of dental and periodontal diseases. Theconcept proposed by Black more than 100 years ago that caries was due wholly to attack fromacids produced by bacteria within dental plaque has stimulated considerable scientificinvestigation on dental plaque. In the past few decades, there has been a tremendous amountof work and numerous exciting discoveries made during these investigations, ranging fromdental plaques microbial composition, structure to its formation process, from its physiologyproperties to different kinds and levels of bacterial interactions within these densely populatedmicrobial community. The virtual explosion of our knowledge regarding dental plaque has hada profound impact on our understanding of the etiology of dental and periodontal diseases(14,3640,43).

Before 1960s, although micro-organisms within dental plaque were held responsible for theetiology of dental and periodontal disease, it was generally believed that diseases were theresult of an increased mass of bacteria rather than qualitative differences in the compositionof the microbiota. This was largely due to the lack of suitable cultivation techniques and thegaps in the knowledge of the taxonomy of the oral microbial flora. This conventional wisdomwas challenged by M. A. Listgaten (46). By analyzing the dental plaque associated withperiodontally healthy and diseased teeth using electron microscopy, he observed that distinct

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qualitative differences exist between supra- and sub-gingival, healthy and disease associatedplaque (46). These original microscopic observations were later confirmed by improvedculture-dependent and non-culture dependent analysis.

In 1978, Dr. J. W. Costerton invented the word biofilm, referring to the matrix-enclosedbacterial community (10). Studies have shown that bacteria within biofilms usually displaydifferent phenotypes compared with their counterparts. They are more resilient and moreresistant to external insults. Within biofilm, there are extensive metabolites exchanges,signaling trafficking and different levels of interactions among different species (9,11,12). Theintroduction of biofilm theory into oral microbiology field provided impetus for researchers totake a closer and thorough look at the dental plaque, the first biofilm described by Antonie vanLeeuwenhoek.

Detailed analysis of a microbial community requires the full knowledge of its endogenousinhabitants. However, for a long time, our understanding of the microbial world has beenhampered by the intrinsic limitation of the conventional culture-dependent methods. Accordingto Staley and Konopka, fewer than 1% of the organisms are able to grow under laboratoryconditions, suggesting that our views of the complexity and genetic diversity of microbialcommunities based on cultivation strategies are severely biased (78). Fortunately, Woese andcoworkers discovered three decades ago that the conservation as well as the variation withinthe sequences of 16S rRNA-encoding genes allows for the construction of evolutionary treesfor bacteria to the species level (70,85). This finding in combination with the development ofPCR methods in 1985 (70) opened the door for culture-independent analyses and classificationof previously unknown members of many different microbial communities, including theresident flora of oral biofilms. The past two decades has witnessed the development of high-throughput tools for microbial community analysis. Some of these methods directly examinenucleic acids isolated from samples of microbial communities, such as microarrays (73) andcheckerboard DNA-DNA hybridization method (77), while others are PCR based, such asdenaturing gradient gel electrophoresis (DGGE) (57) or temperature gradient gelelectrophoresis (87), terminal restriction fragment length polymorphism (T-RFLP) (47,81),automated ribosomal intergenic spacer analysis (5), and denaturing high-performance liquidchromatography (DHPLC) (15). Most of these approaches were originally developed foranalyzing environmental microbial communities and have been used to study human microbialecology, including oral microbial analysis. These culture-independent techniques haverevealed a whole new microbial world within dental plaque with great genetic diversity. Over700 bacterial species (gram-positive, gram negative bacteria and archaea) have been identifiedform the human oral cavity, majority of them are associated with dental plaque, making theoral microbial community one of the most complex microbial floras in the human body (1,64,65). Based on our current knowledge, supragingival plaque is dominated by gram-positivebacteria, including Streptococcus sanguinis, S. mutans, Streptococcus mitis, Streptococcussalivarius, and lactobacilli, while the subgingival plaque is made up primarily by gram-negative anaerobic bacteria, such as Aggregatibacter (Actinobacillus)actinomycetemcomitans, Tannerella forsythia, Campylobacter spp., Capnocytophoga spp.,Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotellaintermedia, and oral spirochetes such as Treponema denticola. In both cases, the microbialcommunities on teeth and gingival tissues can accumulate high concentrations of bacterialmetabolites (e.g., fatty acid end products, ammonia, hydrogen peroxide, oxidants, and carbondioxide) in their local environments, which influence the bacterial species within the microbialcommunity, as well as the host (6).

