Introduction

The most common diseases of the periodontal tissues are inflammatory processes of the gingiva and attachment apparatus of the tooth. The periodontal diseases are polymicrobial infections associated with local accumulation of dental plaque, a subgingival pathogenic periodontal flora, and calculus.

Periodontitis is an important global public health problem which involves mostly the adult population over 35-40 years of age. It begins with gingivitis and without therapy leads to a progressing destructive periodontitis. The variance and severity of this disease is influenced up to 90% by age and oral hygiene (1, 2). However, in only about 10% of the population severe forms of periodontitis occur where no correlation exists between the supragingival plaque index and the severity of the disease. The proportion of these patients increases with age and reaches the highest prevalence at the age of 40-50 years (3-5).

The aggressive periodontitis, a rare form of periodontitis, which often begins in childhood, is characterized by severe, rapidly progressing tissue destruction. The prevalence of juvenile periodontitis in 13-20 year olds is 0.1-0.5% (6-8).

Classification of periodontal diseases

According to the new classification, introduced in 1999 by the World Workshop of Periodontics (9), the following forms of periodontal diseases exist:

Gingival diseases
Chronic periodontitis (localized, generalized)
Aggressive periodontitis (localized, generalized
Periodontitis as a manifestation of systemic diseases
Periodontitis associated with hematological disorders
Periodontitis associated with genetic disorders
Not otherwise specified (NOS)
Necrotizing ulcerative gingivitis (NUG)
Necrotizing ulcerative periodontitis (NUP)
Abscesses of the periodontium
• Gingival abscess
• Periodontal abscess
• Pericoronal abscess
Periodontitis associated with endodontic lesions
Developmental or acquired deformities and conditions

The recent classification has united a broad panel of severe forms of periodontitis that are independent of age into a group of "aggressive periodontitis". In earlier literature this clinical entity is referred to as "severe periodontitis in young adults" (10), "advanced destructive" (11), "postjuvenile" (12), "early-onset" (13, 14) and "rapidly progressive periodontitis (RPP)" (15).
The present study included patients with aggressive periodontitis, who according to the classification from 1989 belonged to a group of "early-onset periodontitis (EOP)" (16). The term was used as a collective designation for a group of dissimilar destructive periodontal diseases that affected young patients, i.e. prepubertal, juvenile and RPP.

Bacterial etiology of periodontal diseases

From earlier studies there is evidence for the primary role of bacteria in the etiology of destructive periodontal diseases (18-21).

It has been previously estimated that about 500 bacterial species colonize the human oral cavity (17-19). The majority of these organisms are commensals and live in complex communities forming oral biofilms on tooth surfaces. In general these microorganisms live in harmony with a host, but under certain circumstances this dynamic interaction may lead to opportunistic infections resulting in breakdown of periodontium. The evidence for the infectious nature of periodontal disease comes from several sources, including:

Studies which correlate most forms of gingivitis and periodontitis with accumulated dental plaque.
Treatment studies which demonstrate that elimination of plaque microorganisms can be correlated with clinical improvement.
In vivo and in vitro studies demonstrating the relative virulence of different plaque bacteria.

It has been observed that clinically most periodontal sites in most subjects do not appear to be undergoing breakdown at any given time, even though they are continuously colonized by varying numbers and species of bacteria. This suggests that there are remarkably effective host defense mechanisms to be overcome and that only specialized uniquely talented bacterial species (with multiple virulence factors) may have the requisite set of properties to cause tissue damage. Thus, bacteria are important in the etiology of disease, but the outcome, protection or tissue damage, is affected by the nature and level of the immune response (22).

While the infectious etiology of periodontal diseases is generally accepted, there is an ongoing discussion as to the relative importance of individual bacterial species within dental plaque. This is reflected in the distinction between the non-specific and the specific plaque hypothesis.

