Chapter Abstracts: Oral Bacterial Ecology: The Molecular Basis

Oral Bacterial Ecology: The Molecular Basis Chapter Abstracts

Chapter 1.

Oral ecology and its impact on oral microbial diversity.
Philip D. Marsh

Chapter 1 Contents

Introduction
Ecological terminology
The resident human microflora
The mouth as a microbial habitat
Factors affecting the growth of micro-organisms in the oral cavity
        Temperature
        Redox potential/anaerobiosis
        pH
        Nutrients
         (i) endogenous nutrients
         (ii) exogenous (dietary) nutrients
        Host genetics and social behaviour
Acquisition of the resident oral microflora
        Pioneer community and ecological succession
        Allogenic and autogenic succession
Distribution of the resident oral microflora
        Lips and palate
        Cheek
        Tongue
        Teeth
        Fissure plaque
        Approximal plaque
        Gingival crevice plaque
        Denture plaque
        Dental plaque from animals
Dental plaque as a microbial biofilm
        Dental plaque formation
        Microbial homeostasis in dental plaque
        Microbial interactions in dental plaque
Dental plaque as a microbial community
Ecological perturbations in dental plaque and their relationship to disease
Concluding remarks
References

Introduction

In order to introduce the concept of ecology to the microbiology of the oral cavity, the reader should consider the following question:

"Which of the following events is the odd one out?":

(1) The growth of algae in rivers and ponds following the leaching of nitrogenous fertilisers from farm land into water.

(2) The loss of marine life around shores following an oil spillage.

(3) The extinction of the dinosaurs due to climatic changes in the Cretaceous period. (4) The development of caries and periodontal diseases in the mouth!

It is the contention of the authors of this volume that the processes underlying all of the above events are similar, and that it is possible to explain all of these four diverse biological events through an understanding of ecological principles. Indeed, a willingness to apply such principles can lead to several tangible benefits which include (1) a clearer understanding of the apparently complex relationship between the host and its resident oral microflora in health and disease, and (2) the identification of novel routes for disease prevention. A further benefit of an application of this ecological approach to understanding the cause of oral disease is that the principles can continue to be applied validly irrespective of subsequent changes to the nomenclature of the microflora or the discovery of "new" organisms. This is because emphasis is placed on determining the properties, and hence the function, of specific organisms in the disease process. This approach also seeks to relate microbiological observations to changes to the life-style and/or medical history of the patient.

Concluding Remarks

The use of rigorous isolation techniques, coupled with improvements in microbial taxonomy, has led to the recognition of a highly diverse microflora that inhabits the various surfaces of the normal mouth. This diversity is particularly apparent in dental plaque where the spatial heterogeneity of biofilms ensures that species can co-exist despite possessing conflicting nutritional and atmospheric requirements.

The recent application of molecular approaches to microbial identification has facilitated the identification of species and taxa not previously described, and which cannot as yet be cultivated in the laboratory. Such taxa are being found commonly in healthy individuals, implying that the resident oral flora has an even greater complexity than hitherto imagined. The continued use and refinement of these powerful techniques will be necessary to fully describe these microbial communities. A likely outcome of these studies will be the recognition that the mouth in general, and dental plaque in particular, can act as a significant reservoir for many medically-important pathogens. Such findings may open up further opportunities for the control of these opportunistic pathogens.

Relatively little is known of the true architecture of dental plaque, of the physical location of particular species within these biofilms, nor how the biofilm "mode of life" affects microbial gene expression. The availability of new techniques such as confocal microscopy, species-specific probes, reporter genes, differential display of mRNA profiles, and DNA chip technology will help resolve many of these fundamental issues. Another important area of study, which is just beginning, is to understand the "cross-talk" that may occur both among the component species of the resident microflora, but also between these communities and the host tissues. Advances in knowledge in these areas offer exciting opportunities for the control and prevention of dental diseases in the future, while still retaining the beneficial properties afforded to the host by a resident microflora.

Chapter 2.

Growth and nutrition as ecological factors.
Jan Carlsson.