To study the formation and architecture of dental plaque, several experimental designs havebeen developed. These include in vitro systems ranging from simple batch culture system (e.g.,the Zurich Biofilm Model) (25), constant depth film fermentor (CDFF) (34), saliva-

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conditioned flow cell (21), to artificial mouths (66,69), as well as in situ device for the invivo generation of intact dental plaque biofilms on natural tooth surface in human subjects(86). Using imaging tools such as the scanning electron microscope (46,80) and, more recently,the confocal laser scanning microscope (60,86), and coupled with variety of stainingtechniques, such as fluorescence in situ hybridization (FISH) (21,83), oral microbiologist nowrealized that dental plaque are not unstructured, homogeneous deposits of cells, but complex,well organized communities of surface-associated cells enclosed in a polymer matrixcontaining open water channels and fluid-filled voids. In vitro and in vivo study on theformation and development of dental plaque revealed that during the formation process, someoral bacteria are early colonizers that express biochemical components allowing them toeffectively adhere to targeted tissues (teeth or periodontal tissue). The later colonizers oftencontain components that enable them to adhere to the early colonizers, often bringing metabolicor other competitive advantages. Within an established dental plaque, specific bacterial speciesare often found located adjacent to each other or mixed together to form unique structures thatoften confer adherence or growth advantages. Previous comprehensive reviews by P. E.Kolenbrander et al should be consulted for assessment of these important properties (36,37,3941).

Community Based Microbial PathogenesisThe improved cultivation technique allows successful isolation and characterization ofmicrobial species from dental plaque, which naturally prompted oral microbiologists to connectspecific bacterial species to certain diseases, according to Koch's postulates. For example, asa result of such strategies, the acid-producing oral bacterium S. mutans present in supra-gingival plaque was selected for its positive correlation with dental caries, animal study alsodemonstrated its ability to induce dental caries. And this has become the base for specificplaque hypothesis proposed by W. J. Loesche, which proposed that only a few specific species,such as S. mutans and Streptococcus sobrinus, are actively involved in the disease (49).However, additional scientific data have suggested that such a simple correlation may be anoversimplification. Unlike many known medical pathogens that are "foreign invaders withspecific virulence factors," the oral "pathogens" such as S. mutans are part of the normal flora(1). While they express certain pathogenic factors (such as acid production in this case), adynamic balance of both synergistic and antagonistic interactions with its neighboring bacteriaplays an essential role in determining whether these pathogenic factors cause damage or not(35,51). As proposed by Marsh in his ecological plaque hypothesis, in the case of complexbiofilms, it is not merely the presence of a single organism in a complex community whichdetermines the properties of a biofilm, but it is the interactions between the biofilm residentswhich is crucial (51). As an example, in the presence of nearby base-producing bacteria, S.mutans in dental plaque may not be as pathogenic to the host. Thus, for dental caries, it is nowgenerally recognized that this disease results not solely because of the presence of S. mutansor any single organism in dental plaque. Rather, it is the result of the interaction of multipleacid-producing organisms such as S. mutans with other biofilm residents (35,51). Such acommunity- and microbial ecology-based pathogenic theory serves as a new concept forunderstanding the relationship between dental plaque and the host in health or disease, as wellas suggesting new strategies for disease treatment and prevention.

Dental plaque is a microbial community with great structural complexity and genetic diversity.Its bacterial composition remains relatively stable despite regular exposure to minorenvironmental perturbations. This stability (microbial homeostasis) is due in part to a dynamicbalance of both synergistic and antagonistic microbial interactions (51,52). This suggests thatthe residents in this community should display extensive interactions while forming biofilmstructures, carrying out physiological functions, and inducing microbial pathogenesis.Extensive in vitro and animal studies have revealed a multitude of bacterial interspecies

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interaction as described in resent reviews. These interactions include (i) competition betweenbacteria for nutrients, (ii) synergistic interactions which may stimulate the growth or survivalof one or more residents, (iii) production of an antagonist by one resident which inhibits thegrowth of another, (iv) neutralization of a virulence factor produced by one organism by anotherresident, and (v) interference in the growth-dependent signaling mechanisms of one organismby another (37,40,41,43,51,52). In a micro-Gaia community, these interactions could beenvisaged as forms of "war and peace" among the bacterial residents of a biofilm.

