Oral Mycoses	Etiologic Agent
Candidiasis	Candida albicans, C. glabrata, C. tropicalis, C. dubliniensis, C. krusei, etc
Aspergillosis	Aspergillus fumigatus
Blastomycosis	Blastomyces dermatitidis
Coccidioidomycosis	Coccidioides immitis
Cryptococcosis	Cryptococcus neoformans
Fusariosis	Fusarium moniliforme
Geotrichosis	Geotrichum candidum
Histoplasmosis	Histoplasma capsulatum
Mucormycosis	Order Mucorales
Paracoccidiomycosis	Paracoccidioides brasiliensis
Penicilliosis	Penicillium marneffei
Sporotrichosis	Sporothrix schenckii

Microbiologic Specificity of Periodontal Diseases

Nonspecific Plaque Hypothesis

In the mid1900s, periodontal diseases were believed to result from an accumulation of plaque over time, eventually in conjunction with a diminished host response and increased host susceptibility with age.

This theory, termed the nonspecific plaque hypothesis, was supported by epidemiologic studies that correlated both age and the amount of plaque with evidence of periodontitis.^223,^322,³³⁶

The nonspecific plaque hypothesis maintains that periodontal disease results from the “elaboration of noxious products by the entire plaque flora.”²¹⁸ According to this thinking, when only small amounts of plaque are present, the noxious products are neutralized by the host. Similarly, large amounts of plaque would produce large amounts of noxious products, which would essentially overwhelm the host’s defenses.

Several observations contradicted these conclusions. First, some individuals with considerable amounts of plaque and calculus, as well as gingivitis, never developed destructive periodontitis. Furthermore, individuals who did present with periodontitis demonstrated considerable site specificity in the pattern of disease. Some sites were unaffected, whereas advanced disease was found in adjacent sites. In the presence of a uniform host response, these findings were inconsistent with the concept that all plaque was equally pathogenic. Recognition of the differences in plaque at sites of different clinical status (i.e., disease versus health) led to a renewed search for specific pathogens in periodontal diseases and a conceptual transition from the nonspecific to the specific plaque hypothesis.^216,^354,³⁶⁰

Inherent in the nonspecific plaque hypothesis is the concept that control of periodontal disease depends on control of the amount of plaque accumulation. The current standard treatment of periodontitis by debridement (nonsurgical or surgical) and oral hygiene measures still focuses on the removal of plaque and its products and is founded on the nonspecific plaque hypothesis. Thus, although the nonspecific plaque hypothesis has been discarded in favor of the specific plaque hypothesis or the ecologic plaque hypothesis, much clinical treatment is still based on the nonspecific plaque hypothesis.

Specific Plaque Hypothesis

The specific plaque hypothesis states that only certain plaque is pathogenic, and its pathogenicity depends on the presence of or increase in specific microorganisms.²¹⁸ This concept predicts that plaque harboring specific bacterial pathogens results in a periodontal disease because these organisms produce substances that mediate the destruction of host tissues. The association of specific bacterial species with disease came about in the early 1960s, when microscopic examination of plaque revealed that different bacterial morphotypes were found in healthy versus periodontally diseased sites (see Video 23-4: Phase Contrast online). At about the same time, major advances were made in techniques used to isolate and identify periodontal microorganisms. These included improvements in procedures to sample subgingival plaque, handling of samples to prevent killing the bacteria, and media used to grow the bacteria in the laboratory.³⁶² The result was a tremendous increase in the ability to isolate periodontal microorganisms and considerable refinement in bacterial taxonomy.¹⁸⁵ Acceptance of the specific plaque hypothesis was spurred by the recognition of A. actinomycetemcomitans as a pathogen in localized aggressive periodontitis.^254,³⁵⁴ These advances led to a series of association studies focused on identifying specific periodontal pathogens by examining the microbiota associated with states of health and disease in cross-sectional and longitudinal studies.

The introduction of molecular methods for bacterial identification has greatly increased the power of association studies, since we are no longer constrained to analyzing those bacteria that can be cultured (<50% of the total oral microflora). For example, specific members of the phylum Synergistetes that have never been cultured in isolation show a clear association with periodontal disease.⁴⁰⁴ Molecular identification techniques such as DNA checkerboard hybridization can identify and enumerate many different organisms simultaneously and are thus well-suited to high-throughput studies. (see Figure 23-20 online) The association of Socranksy’s “red complex” bacteria, P. gingivalis, T. forsythia, and T. denticola with periodontal disease was based on the analysis of 40 different bacteria in >13,000 plaque samples.³⁶⁴ Nevertheless, disease association studies do not reveal whether the presence of specific bacteria causes or correlates with the presence of disease. In addition, these studies have shown that periodontal disease can occur even in the absence of defined “pathogens,” such as red complex bacteria, and conversely that “pathogens” may be present in the absence of disease.

Ecologic Plaque Hypothesis

In the 1990s, Marsh and co-workers developed the “ecologic plaque hypothesis” as an attempt to unify the existing theories on the role of dental plaque in oral disease (Figure 23-33). According to the ecologic plaque hypothesis, both the total amount of dental plaque and the specific microbial composition of plaque may contribute to the transition from health to disease. The health-associated dental plaque microflora is considered to be relatively stable over time and in a state of dynamic equilibrium or “microbial homeostasis.” The host controls subgingival plaque to some extent by a tempered immune response and low levels of GCF flow. Perturbations to the host response may be brought about by the excessive accumulation of (nonspecific) dental plaque, or by plaque-independent host factors (e.g., the onset of an immune disorder, changes in hormonal balance such as in pregnancy), or environmental factors (e.g., smoking, diet). Changes in the host status, such as inflammation, tissue degradation, and/or high GCF flow, may lead to a shift in the microbial population in plaque, culminating in periodontal disease. The ecologic plaque hypothesis is entirely consistent with observations that disease-associated organisms are minor components of the oral microflora in health; these organisms are kept in check by interspecies competition during microbial homeostasis. Disease is caused by the overgrowth of specific elements of dental plaque when the local microenvironment changes, but it is not necessarily the same species in each case. An important consideration of the ecologic plaque hypothesis is that therapeutic intervention can be useful on a number of different levels. Eliminating the disease-inducing stimulus, whether it is microbial, host, or environmental, will help to restore microbial homeostasis. Targeting specific microorganisms may be less effective since the conditions for disease will remain. One pathogen will simply be replaced by another.

Figure 23-33 Ecologic plaque hypothesis in relation to periodontal diseases: gingivitis and periodontitis. Accumulation of plaque causes inflammation of adjacent tissues (gingivitis) and other environmental changes that favor the growth of gram-negative anaerobes and proteolytic species, including periodontopathogens. The increased proportions of such species results in destruction of periodontal tissues (i.e., periodontitis). Eh, Redox-potential; GCF, gingival crevicular fluid; gram +ve, gram-positive; gram –ve, gram-negative.

(Adapted from Marsh PD: Adv Dent Res 8:263, 1994.)