Chapter 2 Contents

Introduction
Microbial growth
        Kinetics of microbial growth
        Conditions for growth in saliva
        Initial growth and replication of microorganisms on tooth surface
        Multi-nutrient limitation of growth
Microbial recognition of the environmental situation
        Sensing mechanisms
        Competition for nutrients and kinetics of nutrient uptake
Stress handling by individual microbial cells
        Coordination of the responses in individual cells (global regulation)
        Starvation
        The anaerobic-aerobic interface
        Reactive oxygen species
        Damaged macromolecules
Stress handling by microbial communities
        Peptides as pheromones
        N-Acyl-homoserine lactones as pheromones
        Killing of neighbors
        Altruistic cell death
Nutrients
        Sugars
        Amino acids and peptides
        Nutrition and energetics of anaerobic microbial communities
Perspective
References

Introduction

Six billion human individuals inhabit our planet, and this is also the number of new microbial cells that may be produced in one to two hours in the mouth of each individual. The latter estimate refers to a study in which water, a glucose solution, or a peptone-yeast-extract-glucose solution were siphoned into the mouths of six individuals for 4 hours; all the while the subjects were chewing a piece of paraffin wax (Carlsson and Johansson, 1973). The fluid accumulated in the mouth was continuously collected. At various times after the start of each experiment the number of viable microorganisms in the fluid was determined. When water was pumped into the mouth, the microbial density of the fluid decreased during the first hour and then remained at a steady level throughout the rest of the experiment (Figure 1). The number of microorganisms in the fluid during the last two hours of the experiment was considered to mirror the yield of new microorganisms in the mouth. The glucose solution did not increase the yield of microorganisms, but the peptone-yeast-extract-glucose solution did. On our planet most people live in poverty struggling for survival. This is also the case with the microbial cells of the mouth. In the present chapter it will be discussed how microorganisms manage to survive and grow in the harsh environment of the oral cavity.

Perspective

During the 20^th century investigators reduced the biofilm on teeth into the smallest possible functional pieces. There is today more knowledge about the indigenous microbial species in the oral cavity than in any other site of our body. We also have impressive knowledge about adherence mechanisms of these microorganisms, and some information on microbial physiology in relation to caries and periodontal disease. However, all of this is inadequate unless an appreciation is developed for interactions within the microbial communities colonizing the oral surfaces and between the microbial communities and their environment in the oral cavity. It is now time to use the knowledge, apply new technologies and consider biofilms as functional units.

This has to start with a more holistic view on the functions of the microbial cell as such. Biofilms develop until the environment no longer fully supports growth. In this review a few examples are presented describing how an organism prepares itself in an environment that is not optimal for growth. A picture emerges where it is clear that a significant part of the genome of an organism is devoted to handle such situations. It is the struggle for nutrients and protection against changes in temperature, osmolarity, pH, and oxygen levels that characterizes everyday life. Most of our present knowledge on the adaptation of an organism to its environment derives from studies where growth and survival of a wild-type microbial strain (mostly of E. coli) are compared to that of a strain with a knockout of a single gene. However, one does not have to take for granted that the product of this gene has the same effect on that organism when it is living in its natural 'real life' environment under multi-nutrient limitation. This calls for more holistic experimental approaches. The basis for such studies will be provided by the knowledge of the entire genome sequences of a representative number of organisms and the interpretation of these sequences into microbial physiology, i.e., functional genomics. A further step to obtain a more holistic view of microbial physiology and ecological adaptation is to follow the changes in protein expression and concentrations of intracellular metabolites in response to alterations in the environment. Combining these approaches will allow a correlation of gene activity and metabolic activity in a cell at any one moment.

All the proteins in a translated microbial genome is called proteome, and the actual goal of the science of proteomics is a quantitative description of protein expression and its changes under the influence of biological perturbations (Humphery-Smith et al., 1997; Blackstock and Weir, 1999). Powerful analytical tools have paved the way for proteomics. A significant beginning was the introduction of two-dimensional polyacrylamide gel electrophoresis by O'Farrel (1975). The next step was when N-terminal amino acid sequencing could identify the separated polypeptides in the gel. A substantial increase in speed and sensitivity of peptide analysis has since been gained by the introduction of matrix assisted laser desorption and ionization in time of flight mass spectrometers (MALDI-TOF/MS) and triple-quadruple and ion trap mass spectrometers (Humphery-Smith et al., 1997; James, 1997; Hochstrasser, 1998).