Multispecies communities like dental plaque can produce polymicrobial infections in whichmicroorganisms interact in a synergistic fashion, leading to pathogenesis. Periodontal diseasesrepresent one of the best-documented polymicrobial infections. Porphyromonas gingivalis,Treponema denticola, and Tannerella forsythia are strongly implicated clinically as apathogenic consortium in the etiology of adult periodontitis (19,75). It is anticipated that abetter understanding of the mechanisms involved in periodontitis may provide opportunitiesto more critically evaluate the role of different virulence factors involved in these mixedinfections. This may provide useful general information for investigators in the developmentof novel diagnostic, preventive, and treatment strategies against polymicrobial infections

Oral microbiology---FutureThe Future Direction of Oral Dental Plaque Study---Metagenomics

The aforementioned high-throughput tools for microbial community analysis are largely basedon PCR amplification of 16s rRNA sequences from microbial communities, which arerelatively short, often conserved but varied enough to differentiate bacteria at species level.Although these approaches can provide us with the microbial composition within thecommunity, unless we have genomic or other research data on those identified species, itreveals very limited information regarding what functions they might carry out within the flora.The introduction and application of metagenomics approach has greatly enhanced and willcontinue to increase our ability to study microbial community, including dental plaque, ingreater detail.

The term "Metagenomics" was first invented by Jo Handelsman (27), and it is defined as "theapplication of modern genomics techniques to the study of communities of microbial organismsdirectly in their natural environments, bypassing the need for isolation and lab cultivation ofindividual species. The advances in refinements of DNA amplification, bioinformatics, andenhanced computational power for analyzing DNA sequences have enabled the adaptation ofshotgun sequencing, such as chip-based pyrosequencing, to metagenomic samples (3,18). Theapproach randomly shears DNA, sequences many short sequences, and reconstructs them intoa consensus sequence (3).

By performing metabolic function analyses on genes identified via metagenomic approach,researchers are able to retrieve information both on which organisms are present and moreimportantly, what functions or metabolic processes are possible in that particular community(23). Using comparative genetic studies coupled with expression experiments such asmicroarray and proteomics, microbiologist will be able to piece together a metabolic networkthat goes beyond species boundaries, and gain valuable insight into the metabolism within thecommunity. Recently, comparative metagenomic study has been initiated to try to compare themicrobial community within dental plaque associated with healthy and diseased sites. Theresults should become available in the next few years. It is anticipated that such comparisonwill assist in identifying potential pathogenic organisms which may not have been detectedusing currently available technologies (42).

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Evidence Based Dental Caries DiagnosisAlthough the infectious nature of dental caries has been proved for more than 100 years, insteadof treating it as infectious disease, traditional dentistry still holds sway and focuses on treatingthe symptom (repair the damaged tooth) via surgical approaches. The recent advancedknowledge we have gained about caries pathogenesis allows us to understand that acomprehensive analysis of dental caries should be more than detecting tooth demineralizationsites and repairing damaged teeth with surgical approaches. Instead, it should include thedetection of cariogenic bacteria and plaque acidogenicity, followed by a comprehensivetreatment of dental caries that includes the elimination of cariogenic bacteria, the reduction ofplaque acidogenicity and the enhancement of tooth re-mineralization (82). The combinationof accurate detection of oral bacteria and in situ monitoring of plaque pHsuch as thepolyaniline-based planer pH sensor developed by Scientists at Jet Propulsion Laboratory, andcombined NMR confocal microscopy from Pacific Northwest Laboratory which can monitorpH gradient within dental plaque with high sensitivity and in real time (50), is opening a newchapter for cariology. Early detection and quantification of cariogenic bacteria in plaque orsaliva samples can help clinicians take preventive measures to stop caries development, muchlike early detection of cancer markers can be detected before overt/detectable cancerous lesionsdevelop. New antibody or nucleotide based bacterial detection techniques have been developedfor the detection of cariogenic bacteria in chair-side or laboratory settings (71). In conjunctionwith nanotechnology development, there tests can be further developed into different forms ofnano-chips for detection of multiple pathogens in the clinical settings (45).