Complicating Factors

The identification of bacterial pathogens in periodontal diseases has been difficult because of a number of factors.³⁶² The periodontal microbiota is a complex community of microorganisms, many of which are still difficult or impossible to cultivate or identify. The chronic nature of periodontal disease has complicated the search for bacterial pathogens. It was previously thought that periodontal diseases progressed at a slow but steady rate.²¹⁴ However, epidemiologic studies established that the disease progresses at different rates, with alternating episodes of rapid tissue destruction and periods of remission (Figure 23-34).³⁶⁵ At this moment, by means of multilevel modelling, the linear and burst theories of periodontitis progression are considered to be a manifestation of the same phenomenon: some sites improve while others progress, in a cyclic manner.¹⁰⁷ Identification of the microorganisms found during the different phases of the disease progression is technically challenging. The interpretation of microbiologic data is further complicated by the clinical classification of the disease status, an area that has undergone a number of recent revisions.^15,³⁹⁶ Previous and perhaps current classifications involve the grouping of potentially different disease states because of difficulties in accurately distinguishing them clinically. It is important to recognize that groupings such as this may obscure microbiologic associations.

Figure 23-34 Diagrammatic representation of possible modes of progression of chronic destructive periodontal diseases. Sites on the x-axis are plotted against time on the y-axis, and activity is shown on the z-axis. A, Some sites show progressive loss of attachment over time, whereas others show no destruction. The time of onset and the extent of destruction vary among sites. B, Random burst model. Activity occurs at random at any site. Some sites show no activity, whereas others show one or several bursts of activity. The cumulative extent of destruction varies among sites. C, Asynchronous multiple burst model. Several sites show bursts of activity over a finite period, followed by prolonged periods of inactivity. Occasional bursts may occur infrequently at certain sites at later periods. Other sites show no periodontal disease activity at any time. The difference from the model shown in B is that in C, the majority of destructive disease activity takes place within a few years of the individual’s life.

(Courtesy Drs. S. Socransky, A. Haffajee, M. Goodson, and J. Lindhe, Boston, and Göteborg, Sweden.)

Currently, periodontitis is considered a mixed infection. This has significant implications for both the diagnosis and treatment of periodontitis. In the diagnosis, some evaluate the presence of up to 40 species, but it still is unclear whether some combinations of species are more pathogenic than others. The treatment is directed to the eradication/reduction of the number of all key periodontopathogens. Since several species might be involved, the appropriate use of antimicrobials (especially antibiotics) is extremely difficult to define because not all expected periodontopathogens are equally susceptible to the same antibiotic.

Recent microbiologic diagnostic tests indicated that the presence of periodontopathogens alone is not sufficient for the development of periodontitis. Because of the high sensitivity of these tests, several pathogens have been detected in periodontitis-free patients. Thus merely the presence of pathogens is not sufficient for disease and the amount of the pathogens plays a key role in relation to disease.³²¹ These observations have major clinical implications. First, the specificity of microbial detection is dramatically reduced (specificity = presence of pathogen means periodontitis). In other words, even though a microbiologic analysis is positive, the patient can be without disease.²²² Second, the understanding of the etiology becomes more complicated because the threshold level for periodontopathogens between health and disease is unknown and obviously subject dependent. Third, for several species, large intrastrain variations in genetic information have been detected (different genotypes) so that, in fact, information on the genotype is needed before the pathogenicity of the strain can be estimated.^55,^72,^115,²⁷² The quality of the host response also plays an essential role, but this cannot yet be correctly estimated. Finally, one can question whether periodontopathogens are indigenous species or exogenous. Indeed, the newer techniques have reported high detection frequencies of all pathogens in healthy subjects.^222,³³² This has significant impact on the treatment strategies. For indigenous species the endpoint of a therapy is the reduction of the species, whereas for exogenous species the endpoint is eradication and prevention of reinfection.

Currently, it is impossible to alter the susceptibility of the host, so periodontal therapy must be focused on the reduction/elimination of periodontopathogens in combination with the reestablishment, often by surgical pocket elimination, of a more suitable environment (less anaerobic) for a more beneficial microbiota. Several studies indicate that the presence of the previously mentioned periodontopathogens (persisting or reestablished after treatment) is associated with a negative clinical outcome of periodontal treatment.^120,^311,³¹² Because several species might be involved, the use of antimicrobials (especially antibiotics) is extremely difficult since not all expected periodontopathogens are susceptible to the same antibiotic and susceptibility depends on the geographic region.^300,⁴⁰²

It is obvious that several key questions still remain unanswered. Some researchers question whether the presence of specific microorganisms in the periodontal pocket is the cause or a consequence of the disease.³⁶⁶ Since most periodontopathogens are fastidious strict anaerobes, they may contribute little to the initiation of disease in shallow gingival pockets but are found only in deep periodontal pockets, their preferred habitat.

Criteria for Identification of Periodontopathogens

In the 1870s, Robert Koch developed the classic criteria by which a microorganism can be judged to be a causative agent in human infections. These criteria, known as Koch’s postulates, stipulate that the causative agent must do the following:

1 Be routinely isolated from diseased individuals.

2 Be grown in pure culture in the laboratory.

3 Produce a similar disease when inoculated into susceptible laboratory animals.

4 Be recovered from lesions in a diseased laboratory animal.

Difficulties exist in the application of these criteria to polymicrobial diseases, and the applicability of Koch’s postulates has been increasingly challenged in recent years. In the case of periodontitis, three primary problems are (1) the inability to culture all the organisms that have been associated with disease (e.g., many of the oral spirochetes), (2) the difficulties inherent in defining and culturing sites of active disease, and (3) the lack of a good animal model system for the study of periodontitis.³⁶² In fact, if the ecologic plaque hypothesis proves correct, it must be inherently impossible to fulfill Koch’s postulates, since no single organism or group of organisms is responsible for all cases of disease.

Sigmund Socransky, a researcher at the Forsyth Dental Center in Boston, proposed criteria by which periodontal microorganisms may be judged to be potential pathogens.³⁶² According to these criteria, a potential pathogen must do the following:

1 Be associated with disease, as evident by increases in the number of organisms at diseased sites

2 Be eliminated or decreased in sites that demonstrate clinical resolution of disease with treatment

3 Induce a host response, in the form of an alteration in the host cellular or humoral immune response

4 Be capable of causing disease in experimental animal models

5 Produce demonstrable virulence factors responsible for enabling the microorganism to cause destruction of the periodontal tissues

Data supporting the role of A. actinomycetemcomitans and P. gingivalis as periodontal pathogens, based on these criteria, have been presented earlier. The association and elimination criteria are discussed in the preceding sections. The latter three criteria focus on the host parasite interaction, which is discussed in Chapter 25.

The Transition from Health to Disease

The oral microbial ecology is dynamic. The presence and numbers of particular microorganisms, even as a part of a community colonizing a niche in a human being, are controlled by the type and quantity of nutrients present (nutritional determinant), their ability to tolerate the physicochemical factors operating there (physicochemical determinants), and their ability to withstand any antimicrobial compounds (biological determinants) or mechanical removal forces (mechanical determinants).⁴²⁴ Although these determinants are well-defined from a theoretic point of view, they overlap each other, especially in more complex environments (i.e., multispecies environments). Bacteria are living species not passive bystanders. They will interact with their environment and vice versa. Therefore the microbial ecology will change its composition or the composition of the microbial ecology will be changed when transitioning from a healthy status to a diseased status or vice versa. A change in the composition of a bacterial community as the result of external, nonmicrobial factors is termed allogenic succession. Smoking is a good example of such interaction. In autogenic succession (i.e., a change in the composition of a microbial community arising from microbial activities), interbacterial and viral-bacterial interactions are involved.