In the elucidation of microbial physiology under various environmental situations genomics and proteomics may fall short if not combined with information about concentrations of intracellular metabolites and kinetic properties of involved enzymes (Tweedale et al., 1998). To analyze the metabolome (i.e., the total intracellular pool of metabolites) there are basically two different approaches: non-invasive and invasive techniques. Nuclear magnetic resonance spectroscopy (NMR) is non-invasive and has high sensitivity. With ³¹P-NMR and ¹³C-NMR spectroscopy such high in-vivo sampling rates as twice per second can be obtained (Weuster-Botz and de Graaf, 1996). If all these techniques are highly standardized with scrupulous documentation of experimental data, microbial activities may be successfully analyzed by powerful bioinformatics in 'virtual laboratories' (Hochstrasser, 1998).

In oral microbiology the use of these techniques is in its infancy. The whole genome sequence of an oral microorganism has not yet been published but several of these projects are nearing completion. Changes in protein profiles using two-dimensional polyacrylamide gel electrophoresis have been studied in Candida albicans upon carbon-starvation at acid pH (Niimi et al., 1996). Changes in protein profiles upon exposure to acid have also been studied in members of the genera Lactobacillus, Streptococcus and Actinomyces (Hamilton and Svensäter, 1998). For a long time, glycolytic intermediates of the metabolome in oral organisms have been studied with invasive techniques, and the findings have been very useful in elucidating the regulation of glycolysis and acid production in cariogenic organisms (Yamada and Carlsson, 1975; Iwami and Yamada, 1985: Takahashi et al., 1991). With the rapid development of functional genomics (Martzen et al., 1999) oral microbiologists can look into the future with confidence. However, there is one major obstacle in oral microbiology as well as in other areas of microbiology. Microbial biochemical physiology has not developed in pace with genomics. During the last few decades this field has actually been neglected in lieu of genomics. Without a thorough knowledge of the enzymes and their regulatory mechanisms at the protein level, genomics alone may be inadequate. Fortunately, many cellular functions are highly conserved among microorganisms. However, it is not too uncommon that one is surprised when one wants to confirm that an organism has solved a specific problem in the same way as others. For example, who would have imagined that GTP and pyrophosphate, and not ATP, is the 'energy currency' of glycolysis in oral members of Actinomyces (Takahashi and Yamada, 1999)?

Current methodologies can thus give us detailed information on global shifts in cellular functions of a microbial population under different environmental conditions (VanBogelen et al., 1999). The real challenge of today is to extract similar information on microbial communities in biofilms. One has then to know the relationship between structure and function, and how the populations communicate and compete for nutrients (see "Microbial Recognition of the Environmental Situation" and "Stress Handling by Microbial Communities"). In this approach of 'putting the pieces together' scanning confocal laser microscopy and various molecular methodologies are indispensable tools (Møller et al., 1998). rRNA probes can be used to identify and quantify defined populations, and monitor the dynamics of the populations (Head et al., 1998). By combining rRNA probes and confocal microscopy, syntrophic partners (see "Nutrition and Energetics of Anaerobic Microbial Communities") in complex microbial communities can be identified (Harmsen et al., 1996). With fluorescent molecular probes and confocal microscopy, variations in oxygen levels or pH within a biofilm can be analyzed (Caldwell et al., 1992). New techniques are continuously being introduced and our lack of imagination may be the only limitation in unraveling the growth and nutrition of the microbial communities.

To understand how the microbial communities handle the situation on tooth surfaces, we will have to use the knowledge of the entire microbial world as our reference. In the present chapter it was discussed how microorganisms grow, how they inform themselves about the situation in their environment, and how they survive under starvation and shifts in aerobic and anaerobic conditions. The day we decipher the whole genomes of a representative number of oral microorganisms, we may have answers to some of these questions. The remaining questions about their physiology and ecology can then be explored from a more knowledgeable basis.

Chapter 3.