New Approaches for Controlling Dental and Periodontal DiseasesCurrent preventive dental therapy is primarily focused on removing dental plaque. Since it isnow known that dental plaque is made of large numbers of commensal bacteria together witha limited number of pathogens (1,65), such an approach may not be effective since the removeall or kill all approach creates open, non-competitive surfaces for pathogens to repopulate theoral cavity. With our new understanding of the oral microbial community interactions, and thewide recognition of microbial ecology based theory on dental and periodontal diseases, thereis now interest in approaches that selectively inhibit oral pathogens or modulate the microbialcomposition of dental plaque to control community based microbial pathogenesis. Amongthem, the probiotic approach has been a popular methods used to affect microbial communities.

The term probiotics refers to live microorganisms, which when administered in adequateamount, confer a health benefit on the host (24). Recently, more evidence suggested thatprobiotic therapy might be applied to the maintenance of oral health (4,54). Using randomizedcontrolled trials, Meurman et al demonstrated that long-term consumption of milk containingthe probiotic Lactobacillus rhamuosus GG strain reduced initial caries in kindergarten children(58). Hillman and colleagues introduced a non-acid producing S. mutans strain that produce abacteriocin active against other S. mutans strains into the oral cavity to replace the naturallyoccurring cariogenic strains. Both in vitro and animal model assessment suggested its potentialin reducing S. mutans colonization. This approach is awaiting evaluation for its efficacy inhumans (29).

Since the beneficial effects of probiotic therapy are mainly achieved through modulatingexisting microbial flora associated with the host, thus obtaining a balanced and healthymicrobe-host relationship, instead of using live organisms, microbiologists are now developingnovel techniques and products that do not involve live bacteria, yet generate targeted effectsagainst pathogenic factors or organisms, thus, achieving similar probiotic effects (28). Onegood example is the Targeted antimicrobial therapy via a novel specifically targeted-anti-microbial peptides (STAMPs) technology (17). A STAMP is a fusion peptide with twomoieties: a killing moiety made of a non-specific anti-microbial peptide and a targeting moiety

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containing a species-specific binding peptide. The targeting moiety provides specific bindingto a selected pathogen and facilitates the targeted delivery of an attached antimicrobial peptide.In one of their published papers, Eckert et al explored a pheromone produced by S. mutans,namely competence-stimulating peptide (CSP), as a STAMP targeting domain to mediate S.mutans-specific delivery of and killing domain. They discovered that such STAMPs werepotent against S. mutans grown in liquid as well as in biofilm states (17). The STAMPs werecapable of eliminating S. mutans from multispecies biofilms without affecting closely relatednon-cariogenic oral streptococci, indicating the potential of these molecules to be developedinto probiotic antimicrobials that may selectively eliminate pathogens while preserving theprotective benefits of the normal flora. This proof-of-principle demonstration using S.mutans suggests that it may be possible to develop other STAMPs which are specificallytargeted to other pathogens, including periodontal pathogens within oral cavity.

Concluding remarksSince the initial observations of bacteria within dental plaque by van Leeuwenhoek using hisprimitive microscopes in 1680, an event that is generally recognized as the advent of oralmicrobiological investigation, our ability to identify the resident organisms in dental plaqueand decipher the interactions between key components has rapidly increased, particularlyduring the past decade. Continued expansion of such information in the future will have a greatimpact on oral microbiology and dentistry as well. We envision that in the future, empoweredwith the detailed knowledge of the microbial community ecology within dental plaque, theinvention and application of new diagnostic tools, dentistry will be an evidence-based dentalpractice emphasizing the triple-pronged approach of early detection, effective and sustainabletreatment, and prevention. Specifically, pathogen-based early detection will become routine,which will allow instantaneous chair-side quantification of cariogenic bacteria in plaque orsaliva samples. This clinical adjunct will help the clinician reinforce the concept of dental cariesas an infectious process and will facilitate immediate, evidence-based treatment decisions.