Early microscopy studies clearly demonstrated that subgingivally the number and proportions of different bacterial morphotypes differ between healthy and diseased sites (Figures 23-35 and 23-36; see Video 23-4: Phase Contrast online).^210,^354,³⁶⁰ The total number of bacteria, determined by microscopic counts per gram of plaque, was twice as high in periodontally diseased sites than in healthy sites.³⁶⁰ Because considerably more plaque is found at diseased sites, this suggests that in general the total bacterial load in diseased sites is greater than that at healthy sites. Fewer coccal cells and more motile rods and spirochetes are found in diseased sites than in healthy sites.²¹⁰ Although this morphologic criterion can be of diagnostic value, one should remember that nearly all key periodontal pathogens except Campylobacter rectus and spirochetes are immobile rods, which adds to the confusion regarding the bacterial etiology of periodontal diseases. By culturing it appears that the microbiota in periodontally healthy sites consist predominantly of gram-positive facultative rods and cocci (approximately 75%).³⁵⁴ The recovery of this group of microorganisms is decreased proportionally in gingivitis (44%) and periodontitis (10% to 13%). These decreases are accompanied by increases in the proportions of gram-negative rods, from 13% in health to 40% in gingivitis and 74% in advanced periodontitis.

Figure 23-35 Pie charts based on culturing studies, representing the relative proportion of different morphotypes in subgingival samples in cases of periodontal health, gingivitis, and periodontitis. A clear distinction is made between facultative species and obligate anaerobic species. Spirochetes are not included.

Figure 23-36 Phase-contrast micrograph of a plaque sample from a healthy (A) and a periodontitis patient (B). Note the large rods and spirochetes in the periodontitis plaque sample.

The current concept concerning the etiology of periodontal diseases considers 3 groups of factors that determine whether active periodontal destruction will occur in a subject: a susceptible host, the presence of pathogenic species, and the absence/small proportion of so-called beneficial bacteria.^12,^359,^362,^369,⁴²⁷ The clinical manifestations of periodontal destruction thus result from a complex interplay between these etiologic agents. In general, small amounts of bacterial plaque can be controlled by the body’s defense mechanisms without destruction, but when the balance between bacterial load and host response is disturbed, periodontal destruction might occur. The latter can be the case when the subject is very susceptible to periodontal infections, and/or when he or she is infected by a large amount of bacteria and/or by a very pathogenic microbiota.

Host Susceptibility

The susceptibility of the host is determined in part by genetic factors but can be influenced by environmental/behavioral factors such as smoking, stress, and viral infections. Genetic factors are known to play a role, especially in patients with so-called early onset periodontitis, now referred to as aggressive periodontitis.²³⁷ Reports on the familial nature of chronic forms of periodontitis are less conclusive. Aggregation of aggressive periodontitis within families is consistent with a genetic predisposition, although common environmental factors cannot be excluded.

Hart and co-workers even suggested autosomal modes of transmission.¹³⁷ Some IL-1 gene polymorphisms are known to be positively associated with periodontitis but genetic-environmental interactions (e.g., IL-1 genotype positive and smoking) are more important than genetic factors alone.³⁷⁷ Receptors for the Fc domain of immunoglobulin G (IgG), FcαR provide a critical link between specific humoral and the cellular branches of the immune system. Polymorphisms of this FcαR gene tend to be associated with both aggressive and chronic forms of periodontitis. Hewitt and co-workers recently reported the association between a decreased activity of the lysosomal protease cathepsin C and chronic periodontitis.¹⁴³ Mutations in the cathepsin C gene result in Papillon-Lefèvre syndrome associated with an aggressive periodontitis that is difficult to control.^23,^98,²²⁵

Smoking is considered a behavioral or eventually an environmental factor. It dramatically increases host susceptibility to periodontal breakdown.

In one of the largest studies on risk factors for periodontitis, it was shown that smokers were at greater risk for experiencing severe bone loss than nonsmokers, with odds ratios ranging from 3.25 (95% confidence interval [CI]: 2.33 to 4.54) to 7.28 (95% CI: 5.09 to 10.31) for light and heavy smokers, respectively. Moreover, a direct and linear dose-response relationship between the levels of tobacco smoking and attachment loss was found by Grossi and co-workers.^116,¹¹⁷ The smoking pack-years or years of exposure to tobacco products are significant risk factors for periodontitis, regardless of other social and behavioral factors. ^9,¹⁵⁸ There is also increasing evidence that smokers heal less satisfactorily after periodontal therapy than nonsmokers.³¹² This also seems to be dose-dependent.¹⁶¹ Smoking negatively influences the results of root coverage by plastic surgery, guided tissue regeneration, and even increased failure rates for osseointegrating oral implants.^208,^242,³⁸⁹

Nevertheless, the exact nature of the relationship between periodontitis and smoking is unclear.^21,¹⁵⁹

Recent studies indicated that smoking impairs the immune response to periodontal pathogens by a decreased chemotaxis, a decreased phagocytic capacity of polymorphonuclear leukocytes and by decreased levels of IgG and IgA.^90,^226,^286,²⁸⁷

On the other hand, there are conflicting reports regarding the effects of smoking on the oral microbiota. Some are in favor of an increased prevalence of P. gingivalis, A. actinomycetemcomitans, T. forsythia, E. coli, and C. albicans in smokers, while others report no difference.* Recently it has been shown that smoking cessation can alter the subgingival biofilm and suggest a mechanism for improved periodontal health associated with smoking cessation.¹⁰⁰ There is some evidence that epithelial cells of smokers are more prone to colonization by respiratory pathogens. Depending on the bacterial strain, the relative increase in the number of adhering pathogens in smokers versus nonsmokers ranged from 40% up to 150%.^91,³²⁵ Interestingly, cigarette smoke has recently been shown to modify the expression of P. gingivalis genes directly, including several virulence-associated genes.²⁰ After exposure to cigarette smoke extract, P. gingivalis cells induce a lower proinflammatory response than unexposed P. gingivalis cells.

Recently, viral infections have been added to the list of factors that modify host susceptibility. HIV and herpesviruses are often associated with periodontal infections.

Several studies show a clear association between HIV infection and some distinct forms of periodontal infection (i.e., necrotizing lesions).^270,^279,³⁴⁰ The increased prevalence and severity of chronic periodontitis in HIV-positive subjects suggest that HIV infection predisposes to chronic periodontitis.³²⁴ A similar association has been shown for herpesviruses and periodontitis.³⁵⁵ Human cytomegalovirus (hCMV) was positively associated with severe periodontitis (odds ratio: 4.65; CI: 1.12-19.30).⁶³ Moreover, active hCMV replication in periodontal sites may suggest that hCMV reactivation triggers periodontitis activity.⁶¹ However, caution is warranted in view of concerns regarding sampling, methods, and interpretation.⁴⁶

It is believed that virus-associated periodontal destruction originates from virus-induced changes in the host response to the local subgingival microbiota.³⁵⁵

The influence of viral infections and bacterial colonization of cells was investigated by Sanford and co-workers, who first described an increased adhesion of bacteria to virally infected cultured cells.³³¹ Similar observations have been made since then for a wide variety of viruses, cells, and bacteria.³⁴ Viral infections relatively increase bacterial adhesion to cells by 20% to 362%.^85,¹²⁵

In relation to periodontopathogen adhesion to virally infected cells, Teughels and co-workers showed recently a 100% increase in A. actinomycetemcomitans colonization when epithelial cells were infected with hCMV.³⁸⁵

Increasing evidence suggests that emotional stress and/or negative life events can play an important role in the development and progression of periodontitis and may also modify the response to periodontal treatment.