The non-specific plaque hypothesis implicates the mere quantity of dental plaque, with any species having the possibility of causing disease. The bacterial mass causes a periodontal disease when it accumulates to the point of exceeding host-defense mechanisms. Variability was recognized, but the true extent of differences in bacterial composition was not acknowledged. This hypothesis is supported by studies of experimental gingivitis, e.g. by Löe et al. (23) showing that the cessation of oral hygiene is associated with the development of gingivitis. Resumption of oral hygiene resolved the gingivitis.
Until as recently as the early 1970s, it was thought that any organism present in subgingival plaque contributed to periodontal destruction (23, 24).

Studies carried out over the last 20 years questioned this hypothesis and contrasted it with the specific plaque hypothesis (25). It proposes that dental plaque isolated from periodontitis lesions is qualitatively distinct from that isolated from healthy sites. It assumes that clinically different forms of periodontitis are associated with distinct species and colonization patterns. For example, the composition of subgingival plaque from lesions of juvenile periodontitis is markedly different from that found in patients with adult periodontitis (26). However, the diversity of bacterial complexes, as well as the variation in host response to bacterial species are some of the major reasons that the specific etiology of periodontal disease has not been definitely established (27, 28).

Originally the concept of "pathogen" was devised for specific microorganisms which caused specific diseases. This cannot be applied to periodontal diseases. At present, it appears unlikely that one single species is a sole agent of periodontal destruction, since no single species occurs as an important part of the flora in all cases of gingivitis or periodontitis (17). The more suitable term "pathogenic microbiota" acknowledges the fact that periodontal diseases have a polymicrobial etiology, and that a multitude of defense systems have to be mobilized by the host against these infections. However, the designation "periodontal pathogen" can be applied to those bacteria which have specific mechanisms to perturb the host defense and in that way cause an accelerated destruction of periodontal tissues. Such a pathogen will never work in isolation, it is always a member of a complex bacterial community.

A set of criteria, derived from Koch's postulates of a monoinfection, has been developed to identify these specific microorganisms in a mixed microflora and apply to an opportunistic infection (29). These criteria are as follows:

The presence of high numbers of putative periodontal pathogens in periodontal lesions compared to either their absence or presence in low numbers in healthy or non-progressing sites
Elimination of the microorganisms from periodontal lesions should result in clinical improvement
Induction of active immune responses in the host
Presence of virulence factors
Appropriate animal models demonstrating tissue destruction in the presence of the microorganisms.

Periodontal research has attempted to define periodontopathic bacteria that induce periodontitis (20, 30-33). Relatively few, about 10-20 species, may play a causal role in the pathogenesis of destructive periodontal diseases. However, it is assumed that about 50 percent of the total oral bacterial flora is still unknown. An attempt made by Paster et al. (19) to investigate the bacterial diversity in subgingival plaque using culture-independent molecular methods revealed 215 novel phylotypes. It is likely that yet-undiscovered bacteria may play a role in the etiology of the disease.

Healthy flora

The dental plaque associated with periodontal health is characterized by predominantly gram-positive facultative coccoid microorganisms, such as Streptococcus and Actinomyces species (17, 18). The presence of gram-negative cocci, rods and filaments is a frequent observation; however, they are in much lower proportions as compared to gram positive flora (30, 34-38). Similar species are associated with healthy subjects in cross-sectional studies, and as "beneficial" species in inactive periodontal pockets (39).

Gingivitis

Without oral hygiene the dental plaque grows in thickness forming a distinct organized structure (40, 41). Subsequent mineralization gives rise to calculus. The undisturbed growth of supragingival plaque results within few days in soft tissue alterations in the adjacent gingiva. It has been hypothesized that the transition between health and gingivitis is due to an overgrowth of gram-positive species (42, 43). However, other investigators have found an increased proportion of gram-negative species as the major determinant (40, 44). In fact, the inflammatory conditions provide a relatively anaerobic environment which favors the colonization by anaerobic motile rods and spirochetes. The species associated with experimental gingivitis include Actinomyces, Streptococcus, Veillonella, Fusobacterium and Treponema species. Additionally Prevotella intermedia and Campylobacter species have been cultivated from the plaque of chronic gingivitis (23, 40, 42).