The progress in oral microbiology will continue to provide in-depth understanding of theecological tug-of-war between the indigenous microbial flora and the cariogenic, periodontalpathogens. And it will become a gateway leading to more specific and proactive therapeuticapproaches in combating dental and periodontal diseases.

References1. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the Normal Bacterial Flora of the Oral

Cavity. J. Clin. Microbiol 2005;43:57215732. [PubMed: 16272510]2. Black GV. Dr. Blacks conclusions reviewed again. Dent. Cosmos 1898;40:440.3. Breitbart M, Salamon P, Andresen B, Mahaffy JM, Segall AM, Mead D, Azam F, Rohwer F. Genomic

analysis of uncultured marine viral communities. Proc. Natl. Acad. Sci. USA 2002;99:1425014255.[PubMed: 12384570]

4. Caglar E, Cilder SK, Ergeneli S, Sandalli N, Twetman S. Salivary mutans streptococci and lactobacillilevels after ingestion of the probiotic bacterium lactobacillus reuteri ATCC 55739 by straws or tablets.Acta. Odontol. Scand 2006;64:314318. [PubMed: 16945898]

5. Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini E, Sorlini C, Corselli C,Daffonchio D. Comparison of different primer sets for use in automated ribosomal intergenic spaceranalysis of complex bacterial communities. Appl. Environ. Microbiol 2004;70:61476156. [PubMed:15466561]

6. Carlsson J. Bacterial metabolism in dental biofilms. Adv. Dent. Res 1997;11:7580. [PubMed:9524445]

7. Clarke JK. On the bacterial factor in the etiology of dental caries. Br. J. Exp. Pathol 1924;5:141147.

He et al. Page 9


NIH


NIH


NIH


8. Cohen SN, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterialplasmids in vitro. Proc. Natl. Acad. Sci. USA 1973;70:32403244. [PubMed: 4594039]

9. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. Bacterial biofilmsin nature and disease. Annu. Rev. Microbiol 1987;41:435464. [PubMed: 3318676]

10. Costerton JW, Geesey GG, Cheng GK. How bacteria stick. Sci. Am 1978;238:8695. [PubMed:635520]

11. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms.Annu. Rev. Microbiol 1995;49:711745. [PubMed: 8561477]

12. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections.Science 1999;284:13181322. [PubMed: 10334980]

13. Dahlen G. Role of Suspected Periodontopathogens in Microbiological Monitoring of Periodontitis.Adv. Dent. Res 1993;7:163174. [PubMed: 8260004]

14. Davey MA, Costerton JW. Molecular genetics analyses of biofilm formation in oral isolates.Periodontology 2000 2006;42:1326. [PubMed: 16930303]

15. Domann E, Hong G, Imirzalioglu C, Turschner S, Kuhle J, Watzel C, Hain T, Hossain H, ChakrabortyT. Culture-independent identification of pathogenic bacteria and polymicrobial infections in thegenitourinary tract of renal transplant recipients. J. Clin. Microbiol 2003;41:55005510. [PubMed:14662931]

16. Dzink JL, Socransky SS, Haffajee AD. The predominant cultivable microbiota of active and inactivelesions of destructive periodontal diseases. J. Clin. Periodontol 1988;15:316323. [PubMed:3292595]

17. Eckert R, He J, Yarbrough DK, Qi F, Anderson MH, Shi W. Targeted killing of Streptococcus mutansby a pheromone-guided "Smart" antimicrobial peptide. Antimicrob. Agents Chemother2006;50:36513657. [PubMed: 17060534]

18. Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson DM, Saar MO,Alexander S, Alexander EC, Rohwer F. Using pyrosequencing to shed light on deep mine microbialecology. BMC Genomics 2006;7:57. [PubMed: 16549033]

19. Feng Z, Weinberg A. Role of bacteria in health and disease of periodontal tissues. Periodontology2000 2006;40:5076. [PubMed: 16398685]

20. Fitzgerald RJ, Keyes PH. Demonstration of the etiologic role of streptococci in experimental cariesin the hamster. J. Amer. Dent. Ass 1960;61:919. [PubMed: 13823312]