Stress associated with financial strain is a significant risk indicator for more severe periodontitis in adults (odds ratio: 2.24; CI: 1.15-4.38).¹⁰⁴

Emotional and psychologic load can influence the immune system and will also alter oral health behaviors.⁶⁸ Both factors increase the susceptibility for periodontitis.

Racial factors also have been implicated in the differences in prevalence of early-onset periodontitis and in the frequency of detection of putative periodontopathogens.^22,²¹⁵

Pathogenic Bacteria

The second essential factor for disease initiation and progression is the presence of one or more pathogens, of the appropriate clonal type and in sufficient numbers. Despite the difficulties inherent in characterizing the microbiology of periodontal diseases, a small group of pathogens is recognized because of their close association with disease. There are obvious data to support the designation of A. actinomycetemcomitans, T. forsythia, and P. gingivalis as key pathogens, since they are strongly associated with the periodontal disease status, the disease progression, and unsuccessful therapy. For the following list of bacteria, moderate evidence for etiology has been reported, at least if their concentration passes a certain threshold level: P. intermedia, Prevotella nigrescens (see Figure 23-1, I), C. rectus, Parvimonas micra (see Figure 23-1, J), F. nucleatum, Eubacterium nodatum (see Figure 23-1, N), and various spirochetes.^12,^359,^362,^369,⁴²⁷ The significance for the role of these key pathogens is largely based on epidemiologic data, the ability of these microorganisms to produce disease when inoculated in animals, and/or their capacity to produce virulence factors. However, the mere presence of putative periodontopathogens in the gingival crevice is not in itself sufficient to initiate or cause periodontal inflammation. An elevation in the relative proportion/number of these pathogens, to reach a critical mass, seems more crucial to mount an effective tissue-damaging process. Indeed, even in health, periodontopathogens are or can be present in the gingival crevice, albeit in low numbers, as members of the normal resident flora.²²² Table 23-10 gives a general overview of the detection frequency for most key pathogens in different forms of periodontal infections. It is immediately obvious that there is no black-and-white situation; most pathogens might (but not necessarily) have to be present for specific forms of periodontitis. This also illustrates that it is most of the time difficult to use the microbial composition to differentiate between different forms of periodontal infections. Table 23-11 further highlights the complexity of the microbiology of periodontitis. Most periodontopathogens can also be detected in healthy subjects with frequencies ranging from 10% to 85%. It is obvious that this automatically reduces the specificity of microbiologic testing in periodontology.

TABLE 23-10 Prevalence of Microbial Species Associated with Various Clinical Forms of Periodontitis

TABLE 23-11 Prevalence of Key Pathogens in Healthy Subjects and Patients with Periodontitis

Beneficial Species

The role of “beneficial” species of the host is less obvious in the development of periodontal diseases.³⁶² Often, bacteria and host cells establish a commensal relationship, beneficial for both.

The host vaginal epithelial cells, for example, supply glucose for colonized lactobacilli, which in turn produce acid. A lowering of the pH prevents the growth of many other species that have deleterious effects on the vaginal environment.³¹⁷ These indigenous bacteria and their products can thus be considered a necessary and beneficial component of a healthy body.

The role of beneficial species of the host is also less obvious in the progression of periodontitis.^317,³⁶² These bacteria can affect the pathogenic species in different ways and thus modify the disease process, as follows: (1) by passively occupying a niche that may otherwise be colonized by pathogens, (2) by actively limiting a pathogen’s ability to adhere to appropriate tissue surfaces, (3) by adversely affecting the vitality or growth of a pathogen, (4) by affecting the ability of a pathogen to produce virulence factors, or (5) by degrading virulence factors produced by the pathogen. The often-used textbook example of such a beneficial action is the effect of S. sanguinis, formerly known as S. sanguis, on A. actinomycetemcomitans.³⁹¹ S. sanguinis produces H₂O₂, which either directly or by host enzyme amplification can kill A. actinomycetemcomitans.¹⁴⁴ Although these so-called beneficial bacteria might be of importance for maintaining a healthy subgingival ecosystem, evidence is limited.³¹⁷ Despite a rapidly increasing knowledge on periodontopathogen-host interactions, the role of beneficial microbiota in this crosstalk remains obscure. In periodontal microbiology, a bacterial strain is considered beneficial when its prevalence is high in periodontal health and low in diseased situations. It is shown for other fields in microbiology that direct and indirect microbial interactions can suppress the emergence of pathogens.³⁵ The importance of the latter is evidenced within the oral cavity by the development of yeast infections when the normal oral flora is reduced, for instance after a period of systemic antibiotic usage.⁴¹⁰

Since it is impossible so far to alter the susceptibility of the host, periodontal therapy is necessarily focused on the reduction/elimination of periodontopathogens in combination with the reestablishment, often by surgical pocket elimination, of a more suitable environment (e.g., less anaerobic) for a more beneficial microbiota. Several studies indeed indicated that the presence of the previously mentioned periodontopathogens (persisting or reestablished after treatment) is associated with a negative clinical outcome of periodontal treatment.^67,^121,^311,^312,³⁶⁴

Taking all of this together, it is clear that the periodontal microbiota will shift when going from a periodontally healthy situation to a periodontally diseased situation (Figure 23-37).

Figure 23-37 Vitality stain of subgingival plaque. A, Plaque derived from a healthy patient and primarily consists of cocci. B, Plaque derived from a periodontitis patient. Note the important morphologic difference with A. Green bacteria are alive; red bacteria are dead.

Comparing the microbiota between health, gingivitis, and periodontitis, the following microbial shifts can be identified as health progresses to periodontitis:

• From gram-positive to gram-negative

• From cocci to rods (and at a later stage to spirochetes)

• From nonmotile to motile organisms

• From facultative anaerobes to obligate anaerobes

• From fermenting to proteolytic species

Periodontal Health

The recovery of microorganisms from periodontally healthy sites is lower when compared with that from diseased sites (Table 23-12). The bacteria associated with periodontal health are primarily gram-positive facultative species and members of the genera Streptococcus and Actinomyces (e.g., S. sanguinis, S. mitis, A. oris, A. israelii, A. gerencseriae, A. viscosus (see Figure 23-1, C), and A. naeslundii). Small proportions of gram-negative species are also found, most frequently P. intermedia, F. nucleatum, F. nucleatum ssp. polymorphum, F. periodonticum, Capnocytophaga spp. (C. gingivalis, C. ochracea, and C. sputigena), Neisseria spp., and Veillonella spp. Microscopical analyses indicate that a few spirochetes and motile rods also may be found. Based on checkerboard DNA-DNA hybridization data, Eubacterium saburreum, Propionibacterium acnes, S. anginosus, S. gordonii, and S. oralis can also be considered as health-associated bacteria.^92,^382,^429,⁴³⁰

TABLE 23-12 Summary of Bacterial Species Significantly Associated with Different Clinical Conditions (Based on Culture, Polymerase Chain Reaction (PCR), Checkerboard Data)

Certain bacterial species have been proposed to be protective or beneficial to the host, including S. sanguinis, Veillonella parvula (see Figure 23-1, B), and C. ochracea. They are typically found in high numbers at periodontal sites that do not demonstrate attachment loss (inactive sites) but in low numbers at sites where active periodontal destruction occurs.^81,³⁶² These species probably function in preventing the colonization or proliferation of pathogenic microorganisms. Clinical studies have shown that sites with high levels of C. ochracea and S. sanguinis are associated with a greater gain in attachment after therapy, further supporting this concept.³⁶² A better understanding of plaque ecology and the interactions between bacteria and their products in plaque will undoubtedly reveal many other examples.