Periodontitis

Subgingival plaque develops by apical progression of supragingival plaque. Alterations in the integrity of the junctional epithelium allow a gradual colonization of the tooth surface leading to the formation of periodontal pocket. The culture-based studies indicate that anaerobic microbiota predominate (10, 17, 31, 45). Although the bacterial composition changes, there is still no direct evidence to conclude which bacterial species initiate the first step of pocket development.
Scanning immunoelectron microscopy showed that rods, filaments, and spirochete-shaped bacteria formed small aggregates at the bottom of the periodontal pockets in the so-called "plaque-free zone" (46). Investigations with fluorescence in-situ hybridization assay which analyzed artificial carriers from deep periodontal pockets found that gram-negative rods and treponemes dominate in the deepest part of the pocket, forming a confluent biofilm (47). These findings suggest that bacteria in the "plaque-free zone" may be critical periodontopathogens in the frontier area of apical plaque.
Studies of the predominant subgingival flora in periodontitis lesions have revealed great microbial diversity. Different forms of periodontitis show variations in the microorganisms colonizing periodontal pockets (14, 31, 45, 48). For instance, patients with juvenile periodontitis have shown infection with A. actinomycetemcomitans (26, 49, 50). The frequently detected bacterial species in periodontal lesions in cases of chronic and aggressive periodontitis are listed in Table 1 (51).

Chronic periodontitis

Aggressive periodontitis

Treponema spp.

Porphyromonas gingivalis

Prevotella intermedia

Tannerella forsythensis

Porphyromonas gingivalis

Actinobacillus actinomycetemcomitans

Tannerella forsythensis

Eikenella corrodens

Peptostreptococcus micros

Campylobacter rectus

Campylobacter rectus

Actinobacillus actinomycetemcomitans

Eikenella corrodens

Fusobacterium spp.

Selenomonas spp.

Eubacterium spp.

Table 1. Bacterial species associated with chronic and aggressive periodontitis.

Although there are strong indications that certain species are likely etiological agents of periodontal diseases in distinct subject groups, it is still difficult to determine which of the likely candidates is the critical organism (or a set of bacteria) in a given subject. Recently Mombelli et al. (52) after reviewing cross-sectional and longitudinal studies by stringent criteria questioned the strict distinction of specific microbiota in patients with aggressive and chronic periodontitis. The presence or absence of A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythensis and C. rectus could not discriminate between subjects with different clinical disease entity.

It is apparent that the mere presence of one or more pathogenic species in a subject is insufficient to produce significant deterioration in the periodontal status of the individual. Multiple other factors, such as host and environmental factors, as well as local bacterial interactions that can modulate the virulence of bacterial species contribute to the clinical outcome.

Bacterial consortia

The bacteria in subgingival plaque form an ecosystem where complex relationships exist between population members as well as between bacteria and the host. The microorganisms tend to form coaggregations where bacterial interactions play an important role in species survival (53, 54). So-called "beneficial" species can turn these interactions beneficial to the host by affecting disease progression in a number of ways:

by "passively" occupying a niche which might otherwise be colonized by a pathogen
by actively limiting a pathogen's ability to adhere to appropriate tissue surfaces
by adversely affecting the vitality or growth of a pathogen
by affecting the ability of a pathogen to produce virulence factors
by degrading virulence factors

It has been observed that high levels of both P. gingivalis and C. ochracea diminish the risk of new attachment loss (27). In contrast, in subjects with high levels of P. gingivalis, but low levels of C. ochracea there was a tendency for disease progression. It has been shown that subjects who received an adjunctive antibiotic therapy showed a higher percentage of sites with attachment level gain and higher levels of the suspected beneficial species like C. ochracea and S. sanguis II post-therapy than subjects who did not get antibiotics (55). Therefore, the aim of the therapy must not be exclusively the reduction or elimination of pathogens, but also supporting colonization shift to high levels of beneficial species.
The differences in subgingival microbial constellations between patients with similar clinical signs as well as spatio-temporal variations have been acknowledged (45).
As yet it is not clear whether the individual differences in flora composition are controlled primarily by genetic disposition of the host or by environmental influences. Moore et al. found that the periodontal flora of twin children were significantly more similar than those of unrelated children of the same age, indicating that genetics as well as environment influence the composition of the flora (56). Once the oral microbiota is established in an individual, it may be more difficult to introduce new species or clonal types (57).