Adhesion as an ecological determinant in the oral cavity.
Richard J. Lamont and Howard F. Jenkinson

Chapter 3 Contents

Introduction
Acquisition of the oral bacterial microbiota
        Environmental factors influencing adhesion
Mechanisms of oral bacterial adhesion
        Cell surface hydrophobicity
        Lectin-like adhesion
Bacterial surface structures and adhesion
        Fimbriae and fibrils
                Streptococcus fibrils
                Streptococcus fimbriae
                Actinomyces naeslundii fimbriae
                Porphyromonas gingivalis fimbriae
                Actinobacillus actinomycetemcomitans fimbriae
Repertoire of adhesin functions
        Fimbrial adhesins
        Non-fimbrial adhesins
        Adhesins with alternate functions
        Adhesins and nutrition
Bacterial cell-cell interactions
        Mechanisms of bacterial coadhesion
        Role of bacterial coadhesion
        Glucan mediated coadhesion
Consequences of adhesion
        Invasion
        Host-signaling without invasion
        Bacterial cell signaling
Concluding remarks
References

Introduction

The oral cavity harbors a diverse, abundant and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in a sessile biofilm. These organisms engage the host in an intricate cellular and molecular dialogue, the outcome of which normally serves to constrain the bacteria in a state of commensal harmony. Under certain circumstances, however, the oral microbiota can be directly or indirectly responsible for disease (see Chapters 5 and 6). An understanding of the basis of microbial colonization thus provides insight into both oral ecology and the underlying pathogenic mechanisms of oral bacteria. This chapter will focus on oral microbial adhesion, which, as depicted in Figure 1, is the underlying process that drives colonization and ultimately disease progression.

Concluding Remarks

Bacterial adhesion can be seen as a defining event for oral bacteria. Once firmly anchored to oral surfaces, bacteria can be expected to remain at that site for a long period (in relation to bacterial doubling time) and may alter their phenotypic properties accordingly. The multiplicity of adhesins that bacteria produce is testimony to the ecological importance of adhesion and the opportunities available to sessile organisms. The specificity and expression of these adhesins determines, to a large extent, the ecological niche of individual species within the oral cavity. Accumulation into a complex plaque biofilm provides for nutritional and protective advantages not afforded by planktonic growth. These accumulations are responsible for superficial tissue damage, such as that associated with gingivitis, and if unchecked can lead to permanent loss of enamel and dentin (caries), or periodontal tissues (periodontal diseases) (see Figure 1). For some species of bacteria, adhesion to host tissues is the prelude to internalization within host cells, and the development of systemic disease conditions (Figure 1). Since adhesion is of paramount ecological significance in oral infections, strategies aimed at blocking adhesin functions and/or subsequent signaling through immunization or drug therapies may receive greater attention in oral disease prevention in the future.

Chapter 4.

Oral innate host defense responses: interactions with microbial communities and their role in the development of disease.
Richard Darveau

Chapter 4 Contents

Introduction
The dynamic between the microbial community and the host in the periodontium is one of constant communication
        Dental plaque is a microbial biofilm community which forms on the tooth and tooth root surface
        Bacterial shedding informs the host of the colonization status of the biofilm
Innate Host defense recognition and response
        Pattern recognition provides a framework to understand innate host defense responses
        The LBP/CD14 system
        Mechanism of multiple ligand binding for pattern recognition receptors
        Toll-like receptors (TLR) represent a new family of pattern recognition innate host defense proteins
        Where is non- self recognized?
Clinically healthy status
        Control of supragingival plaque growth in the clinically healthy individual
        Control of subgingival plaque growth in the clinically healthy individual
        Relationship between microbial colonization and innate host defense status
        Is the oral ecosystem associated with clinical health a symbiosis with the host?
Influence of impaired innate host response on the development of disease
        Immunosuppressive therapy
        Leukocyte adhesion deficiency
        Chediak-Higashi Syndrome
        Chronic neutropenia
        Papillon-Lefevre Syndrome
        Diabetes
        Cigarette smoking
        Human Immunodeficiency Virus
        Congenital or induced immune host defects generally not associated with the development of periodontitis
        Chronic granulomatous disease
        Complement
        IgA deficiency and agammaglobulinemia
        Summary of the effects of induced and congenital deficiencies on the oral microbiota
Gingivitis and periodontitis represent two clinical conditions where the microbial compositions are linked to an altered innate host response
        Gingivitis
        Periodontitis
        Dysfunctional inflammatory response
        Role of periodontopathogens in the innate host response
Conclusions
References