Gingivitis

The development of gingivitis has been extensively studied in a model system referred to as experimental gingivitis and initially described by Harald Löe and co-workers.^216,³⁸⁷ Periodontal health is first established in human subjects by cleaning and rigorous oral hygiene measures, followed by abstinence from oral hygiene for 21 days. After 8 hours without oral hygiene, bacteria may be found at concentrations of 10³ to 10⁴ per square millimeter of tooth surface and will increase in number by a factor of 100 to 1000 in the next 24-hour period.³⁶⁷ After 36 hours, the plaque becomes clinically visible (see Video 23-1 online). The initial microbiota of experimental gingivitis (see Figure 23-21) consists of gram-positive rods, gram-positive cocci, and gram-negative cocci. The transition to gingivitis is evident by inflammatory changes and is accompanied first by the appearance of gram-negative rods and filaments, then by spirochetal and motile microorganisms.³⁸⁷

The subgingival microbiota of dental plaque-induced gingivitis (chronic gingivitis) differs from that of both health and chronic periodontitis (see Table 23-12). It consists of roughly equal proportions of gram-positive (56%) and gram-negative (44%) species, as well as facultative (59%) and anaerobic (41%) microorganisms.³⁵⁴ It should be noted that the majority of predominant species in chronic periodontitis are already present in the gingivitis state, however, mostly in small numbers.

Predominant gram-positive species include Streptococcus spp. (S. sanguinis, S. mitis, S. intermedius, S. oralis, and S. anginosus), Actinomyces spp. (A. viscosus, A. naeslundii), Eubacterium nodatum, and Parvimonas micra. The gram-negative microorganisms are predominantly Capnocytophaga spp., Fusobacterium spp., Prevotella spp., Campylobacter gracilis, Campylobacter concisus, V. parvula, Haemophilus spp., and E. corrodens. Both groups are commonly associated with gingivitis. However, the following gram-negative species are commonly found in periodontitis but are also associated with gingivitis but in fewer numbers: P. gingivalis, T. forsythia, P. intermedia, Campylobacter rectus, Treponema spp., and A. actinomycetemcomitans serotype a.^244,^250,³⁵⁴

Pregnancy-associated gingivitis is an acute inflammation of the gingival tissues associated with pregnancy. This condition is accompanied by increases in steroid hormones in crevicular fluid and dramatic increases in the levels of P. intermedia and C. rectus, which use the steroids as growth factors.^181,⁴³³

Studies of gingivitis support the conclusion that disease development is associated with selected alterations in the microbial composition of dental plaque and is not simply the result of an accumulation of plaque. Gingivitis was generally believed to precede the development of chronic periodontitis; however, many individuals demonstrate long-standing gingivitis that never advances to destruction of the periodontal attachment.^38,²¹³

Chronic Periodontitis

Numerous forms of periodontal disease are found in adult populations, characterized by different rates of progression (see Figure 23-34) and different responses to therapy.¹⁵ Studies in which untreated populations were examined over long time intervals indicate disease progression at mean rates ranging from 0.05 to 0.3 mm of attachment loss per year (i.e., the gradual model).³⁸ When populations are examined over short time intervals, individual sites demonstrated short phases of attachment destruction interposed by periods of no disease activity (i.e., the burst model).¹¹² At this moment, by means of multilevel modelling, the linear and burst theories of periodontitis progression are considered to be a manifestation of the same phenomenon: some sites improve while others progress, in a cyclical manner.¹⁰⁷

Microbiologic examinations of chronic periodontitis have been carried out in both cross-sectional and longitudinal studies; the latter have been conducted with and without treatment. These studies support the concept that chronic periodontitis is associated with specific bacterial agents. Microscopic examination of plaque from sites with chronic periodontitis has consistently revealed elevated proportions of spirochetes (see Video 23-4 online).^210,²²⁰ Cultivation of plaque microorganisms from sites of chronic periodontitis reveals high percentages of anaerobic (90%) gram-negative (75%) bacterial species^353,³⁵⁴ (see Figure 23-27 and Table 23-12).

In chronic periodontitis, the bacteria most often detected at high levels include P. gingivalis, T. forsythia, P. intermedia, P. nigrescens, C. rectus, Eikenella corrodens (see Figure 23-1, S), F. nucleatum, A. actinomycetemcomitans (often serotype b), P. micra, E. nodatum, Leptotrichia buccalis, Treponema (T. denticola), Selenomonas spp. (S. noxia), and Enteric spp.* When periodontally active sites (i.e., with recent attachment loss) were examined in comparison with inactive sites (i.e., with no recent attachment loss), C. rectus, P. gingivalis, P. intermedia, F. nucleatum, and T. forsythia were found to be elevated in the active sites.⁸⁰ Furthermore, detectable levels of P. gingivalis, P. intermedia, T. forsythia, C. rectus, and A. actinomycetemcomitans are associated with disease progression and their elimination via therapy is associated with an improved clinical response.^59,^80,^120,^358,⁴¹⁹ Both P. gingivalis and A. actinomycetemcomitans are known to invade host tissue cells, which may be significant in aggressive forms of periodontitis.^52,^56,³²⁷

Recent studies have documented an association between chronic periodontitis and viral microorganisms of the herpesviruses group, most notably EBV type 1 (EBV-1) and hCMV.⁶² Further, the presence of subgingival EBV-1 and hCMV are associated with high levels of putative bacterial pathogens, including P. gingivalis, T. forsythia, P. intermedia, and T. denticola. These data support the hypothesis that viral infection may contribute to periodontal pathogenesis, but the potential role of viral agents remains to be determined.

Localized Aggressive Periodontitis

Several forms of periodontitis are characterized by rapid and severe attachment loss occurring in individuals during or before puberty. Localized aggressive periodontitis, previously referred to as localized juvenile periodontitis (LJP), develops around the time of puberty, is observed in females more often than in males, and typically affects the permanent first molars and incisors (Figure 23-38). This condition is almost uniformly seen in individuals who demonstrate some systemic defect in immune regulation, and often affected individuals demonstrate defective neutrophil function. Without treatment, the local form often extends to a more generalized form with severe attachment loss around many teeth. The first symptoms of LJP are already detectable in the deciduous dentition, especially via periodontal destruction around the canines and second molars.⁴⁸

Figure 23-38 Clinical picture (A) and intraoral radiograph (B) of the dramatic bone destruction in adolescent with localized aggressive periodontitis.