Biofilm

When supragingival hygiene is not maintained, dental plaque immediately develops by a dynamic intraplaque interaction, which climaxes with the establishment of a well-structured community of microflora - a biofilm (58, 59). The formation of biofilm begins with attachment of planctonic cells (mostly gram-positive cocci) on the pellicle of the tooth surface. Auto-aggregation with each other or coaggregation with other planctonic cells or neighbors begins. Gradually the microenvironment of the inner community changes from aerobic/capnophilic to facultative anaerobic and to anaerobic. The community is re-organized, new ecological niches are involved by spirochetes and motile organisms.
The biological characteristics and growth rate of bacteria in a biofilm are different from their planctonic counterparts, and thus the gene expression of virulence factors may be different according to their living conditions (60). The biofilm mode of growth seems to be advantageous for microorganisms. These three-dimensional structured communities contain fluid channels for transport of substrates, waste products and signal molecules (59). Glycogalyx, the polymeric substances that make up the matrix of a biofilm, retard the diffusion of antibiotics and host-driven antimicrobial factors (58, 61). Thus, biofilms are more resistant to immune defense mechanisms, less susceptible to antibiotic therapy and even not easily controlled by mechanical means (62).

Virulence factors

The expression of virulence factors may be an important indicator of the potential of a species to contribute to disease progression; however, the virulence traits of individual species in vitro might bear little resemblance to their behavior in a microbial community and, indeed, in vivo (63). It is unlikely that a single virulence factor will be responsible for tissue damage. Often a series of virulence factors is expressed under coordinate regulation. Very often the pathogen's environment appears to regulate the expression of these virulence factors (53, 64). Environmental factors such as temperature, osmolarity, iron and Mg levels have been shown to affect the expression (63, 65, 66). Interbacterial relationships play an important role in species survival. Some relationships are favorable and others are antagonistic. The interspecies aggregation can enable attachment of some species. Co-aggregation has been observed between P. gingivalis and F. nucleatum, C. ochracea and S. sanguis, P. gingivalis and A. viscosus, F. nucleatum and S. sanguis (67-69). Antagonistic substances may prevent aggregation or even kill other bacteria. For instance A. actinomycetemcomitans produces bacteriocin which inhibits the growth of S. sanguis. S. sanguis on the other hand produces H2O2 which kills A. actinomycetemcomitans (70). Inhibitory microbiota may help in preventing an infection by oral pathogens.
In order to colonize subgingival sites, a species must be able to attach to available surfaces, multiply, compete against other species in this habitat, and defend itself against host defense mechanisms (53). Bacteria attach to specific receptors on the host cell or tooth pellicle by specific adhesion molecules (71). Fimbriae and other cell-associated proteins have been identified as adhesins in several subgingival species. Attachment of P. gingivalis to epithelial cells, gram-positive bacteria, basement membrane, and type I and IV collagen has been demonstrated (72-76). By using scanning immunoelectron microscopy it could be demonstrated that P. gingivalis participates in biofilm formation in the most apical part of a pocket, in so-called "plaque-free zone" probably using its attachment ability (46). Adhesins of E. corrodens and A. actinomycetemcomitans enable these species to attach to the epithelial cells (53, 77, 78, 88). It has been shown that strains of F. nucleatum adhere to red blood cells, basement membrane, and type IV collagen (53, 72, 73). T. denticola adheres well to fibroblasts, fibronectin, basement membrane, as well as type I and IV collagen (79). Adherence to host cells might be the prerequisite for further invasion of deeper tissues (76, 78, 80).