Introduction

The interactions between the complex oral microbiological community and the host and its succession from health to disease might be best understood in the terms by which defense systems and the oral microbial community recognize each other. The host has evolved mechanisms to recognize non-self as opposed to specific microorganisms, and bacteria have evolved mechanisms to sense their environment and evade or modify the host as needed to produce progeny. Bacteria have evolved such that they occupy the ecological niche provided by both the tooth surface and gingival epithelium as well as the surrounding environmental conditions of the oral cavity. However, a highly efficient innate host defense system constantly monitors the bacterial colonization status and prevents bacterial intrusion into local tissues. A dynamic equilibrium exists between dental plaque bacteria and the innate host defense system. This interaction is not a haphazard process, but represents a highly evolved interaction between bacteria and host. The consortium of bacteria that constitute dental plaque represent highly evolved, highly specialized organisms that have adapted and are still adapting to make the best of their local environment. Under conditions of clinical health they may represent a symbiotic relationship with the commensal bacteria providing the host the appropriate inflammatory stimulus to maintain an effective, non-destructive inflammatory barrier against potential pathogens. In polymicrobic infections, typified by periodontitis, the microbial community has established a new dynamic that results in a pathologic state. The innate host response system, particularly the inflammatory response, is central to both the healthy and diseased environments. This chapter will examine how the host sees these different microbial communities and how its response contributes to health and disease. Recent discoveries on the molecular mediators of inflammation including host factors that recognize bacterial components, combined with the molecular characterization of clinically healthy and diseased periodontal tissue preclude a discussion of the role of adaptive immune response (both cellular and antibody mediated) in regulating oral microbial ecology in this chapter. I refer the reader to a recent review by Hajishengallis and Michalek (1999) for more information on this subject.

Conclusions

The host constantly monitors and responds to the colonization status of the oral cavity. It is clear that subgingival bacterial numbers and compositions have profound effects on the host innate response, whereas provocative data suggest that saliva composition may be altered in response to Candida infection. Studies of individuals with impaired innate host responses clearly demonstrate that a proper innate inflammatory response is key to periodontal health. Examination of the innate host response status in clinically healthy individuals has revealed a low level "inflammatory surveillance" state where the host maintains an effective barrier against bacterial infection. It is possible that the normal oral microflora is not merely a series of non-pathogenic commensal communities, but rather these communities participate in establishing this protective state, elevating them to symbiotic partners with the host. Mechanisms of bacterial recognition are emerging that may help explain how different members of the microbiotia contribute to maintenance of an effective host defense barrier. Likewise, information on the host activation potential of periodontopathogens suggests that dysfunctional host responses may be created. Additional studies examining the bacterial/host dynamic in the clinically normal and diseased host are needed to more fully understand how these different microbial communities are created.

Chapter 5.

Ecological basis for dental caries.
Ian R. Hamilton

Chapter 5 Contents

Introduction
Dental Caries
The Plaque Ecosystem
The Dental Plaque Biofilm
The Bacteria
        Mutans Streptococci
        Other 'Cariogenic' Bacteria
Microbial Metabolism and Caries
        Dental Plaque in situ
        Plaque Acidogenicity
Metabolism by the Acidogenic Microflora
Acid Tolerance
Other Environmental Stress Factors
Base Formation
Caries Lesions - To Be or Not to Be
Control of Caries
Future Directions
References

Introduction

In recognition of W. D. Miller's historic book "The microorganisms of the human mouth" published in 1890, an appropriate sub-title for this Chapter might be "From Miller to the Millenium". This book resulted from Miller's work in Robert Koch's laboratory in Berlin in which he brought together the idea of acid and microorganisms in his "chemicoparasitic theory" of dental caries. While earlier theories had implicated "parasites" in caries etiology, Miller was the first to establish the role of oral bacteria in dental plaque in the demineralizing tooth enamel. He showed that the degradation of carbohydrate-containing foods resulted in acid formation and was able to demonstrate this process in vitro with isolated oral bacteria and extracted teeth. He also visualized that the final destruction of enamel and dentin was accompanied by proteolysis of the organic matrix of enamel. This led to Miller's major conclusion that dental caries was caused by multiple species of oral bacteria, in what Loesche (1982) later described as the "non-specific plaque hypothesis". In fact, it can be argued that the practice of toothbrushing, flossing and professional toothcleaning as a means of preventing dental diseases have their basis in Miller's original 1890 experiments.