The microbiota associated with localized aggressive periodontitis is predominantly composed of gram-negative, capnophilic, and anaerobic rods^254,^255,³⁵⁴ (see Table 23-12). Microbiologic studies indicate that almost all LJP sites harbor A. actinomycetemcomitans, which may comprise as much as 90% of the total cultivable microbiota.^182,²⁴⁷ Other organisms found in significant levels include P. gingivalis, E. corrodens, C. rectus, F. nucleatum, B. capillus, Eubacterium brachy, and Capnocytophaga spp. and spirochetes.^182,^238,^247,²⁴⁹ Recent checkerboard DNA-DNA hybridization studies have also shown high proportions of E. nodatum, P. intermedia, Treponema spp. (T. denticola), L. buccalis, and T. forsythia.^92,⁴²⁹ Herpesviruses, including EBV-1 and hCMV, also have been associated with localized aggressive periodontitis.^62,^238,³⁸⁸

A. actinomycetemcomitans is generally accepted as the primary etiologic agent in most but not all cases of localized aggressive periodontitis.^180,³⁶² A high leukotoxic clone is uniquely associated with aggressive forms of periodontitis. However, the fact that not all aggressive periodontitis patients show this feature points to the possibility that several other factors may provoke this disease.²⁴³ Studies of therapy indicate that mechanical debridement in combination with systemic antibiotic treatment are necessary to control the levels of A. actinomycetemcomitans in this disease.^182,^313,³¹⁴ The failure of mechanical therapy alone may relate to the ability of this organism to invade host tissues.^52,^56,³²⁷

Aggressive Periodontitis

Aggressive periodontitis, also known as generalized aggressive periodontitis, early-onset periodontitis, or rapidly progressive periodontitis, is a severe form of periodontitis that occurs at a relatively young age (between 20 to 40 years). It is characterized by a severe gingivitis, a large number of deepened pockets, and a high tendency for bleeding on probing. In relation to the patient’s age, there is a lot of bone destruction. It is sometimes considered as a generalized form of localized aggressive periodontitis. From a microbiologic point of view, it has a lot of similarity toward localized aggressive periodontitis (see Table 23-12). However, aggressive periodontitis is clearly more dominated by P. gingivalis, P. intermedia, T. forsythia, and Treponema spp.(T. denticola) and less by A. actinomycetemcomitans. Recent checkerboard DNA-DNA hybridization studies indicate also high proportions of E. nodatum, Campylobacter gracilis, Campylobacter showae, and P. nigrescens.^92,⁴²⁹

Necrotizing Periodontal Diseases

Necrotizing periodontal diseases (Figure 23-39) present as an acute inflammation of the gingival and periodontal tissues characterized by necrosis of the marginal gingival tissue and interdental papillae. Clinically, these conditions often are associated with stress or HIV infection. They may be accompanied by malodor, pain, and possibly systemic symptoms, including lymphadenopathy, fever, and malaise. Microbiologic studies indicate that high levels of P. intermedia and especially of spirochetes and F. nucleatum are found in necrotizing ulcerative gingivitis lesions. Spirochetes are found to penetrate necrotic tissue and apparently unaffected connective tissue.^211,²¹²

Figure 23-39 Clinical picture of lower front teeth with necrotizing gingivitis.

Abscesses of the Periodontium

Periodontal abscesses (Figure 23-40) are acute lesions that may result in very rapid destruction of the periodontal tissues. They often occur in patients with untreated periodontitis but also may be found in patients during maintenance or after scaling and root planing of deep pockets. Periodontal abscesses also may occur in the absence of periodontitis; for example, associated with impaction of a foreign object (e.g., a popcorn kernel or dental floss) or with endodontic problems.¹⁴² Typical clinical symptoms of periodontal abscesses include pain, swelling, suppuration, bleeding on probing, and mobility of the involved tooth. Signs of systemic involvement may be present, including cervical lymphadenopathy and an elevated white blood cell count.¹⁴¹ Investigations reveal that bacteria recognized as periodontal pathogens are commonly found in significant numbers in periodontal abscesses. These microorganisms include F. nucleatum, P. intermedia, P. gingivalis, P. micra, and T. forsythia.^124,^141,²⁵⁴

Figure 23-40 Clinical pictures (A, B, and D) of a patient with several periodontal abscesses. The intraoral radiograph (C) shows the severity of the periodontal destruction. Gutta-percha points (B and C) are inserted in the fistulae to show their course.

Periimplantitis

The term periimplantitis refers to an “inflammatory process” affecting the tissues around an already osseointegrated implant resulting in loss of supporting bone.¹⁵ This inflammatory process has been associated (e.g., in animal studies, cross-sectional and longitudinal observations in man) with a microbiota comparable to that of periodontitis (high proportion of anaerobic gram-negative rods, motile organisms, and spirochetes), but this association does not necessarily prove a causal relationship (for review see Quirynen et al).²⁹⁵ Healthy periimplant pockets are characterized by high proportions of coccoid cells, a low ratio of anaerobic/aerobic species, a low number of gram-negative anaerobic species, and low detection frequencies for periodontopathogens.^3,^30,^198,²⁶³ Implants with periimplantitis reveal a complex microbiota encompassing conventional periodontal pathogens (see Table 23-12). Species, such as A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. micra, C. rectus, F. nucleatum, P. intermedia, T. denticola, and Capnocytophaga, are often isolated from failing sites but can also be detected around healthy periimplant sites.³⁴³ Other species, such as Pseudomonas aeruginosa, Enterobacteriaceae spp, C. albicans, or staphylococci are also frequently detected around implants.⁷ These organisms are uncommon in the subgingival area but have been associated with refractory periodontitis.³⁵⁹ High proportions of Staphylococcus aureus and S. epidermidis on oral implants have been reported.³⁰⁸ The relative resistance of these organisms to commonly utilized antibiotics suggests that their presence might represent an opportunistic colonization secondary to systemic antibiotic therapy.³⁵⁸

Some large-scale clinical follow-up studies seem to indicate that implant failures cluster within patients, with increased odd ratios for a second implant failure in patients that have already lost one implant.^399,⁴²⁰ These observations indicate that within patients systemic factors are important in characterization of implant losses.

Virulence Factors of Periodontopathogens

It is clear that some organisms, such as P. gingivalis, A. actinomycetemcomitans, spirochetes, and P. intermedia, are strongly associated with a number of periodontal diseases. However, periodontal disease never occurs in the absence of a complex microbiota and it is often difficult, if not impossible, to determine precisely how different organisms contribute to an individual case of disease. In fact, the contributions of specific bacteria to disease may be unimportant according to the ecological plaque hypothesis. Targeting one or more “pathogens” will not necessarily cure disease, since other organisms with similar activities might take their place. It may make sense therefore to focus on the specific molecules that contribute to disease (virulence factors), rather than on the microorganisms that produce them. In fact, it is often difficult to separate the virulence determinants from the organisms that produce them. For example, adhesins are produced by commensal organisms, as well as by pathogens, yet only those adhesins that promote attachment of a pathogenic organism could be considered virulence determinants. With this in mind, some of the known or putative virulence factors for periodontal disease are described below. It is important to note the following:

1 Only a proportion of periodontal bacteria have ever been isolated, and there are almost certainly many other virulence factors that are currently unknown.

2 Most of our understanding of virulence factors has come from studies on a very limited number of bacterial species and strains. It is far from clear that the molecules that have been studied in the greatest detail are truly representative of their classes.

Virulence factors of periodontal micro-organisms can be subdivided into (1) factors that promote colonization (adhesins), (2) toxins and enzymes that degrade host tissues, and (3) mechanisms that protect pathogenic bacteria from the host.