21. Foster JS, Kolenbrander PE. Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell. Appl. Environ. Microbiol 2004;70:43404348. [PubMed: 15240317]

22. Gest H. The discovery of microorganisms by Robert Hooke and Antoni van Leeuwenhoek, Fellowsof The Royal Society. Notes and Records of the Royal Society of London 2004;58:187201.[PubMed: 15209075]

23. Gill SR, Pop M, DeBoy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman DA, Fraser-Liggett CM, Nelson KE. Metagenomic Analysis of the Human Distal Gut Microbiome. Science2006;312:13551359. [PubMed: 16741115]

24. Guarner F, Perdigon G, Coerthier S, Salminen B, Morelli L. Should yoghurt cultures be consideredprobiotic? Br. J. Nutr 2005;93:783786. [PubMed: 16022746]

25. Guggenheim B, Guggenheim M, Gmur R, Giertsen E, Thurnheer T. Application of the ZurichBiofilm Model to problems of cariology. Caries Res 2004;38:212222. [PubMed: 15153691]

26. Haffajee AD, Socransky SS. Microbial etiological agents of destructive periodontal diseases.Periodontology 2000 1994;5:78111. [PubMed: 9673164]

27. Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM. Molecular biological access to thechemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol 1998;5:245249.

28. He X, Lux R, Kuramitsu HK, Maxwell HA, Shi W. Achieving Probiotic Effects via Modulating OralMicrobial Ecology. Adv. Dent. Res 2009;21

29. Hillman JD. Genetically modified Streptococcus mutans for prevention of dental caries. Antoine vanLeewenhoek 2002;82:361366.

He et al. Page 10


NIH


NIH


NIH


30. Howell AJ, Jordan HV, Georg LK, Pine L. Odontomyces viscosus, gen. nov., spec. nov., aFilamentous Microorganism Isolated from Periodontal Plaque in Hamsters. Sabouraudia 1965;4:6568. [PubMed: 5896131]

31. Jordan HV. Rodent model systems in periodontal disease research. J. Dent. Res 1971;50:236242.[PubMed: 4929694]

32. Keyes PH. Infections and transmissible nature of experimental dental caries. Arch. Oral Biol1960;1:304320. [PubMed: 14408737]

33. Keyes PH, Jordan HV. Periodontal Lesions in the Syrian Hamster. III. Findings Related to anInfectious and Transmissible Component. Arch Oral Biol 1964;9:377400. [PubMed: 14179047]

34. Kinniment SL, Wimpenny JW, Adams D, Marsh PD. Development of a steadystate oral microbialbiofilm community using the constant-depth film fermenter. Microbiology 1996;142:631638.[PubMed: 8868438]

35. Kleinberg I. A mixed-bacterial ecological approach to understanding the role of oral bacteria in dentalcaries causation: an alternate to Streptococcus mutans and the specific-plaque hypothesis. Crit. Rev.Oral Biol. Med 2002;13:108125. [PubMed: 12097354]

36. Kolenbrander PE. Intergeneric coaggregation among human oral baceria and ecology of dental plaque.Annu. Rev. Microbiol 1988;42:627686. [PubMed: 3060002]

37. Kolenbrander PE. Oral microbial communities: Biofilms, Interactions, and Genetic Systems. Annu.Rev. Microbiol 2000;54:413437. [PubMed: 11018133]

38. Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJJ. Communicationamong oral bacteria. Microbiol. Mol. Biol. Rev 2002;66:486505. [PubMed: 12209001]

39. Kolenbrander PE, Andersen RN, Kazmerzak K, Wu R, Palmer RJ. Spatial organization of oral bacteriain biofilms. Methods Enzymol 1999;310:322332. [PubMed: 10547802]

40. Kolenbrander PE, Egland PG, Diaz PI, Palmer JRJ. Genome-genome interactions: bacterialcommunities in initial dental plaque. Trends Microbiol 2005;13:1115. [PubMed: 15639626]

41. Kolenbrander PE, Palmer RJJ, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI. Bacterialinteractions and successions during plaque development. Periodontology 2000 2006;42:4779.[PubMed: 16930306]

42. Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ. New bacterial speciesassociated with chronic periodontitis. J. Dent. Res 2003;82:338344. [PubMed: 12709498]

43. Kuramitsu HK, He X, Lux R, Anderson MH, Shi W. Interspecies Interactions within Oral MicrobialCommunities. Microbiol. Molecul. Biol. Rev 2007;71:653670.