As one would expect, however, a vast amount of newer information is available on the caries process, particularly in relation to the bacteria associated with this disease, including the manner in which they adhere to the tooth surface, the metabolic processes that result in acid formation in the presence of carbohydrate substrates, and the resulting physical-chemical reactions with tooth enamel. Even though the recent studies are providing information on many events associated with caries development in finer detail, some of it at the molecular level, there is still a need on occasion to stand back and view the basic elements of the caries process in light of this new information. This discussion can be enhanced by examining situations where caries has occurred and where it has not occurred. Good examples of this are approximal sites on adjacent teeth where caries has occurred in one surface, but not on an adjacent tooth only a millimeter away. For example, in Figure 1 [A], a lesion is evident on tooth #35 of an 11 year old male patient, while the adjacent site on tooth #36 is free of caries. Another example (Figure 1[B]) is that of two approximal lesions on tooth #45 of a 28 year male patient with no caries on the adjacent teeth (#44 and #46). As we will see later, these two apparently similar situations probably occurred for different reasons, but in order to explain these lesions, we need to understand the nature of the site involved, the bacteria that inhabit that site, and the local environment. In other words, we need to understand the ecology of the specific site in question. Before a discussion of the microbial ecology of dental plaque, it is perhaps useful to review some of the salient features of the caries process.

A considerable literature exists on microbial ecology, dental caries, the oral microflora associated with dental plaque and caries lesions, and the pertinent metabolism associated with these bacteria. Consequently, it will not be possible in this chapter to cite all of the relevant references; however, attempts have been made to cite key papers and reviews, particularly, the more current references which will provide the reader with an avenue to trace the important studies that relate to the ecology of dental caries.

Future Directions

Even with the passage of over 100 years, our understanding of the etiology of dental caries continues to evolve. Organisms such as S. mutans and Lactobacillus spp. have long been associated with the disease; however, the application of more comprehensive and sophisticated microbiological analysis in cross-sectional and longitudinal human studies over the past 20 years has expanded the list of the putative pathogens. Much more information is needed on the contribution of the individual species grouped within the "low pH-non-mutans streptococci" and the Actinomyces. And what of the role, if any, of those organisms not normally associated with caries etiology that assume dominant positions in incipient and advanced root lesions, such as those listed in Table 2. Cariogenic bacteria are generally assumed to be acidogenic with the capacity to metabolize carbohdyrate substrates at low pH values and to possess a degree of acid tolerance that permits them to grow and survive in such environments. If this definition applies to those organisms in Table 2 then one must assume that some genotypic or phenotypic change had occurred to alter cell physiology in a substantial manner to permit growth and survival under acidic conditions.

A variety of environmental signals are received by bacteria in complex communities, including those from other bacteria in the habitat. The signal triggering a change increasing fitness to acid may have been received and acted upon by the entire community, a species population within the community, or even a single cell within a population. Since bacteria in dental plaque are sequestered in the biofilm, we can assume that within a species population, diversity will increase with distinct clones descending from a single ancestor, much like the wedge-shaped, radically oriented sectors seen within colonies of a single organism on agar plates (Shapiro, 1997). Such self-generated changes within bacteria to increase fitness would likely result in alterations in dedicated signalling and regulatory molecules and ultimately gene expression. We already have evidence of differences between members of the same species as exemplified in Table 3 by the variation in glycolytic rates by fresh isolates of S. mutans. Since the domination of lesions by organisms other than S. mutans, Lactobacillus spp. and the non-MS may be a relatively rare events, it is conceivable that there will be a species variation in the potential to receive and act on 'fitness' signals.

These future studies, of course, require that we have a much better understanding of the characteristics and properties of oral biofilms, and the degree to which such microbial communities permit 'biochemical flexibility' and enhance survival from external and internal forces. An array of sophisticated techniques are now being applied to the study of biofilms, including fluorescent microscopy, scanning laser confocal microscopy, attenuated total reflectance infrared spectroscopy, two-photon scanning microscopy and a variety of molecular biological, fluorescent and other methods. Current results clearly show that biofilm formation involves the activation of specific genes (Burne et al., 1997; Davies et al., 1998). Thus, future research will undoubtedly involve the increasing application of molecular biology to oral biofilms, such as the use of reporter genes, differential display polymerase chain reaction, in vivo expression technology, and proteomics, to name a few. Such methods will not only identify biofilm-expressed genes, but permit the identification of the important genes involved in global signalling that enhances survival to environmental stress, such as acid, oxidation and starvation, as well as, the response of biofilm cells to the application of biocides. These methods could also be used to test the effects of interspecies interactions in mixed biofilm model systems. The combined information would provide a clearer picture of dental plaque as an integrated multicellular unit operating with a complex network of intercellular communication that can be involved in gene expression, differentiation and other cellular processes. From this knowledge should come the identification of key molecular events in the plaque microflora that trigger the pathogenic process leading to dental caries.