Adhesive Surface Proteins and Fibrils

To colonize the periodontal pocket, bacteria must adhere to cells or tissues in the region such as teeth, the existing microbial biofilm, or the pocket epithelium. Bacterial cell surface structures provide the points of contact. Often, these structures extend some distance from the cell surface. Fimbriae, or pili, are polymeric fibrils composed of repeating subunits that can extend several microns from the cell membrane. Pili were once thought to be unique to gram-negative bacteria but have now been identified in several gram-positive organisms, including streptococci and actinomyces.²³⁰ Strains of P. gingivalis produce two types of fimbriae, known as the major and minor fimbriae.¹²⁶

The major fimbriae are single-stranded filaments approximately 5 nm in diameter and up to 3 µm in length. The backbone of major fimbriae is a chain of repeating subunits of the 43 kDa FimA protein. Minor fimbriae are composed of a 67 kDa protein, Mfa1, and extend around 0.1 to 0.5 µm from the cell surface.¹⁴ Major and minor fimbriae interact with oral streptococci such as S. gordonii. P. gingivalis FimA binds to glyceraldehyde-3-phosphate dehydrogenase, whereas Mfa1 interacts with the S. gordonii cell surface adhesin SspB.^228,²⁶⁹ Major fimbriae have also been shown to bind host extracellular matrix proteins fibronectin and type I collagen, salivary proline-rich proteins and statherin, and epithelial cells.^11,^127,²⁵⁶ Extensive variation in the fimA gene encoding the FimA fimbrial subunit has been observed, and six different genotypes have been designated (genotypes I, Ib, II, III, IV, and V).⁸⁸ Of these, genotypes II and IV are associated with periodontitis.^10,⁸⁷ However, at present there is no evidence that genotypes II and IV fimbriae are functionally distinct from other major fimbriae.

Adhesion is a fundamental process in the lifestyle of A. actinomycetemcomitans. On solid media, fresh isolates of A. actinomycetemcomitans generally form rough star-shaped colonies embedded in the agar. In broth, these strains form clumps (autoaggregate) and grow firmly attached to the sides of the culture vessel, leaving the bulk fluid almost completely clear. Following several laboratory subcultures, rough strains often revert to “smooth” colony variants. The conversion from rough to smooth forms is associated with the loss of pili. A. actinomycetemcomitans pili are encoded by a region of the chromosome that contains 14 genes, including the structural pilus subunit gene flp-1 and an associated system for secretion and modification of the fibrils. Twelve of these genes have been shown in gene disruption studies to be essential for biofilm formation.^274,²⁷⁸ In a rat model of periodontitis, genes in the A. actinomycetemcomitans pilus locus (flp-1 and tadA) were essential for pathogenesis.³³⁸ Despite these seemingly convincing data, there is still some debate regarding the exact role of pili in A. actinomycetemcomitans adhesion. Carbohydrate appears to be more important for attachment of A. actinomycetemcomitans than protein. Treatment with periodate (which disrupts carbohydrate structures) detaches cells from surfaces, whereas degradation of cell surface proteins by trypsin does not.⁹⁴ During growth, A. actinomycetemcomitans produces a glycosidase enzyme that acts as a “dispersin” and releases even tightly adherent cells from surfaces.¹⁶³ It is not known whether dispersin acts on carbohydrates that decorate pili or on extracellular polysaccharides. However, there is some evidence that production of extracellular polysaccharides may be linked to pilus biosynthesis, since knocking out almost any gene in the pilus locus reduces extracellular polysaccharide production.⁴¹⁴

Several nonpilus proteins from periodontopathogens have been shown to interact with host or bacterial receptors and in some cases with both. A relatively well-studied example is the major sheath protein (Msp) of T. denticola. Msp is a 53-64 kDa protein that was originally thought to assemble into a hexagonal array in the outer sheath. However, careful analysis by electron microscopy indicated that Msp is predominantly a periplasmic protein and that some regions are exposed on the cell surface.⁴⁵ Purified Msp interacts with host extracellular matrix proteins fibrinogen, fibronectin, and laminin.^83,^93,¹¹⁹ In addition, Msp recognizes receptors on P. gingivalis and F. nucleatum and mediates coaggregation.³¹⁸ Therefore Msp is a multifunctional adhesin that promotes binding to host and bacterial receptors.

Factors that Promote Tissue Destruction

Many bacterial proteins that interact with host cells are recognized by the immune system and trigger immune responses. Fimbriae of P. gingivalis and A. actinomycetemcomitans are highly antigenic. Inflammation is a major contributor to tissue destruction in periodontal disease and is considered separately in Chapter 21. However, a number of bacterial products directly promote tissue destruction in addition to modulating host immunity, and the most notable of these are the extracellular proteolytic enzymes.

Bacterial proteolytic activity in dental plaque, and particularly trypsin-like protease activity, is closely correlated with clinical markers of periodontal disease.²⁶⁰

Trypsin-like proteases are produced by P. gingivalis, T. denticola, and T. forsythia, and these molecules have been the subject of a comprehensive review.²⁸⁰ Proteases supply nutrition in the form of peptides or amino acids to the bacteria that produce them. In the process they cause sometimes extensive degradation of host extracellular proteins. Periodontopathogens frequently produce complex proteolytic systems consisting of multiple peptide-degrading enzymes; at least 13 peptidases of P. gingivalis have been characterized and a further 29 peptidases are predicted from the genome sequence of strain W83, for example.^252,²⁸⁰

However, the bulk of the host tissue degrading activity is restricted to a small number of these enzymes. In the case of P. gingivalis, three enzymes known as gingipains are responsible for at least 85% of the total host protein degradation activity.²⁸¹

The gingipains belong to the cysteine protease family, which utilize an active site cysteine residue for catalysis. Gingipains are classed as “Arg-gingipains” (RgpA and RgpB) or “Lys-gingipain” (Kgp), based on their ability to cleave Arg-Xaa or Lys-Xaa peptide bonds (Xaa represents any amino acid).

Gingipains are multifunctional proteins that play important roles in adhesion, tissue degradation, and evasion of host responses.

Protein domains involved in adhesion are located in the C-terminus of pro-RgpA and pro-Kgp and are subsequently removed by posttranslational proteolytic processing. The cleaved protease and adhesin domains sometimes reassociate noncovalently. In contrast, the gene encoding RgpB does not contain the sequences for adhesin domains. The combination of adhesin functions and protease activity together in the same molecule provides an efficient mechanism for targeting substrates. Thus the catalytic/adhesion domain complex of RgpA is more efficient than RgpB at binding and degrading fibrinogen.¹⁵²

Proteases are produced by A. actinomycetemcomitans but appear to be less important for the virulence of this organism than the leukotoxin (LtxA).

LtxA is a member of the repeat in toxin (RTX) family of proteins. These toxins act by delivering an adenylate cyclase domain into cells, which catalyzes the uncontrolled conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP).

Most strains of A. actinomycetemcomitans produce low levels of LtxA. However, some strains are regarded as hyperleukotoxic, since they express high levels of the transcript from the ltxA gene, encoding LtxA. Hyperleukotoxic strains include the JP2 clone of A. actinomycetemcomitans, which is uniquely associated with localized aggressive periodontitis.^138,²⁴³ Two distinct mechanisms that give rise to high levels of ltxA expression have been described.³³⁵ The JP2 clone contains a deletion in a regulatory gene, designated orfA, upstream of the ltxCABD gene locus. Hyperleukotoxic A. actinomycetemcomitans strains isolated in Japan do not contain deletions in orfA but instead harbor a mobile genetic element (transposon), IS1301, upstream of the orfA gene. The sequence of IS1301 contains elements that direct enhanced transcription of the orfA-ltxCABD operon. From a diagnostic point of view, it is important to consider that simply detecting A. actinomycetemcomitans and/or LtxA in the periodontal biofilm is by no means indicative of disease. However, detection of a hyperleukotoxic strain of A. actinomycetemcomitans may be significant.