44. Laidlaw PP. Some simple anaerobic methods. Brit. Med. J 1915;1:497498. [PubMed: 20767540]45. Li Y, Denny P, Ho CM, Montemagno C, Shi W, Qi F. The Oral Fluid MEMS/NEMS Chip (OFMNC):

diagnostic and translational applications. Adv. Dent. Res 2005;18:35. [PubMed: 16000263]46. Listgarten MA. Structure of the microbial flora associated with periodontal health and disease in man:

a light and electron microscopic study. J. Periodontol 1976;47:118. [PubMed: 1063849]47. Liu W, Marsh T, Cheng H, Forney L. Characterization of microbial diversity by determining terminal

restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol1997;63:45164522. [PubMed: 9361437]

48. Loe H, Theilade E, Jensen S. Experimental gingivitis in man. J. Periodontol 1965;36:177187.[PubMed: 14296927]

49. Loesche WJ. The specific plaque hypothesis and the antimicrobial treatment of periodontal disease.Dent. Update 1992;19:6874. [PubMed: 1291362]

50. Majors PD, McLean JS, Pinchuk GE, Fredrickson JK, Gorby YA, Minard KR. NMR methods for insitu biofilm metabolism studies. J. Microbiol. Methods 2005;62:337344. [PubMed: 15936835]

51. Marsh PD. Dental plaque: biological significance of a biofilm and community life-style. J. Clin.Periodontol 2005;32:715. [PubMed: 16128825]

52. Marsh PD. Microbial Ecology of Dental Plaque and its Significance in Health and Disease. Adv.Dent. Res 1994;8:263271. [PubMed: 7865085]

53. McIntosh J, Fildes P. A new apparatus for the isolation and cultivation of anaerobic micro-organisms.Lancet 1916;1:768770.

He et al. Page 11


NIH


NIH


NIH


54. Meurman JH, Stamatova I. Probiotics: contributions to oral health. Oral Dis 2007;13:443451.[PubMed: 17714346]

55. Miller, WD. Graphische Anstalt Schuler AG. Biel, Switzerland: 1890. The micro-organisms of thehuman mouth.

56. Mitchell DF, Johnson MJ. The Nature of the Gingival Plaque in the Hamster-Production, Prevention,and Removal. J. Dent. Res 1956;35:651655. [PubMed: 13345946]

57. Nakatsu C. Soil microbial community analysis using denaturing gradient gel electrophoresis. Soil.Sci. Soc. Am. J 2007;71:562571.

58. Nase L, Hatakka K, Savilahti E, Saxelin M, Ponka A, Poussa T, Korpela R, Meurman JH. Effect oflong-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dentalcaries and caries risk in children. Caries Res 2001;35:412420. [PubMed: 11799281]

59. Navia, JA. Birmingham, Alabama: University of Alabama Press; 1977. Animal Models in DentalResearch.

60. Netuschil L, Reich E, Unteregger G, Sculean A, Brecx M. A pilot study of confocal laser scanningmicroscopy for the assessment of undisturbed dental plaque vitality and topography. Arch. Oral. Biol1998;43:277285. [PubMed: 9839703]

61. Nikiforuk, G. Understanding Dental Caries. Vol. Vol. 1. Basel: S. Karger; 1985. p. 6062. Nishihara T, Koseki T. Microbial etiology of periodontitis. Periodontology 2000 2004;36:1426.

[PubMed: 15330940]63. Noguchi H. A method for the pure cultivation of pathogenic Treponema pallidum. J. Exp. Med

1911;14:99112. [PubMed: 19867465]64. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE.

Bacterial Diversity in Human Subgingival Plaque. J. Bacteriol 2001;183:37703783. [PubMed:11371542]

65. Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacteria diversity in the human periodontalpocket and other oral sites. Periodontology 2000 2006;42:8087. [PubMed: 16930307]

66. Pigman W, Elliott HC. An artificial mouth for caries research. J. Dent. Res 1952;31:627633.[PubMed: 12990724]

67. Rosebury, T. Microorganisms indigenous to man. New York: McGraw-Hill Book Co., Inc.; 1962.68. Rosebury T, Reynolds JB. Continuous anaerobiosis for cultivation of spirochetes. Proc. Soc. Exptl.