Chapter 6.

Periodontitis as an ecological problem.
Daniel Grenier and Denis Mayrand

Chapter 6 Contents

Introduction
Periodontal diseases and periodontopathogens
Bacterial interactions
Virulence factors of periodontopathogens
        Adhesins
        Lipopolysaccharides
        Proteinases
        Outer membrane vesicles
        Other virulence factors of ecological significance
Modulation of periodontal microbiota and expression of virulence factors by environmental parameters
Stress response and pathogenicity
Host-bacteria interactions
Conclusions
References

Introduction

There is now much evidence to suggest that the physiology, biological properties, and pathogenicity of a bacterium are largely influenced by the environment. As a matter of fact, signal transduction systems that recognize and respond to specific environmental cues are often utilized by pathogens to ensure that genes required for survival, multiplication and virulence are appropriately expressed during the infection (DiRita and Mekalanos, 1989; Guiney, 1997; Straley and Perry, 1995). The situation is much more complex when an infectious disease results from a mixed infection i.e. from the interaction of several bacterial species each contributing to cause the disease. Mixed anaerobic infections are generally found to have several characteristics: i) the infectious agents are members of the normal indigenous bacterial microbiota and hence may be regarded as opportunistic pathogens, ii) as individuals, the pathogenic members of such infections are only minimally virulent under most conditions, and iii) predisposing factors may play an important if not essential role in the initiation of such infections, since a change in the environmental conditions enables some members of the ecosystem to become prominent and/or to manifest some of their pathogenic properties.

Virulence promotion by mixed bacterial populations has three underlying features: bacterial interactions, expression of virulence factors and host-bacteria interactions. The oral cavity and more particularly the subgingival environment represent a perfect example of microbial ecology where different bacterial populations cohabitate. Periodontal diseases, which are considered as mixed anaerobic infections, develop when the equilibrium of the community is broken in favor of the pathogenic bacteria that most individuals harbor in low numbers in their subgingival sites. The mechanisms that regulate and influence the proportions of pathogenic and commensal or beneficial bacteria in subgingival plaque are not fully understood. However, it is likely that host, bacterial and environmental factors are of utmost importance for determining the subgingival microbiota composition and modulating the expression of bacterial virulence factors.

Conclusions

Microbiological and immunological studies carried out over the last years suggest the following model for the pathogenesis of periodontal diseases (Figure 4). Bacterial interactions and modification of the environment result in overgrowth of periodontopathogens including P. gingivalis, B. forsythus, A. actinomycetemcomitans and T. denticola. Adhesins expressed by these bacteria favor their establishment in subgingival sites via attachment to other bacteria or host cells. Bacteria produce proteinases that are not neutralized by plasma proteinase inhibitors found in the gingival crevicular fluid. Bacterial proteinases either cell- or vesicle-associated directly destroy tissue components and negatively affect the host defense system, favoring a continuous migration of inflammatory cells at the infection site. Hydrolysis of the host proteins generates nutrients (peptides, iron) to support growth of the periodontopathogens. LPS present in high concentration in diseased sites induce proinflammatory mediators such as cytokines and initiate host-mediated damage. The inability of phagocytic cells to destroy and digest all the bacteria results in a chronic influx of inflammatory cells associated with release of host proteinases including MMPs. Together, the host and bacterial proteinases severely damage the periodontal tissues.

Although the pathogenesis of periodontal diseases is now better known, there are still unresolved issues that require studies. More particularly, do uncultivable bacteria play a significant role in the initiation and progression of periodontitis? Are there uncover virulence factors not yet identified because studies of characterization are performed in vitro? Our improved knowledge of the pathogenesis of periodontitis may help to develop potential new therapies. Two promising approaches in this regard would be i) to favor the establishment and growth of bacteriocin-producing nonpathogenic bacteria in the subgingival microbiota; and ii) to neutralize both bacterial and host proteinase activities with specific inhibitors.

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