Strategies for Evading Host Immunity

Pathogenic bacteria have many and varied strategies for evading or subverting the host immune system, including (1) the production of an extracellular capsule, (2) proteolytic degradation of host innate and/or acquired immunity components, (3) modulation of host responses by binding serum components on the bacterial cell surface, and (4) invasion of gingival epithelial cells. A detailed description of bacteria-host interactions is given in Chapter 25. Selected examples of factors that mediate these processes are given next.

Strains of P. gingivalis produce polysaccharide capsules (Figure 23-41) that surround the outer membrane. Six different antigenic capsule types have been described based on differences in the polysaccharide K antigen.¹⁹⁰ In a mouse model, capsular strains of P. gingivalis produced a spreading type of infection, whereas acapsular strains tended to form more localized abscesses.¹⁹² The majority of P. gingivalis isolates from periodontitis patients are encapsulated.¹⁹¹ It is hypothesized that the capsule protects cells from the host immune system. However, protection against the complement system appears to be mediated primarily by a branched phosphomannan polysaccharide that is independent of the K antigen.³⁵⁰

Figure 23-41 Glycocalyx or polysaccharide capsule of Porphyromonas gingivalis visualized with a East-Indian ink staining (clear halo).

Several periodontopathogens are resistant to complement-mediated phagocytosis, and it is thought that proteolysis of complement components contributes to resistance to some extent. P. gingivalis gingipains have been shown to degrade complement components C3, C4, C5, and factor B.²⁸⁰ More recently, a cysteine protease of P. intermedia termed interpain A has also been shown to degrade C3.²⁸² In vitro, interpain A acts synergistically with gingipains to decrease deposition of complement C3b.²⁸² These data fit with the hypothesis that periodontal disease processes are mediated by polymicrobial consortia rather than by individual periodontopathogens.

A novel mechanism for complement evasion has been identified in A. actinomycetemcomitans. This organism produces a 100 kDa protein on the outer membrane (Omp100 or ApiA), which mediates adhesion to and invasion of host cells.^17,²⁰⁴ Mutants lacking ApiA were sensitized to killing by human serum: wild-type cells were almost completely resistant to 30% or 50% normal human serum, whereas 90% of ApiA mutant cells were killed. ApiA was shown to bind factor H, an inhibitor of the complement cascade. Interestingly, H₂O₂ produced by S. gordonii induces the expression of ApiA and enhances the serum resistance of A. actinomycetemcomitans.³⁰⁹ It has been proposed that H₂O₂ derived from oral streptococci may stimulate the immune response and that early detection of H₂O₂ by A. actinomycetemcomitans may provide an ecologic advantage under these conditions.³⁰⁹ Alternatively, the ability of A. actinomycetemcomitans to detect streptococcal H₂O₂ may be a fortuitous consequence of a system that has evolved primarily to detect and respond to the neutrophil oxidative burst.

Periodontal pathogens are notoriously difficult to eradicate completely. Despite aggressive antibacterial attacks from the host defenses and from clinical treatments, it is often impossible to prevent reinfection. One potential reservoir for periodontopathogens is inside gingival epithelial cells. P. gingivalis and A. actinomycetemcomitans, for example, can invade epithelial cells in vitro.^193,²³⁶ In vivo, P. gingivalis, T. forsythia, P. intermedia, T. denticola, and A. actinomycetemcomitans can be detected within gingival epithelial cells of periodontitis patients before and after periodontal therapy.¹⁶⁰ Adhesion to host cells is critical for invasion. However, it has been suggested that there may be factors that specifically control the ability of bacteria to invade cells. In A. actinomycetemcomitans, a screen for invasion-associated genes identified two loci, encoding ApiA (see previous paragraph) and ApiBC that are related to known bacterial invasion proteins (invasins).²⁰⁴ The ability of bacteria to invade cells may be influenced by their interactions with other organisms in the gingival sulcus. Thus coinfection of an epithelial cell line with P. gingivalis and a coaggregating strain of F. nucleatum resulted in increased invasion by P. gingivalis compared with a monoculture control.³²⁸ Coaggregation with F. nucleatum is mediated by the P. gingivalis capsular polysaccharide and lipopolysaccharide, and therefore these molecules may indirectly contribute to invasion.³¹⁸

Future Advances in Periodontal Microbiology

Scientific progress at the end of the twentieth century, particularly in the field of molecular biology, has led to significant advances in our understanding of periodontal microbiology. DNA-based methodologies for the identification and detection of specific bacteria and viruses offer remarkable time and cost advantages, compared to culturing techniques. A dramatic increase in the number of samples that can be examined and the number of microorganisms enumerated is now possible. Perhaps even more relevant is the present ability to detect microorganisms that cannot be cultivated so far, which has underscored the limitations of our knowledge of this complex ecologic niche. Greater awareness of the role of the host response in periodontal disease will further improve the understanding of the severity and therapy of periodontal infections. Finally, the recognition of the beneficial activity of several groups of commensal species might open new strategies for treating periodontal disease, for example, using probiotics or microbial replacement therapy.³⁸⁶

Science Transfer

Subgingival plaque is an example of a complex biofilm composed of up to 150 bacterial species present at any one site with sharing of nutrients and deoxyribonucleic acid (DNA) and significant interdependencies between species for metabolites and adhesion. Biofilms give bacteria up to 1500 times more resistance to antimicrobial agents than that seen when bacteria exists in a liquid environment. There are significantly more bacteria present in diseased pockets (10⁸ bacteria) compared to healthy gingival crevices (10³ bacteria). One tooth surface may have as many as 10⁹ bacteria in its supragingival biofilms.

After a tooth surface is cleaned during a prophylaxis treatment, a dental pellicle consisting of polysaccharides and glycoproteins from saliva appears on the surface within 1 minute. This is quickly followed within 3 minutes by the first bacterial components of the biofilm. These first bacteria include gram-positive cocci, which can initiate caries. It takes at least 4 to 7 days before the gram-negative anaerobic proteolytic bacteria related to periodontal disease occur. Thus caries can begin immediately, particularly if exposure occurs to sucrose from food. Gingival inflammation has a longer lag period.

There are specific groups of bacteria related to periodontal breakdown. These include the red complex of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The broader group of putative periodontal bacteria can be seen in healthy sites but are only present in larger numbers in periodontal pockets. Viruses, such as human cytomegalovirus, may accentuate the destructive capacity of bacterial plaque.

The most effective method for changing the subgingival biofilm in patients with periodontal disease is the mechanical debridement using curettes and ultrasonic instruments. If this is followed by appropriate periodontal therapy that reduces pocket depths, then the patient is able to restrict the formation of a subgingival pathogenic biofilm with effective oral hygiene techniques.

At present, only a proportion of anaerobic gram-negative bacteria can be cultivated from periodontal pockets and many potential pathogenic species cannot be identified. This restricts the value of diagnostic cultural tests, and in the future, more sophisticated bacterial species identification procedures involving DNA will be needed before a clear picture of the role of periodontal microbiology in human periodontal disease can be substantiated.

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^* References 2, 5, 16, 151, 154, 342, 344, and 345.

^* References 27, 42, 118, 291, 301, and 316.

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Retroviruses

Clinical Manifestations of Oral Viral Diseases

Oral Ulcers

Oral Tumors

Other Oral Pathologies

Periodontitis

Yeasts

Protozoa

Archaea