Biol. Med 1964;117:813815. [PubMed: 14244963]69. Russell C, Coulter WA. Continuous monitoring of pH and Eh in bacterial plaque grown on a tooth

in an artificial mouth. Appl. Microbiol 1975;29:141144. [PubMed: 234712]70. Saiki R, Scharf S, Faloona F, Mullis K, Horn G, Erlich H, Arnheim N. Enzymatic amplification of

beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985;230:13501354. [PubMed: 2999980]

71. Shi W, Jewett A, Hume WR. Rapid and quantitative detection of Streptococcus mutans with species-specific monoclonal antibodies. Hybridoma 1998;17:365371. [PubMed: 9790071]

72. Slots J, Listgarten MA. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillusactinomycetemcomitans in human periodontal diseases. J. Clin. Periodontol 1988;15:15.

73. Small J, Call DR, Brockman FJ, Straub TM, Chandler DP. Direct detection of 16S rRNA in soilextracts by using oligonucleotide microarrays. Appl. Environ. Microbiol 2001;67:47084716.[PubMed: 11571176]

74. Socransky SS. Criteria for the infectious agents in dental caries and periodontal disease. J. Clin.Periodontol 1979;6:1621. [PubMed: 295292]

75. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL. Microbial complexes in subgingivalplaque. J. Clin. Periodontol 1998;25:134144. [PubMed: 9495612]

76. Socransky SS, MacDonald JB, Sawyer S. The cultivation of Treponema microdentium as surfacecolonies. Arch. Oral Biol 1959;1:171172. [PubMed: 13832418]

77. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE. "Checkerboard" DNA-DNAhybridization. BioTechniques 1994;17:788792. [PubMed: 7833043]

78. Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms inaquatic and terrestrial habitats. Annu. Rev. Microbiol 1985;39:321346. [PubMed: 3904603]

He et al. Page 12


NIH


NIH


NIH


79. Suddick RP, Harris NO. Historical perspectives of oral biology: a series. Crit. Rev. Oral. Biol. Med1990;1:135151. [PubMed: 2129621]

80. Theilade J, Fejerskov O, Horsted M. A transmission electron microscopic study of 7-day old bacterialplaque in human tooth fissures. Arch. Oral. Biol 1976;21:587598. [PubMed: 1068650]

81. Thies JE. Soil microbial community analysis using terminal restriction fragment lengthpolymorphisms. Soil Sci. Soc. Am. J 2007;71:57.

82. Tsang P, Qi F, Shi W. Medical Approach to Dental Caries: Fight the Disease, Not the Lesion. Pediatr.Dent 2006;28:188191. [PubMed: 16708795]

83. Wagner M, Assmus B, Hartmann A, Hutzler P, Amann R. In situ analysis of microbial consortia inactivated sludge using fluorescently labelled, rRNA-targeted oligonucleotide probes and confocalscanning laser microscopy. J. Microsc 1994;176:181187. [PubMed: 7532718]

84. Williams JL. On structural changes in human enamel; with special reference to clinical observationson hard and soft enamel. Dental Cosmos 1898;40:505.

85. Woese CR. Bacterial evolution. Microbiol. Rev 1987;51:221271. [PubMed: 2439888]86. Wood SR, Kirkham J, Marsh PD, Shore RC, Naltress B, Robinson C. Architecture of Intact Natural

Human Plaque Biofilms Studied by Confocal Laser Scanning Microscopy. J. Dent. Res 2000;79:2127. [PubMed: 10690656]

87. Yoshino K, Nishigaki K, Husimi Y. Temperature sweep gel electrophoresis: a simple method to detectpoint mutations. Nucleic. Acids. Res 1991;19:3153. [PubMed: 2057372]

88. Zhulin IB. It is computation time for bacteriology! J. Bacteriol 2009;191:2022. [PubMed: 18978045]

He et al. Page 13


NIH


NIH


NIH


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