Earlier the term "invasion" was taken to mean intercellular penetration, i.e. bacteria locating between the host cells. The introduction of optical sectioning by confocal scanning laser microscopy (CSLM) enabled a three-dimensional localization of the bacteria. An intracellular location of A. actinomycetemcomitans and P. gingivalis within buccal epithelial cells of healthy subjects was observed using this technique (81). The authors suggested that intracellular location may be a common ecological niche for these bacterial species in both health and disease. Intracellular invasion has also been documented for Prevotella intermedia (82), Fusobacterium nucleatum (83) and Tannerella forsythensis (84). However, this property is not universal, e.g. Treponema denticola does not invade epithelial cells (85). The closer proximity to host targets allows destructive bacterial products to cause greater havoc upon the structural integrity of the periodontal tissues (86).
Some of the suspected pathogens produce an unusually wide spectrum of proteases including those which degrade collagen (like P. gingivalis. T. denticola and A. actinomycetemcomitans ) (73, 76, 79, 87-91), and fibronectin (like P. gingivalis and P. intermedia) (76, 91, 92). Trypsin-like activity has been demonstrated for P. gingivalis, T. forsythensis, T. denticola and Capnocytophaga spp. (79, 93-95). Also the metabolic end-products (such as volatile sulfur compounds, NH3, fatty-acids and indole), produced by P. gingivalis and some by F. nucleatum, adversely affect mammalian cells (96, 97).
It has been shown that lipopolysaccharide (LPS), the so-called endotoxin, which is an integral component of the cell wall of gram-negative bacteria, induces the production of biologically active molecules, such as IL-1, TNF- and prostaglandins from monocytes or macrophages (98-101). Besides their proinflammatory properties, these cytokines are capable of stimulating bone resorption. Recently, it was discovered that the toll-like receptors play a crucial role in transduction of the signals of LPS (102). Interestingly, LPS from P. gingivalis and C. ochracea showed antagonistic activity by not inducing human TLR4-mediated signaling (103). Antagonistic activity would be of great advantage for the microorganisms to escape from the innate immune system.
Several bacterial species possess mechanisms to overcome the defense of the host's immune system. IgG and IgA proteases of P. gingivalis, P. intermedia and Capnocytophaga spp. are able to specifically destroy antibodies (77, 104). A number of species have developed strategies to interfere with the killing mechanisms of the polymorphonuclear leukocytes and monocytes. These include the production of leukotoxin by A. actinomycetemcomitans (105, 106) and C. rectus (107). Additionally, leukotoxin from A. actinomycetemcomitans can induce apoptosis in a variety of host immune cells (108).

Pathogenesis of periodontitis

The indigenous oral microflora and the host are normally in a state of equilibrium. The interactions between the microorganisms and the host are very dynamic, thus allowing the complex interplay between host molecules and bacterial antigens (109). The exact mechanisms that allow the host to "tolerate" non-pathogenic microorganisms are largely unknown. Any disruption of the "established" state, whether by commensal bacteria, pathogenic bacteria or a compromise in the local or systemic health of the host will lead to an altered host condition, resulting in disease (110). The pathogenesis of periodontitis is thus mediated by interactions between host and microbial factors, complicated by genetic and environmental risk factors.

Mixed consortiums of microorganisms are involved in periodontal disease, which develops as a consequence of imbalances in microbial biofilm inducing an inflammation in host tissues. The environment is altered by increased flow of gingival crevicular fluid and nutrients, as well as a pH rise that favors growth of periodontopathic bacteria (111). The microorganisms increase in number and produce several bioactive end products, endotoxins and exotoxins (58). Protease-producing bacteria, such as P. gingivalis, T. forsythensis and T. denticola, may be involved as initiators of disease activity.
The host has several defense strategies to protect its barriers against bacterial invasion. The defense mechanisms - innate and adaptive immunity - function in a complex way. Innate immunity is responsible for initiation of the inflammation process, acting as the first line of host defense against microbial pathogens (22). Adaptive immunity, mediated by B and T lymphocytes, which carry immunoglobulins and T-cell receptors, respectively, present a more effective defense against specific bacterial species, however, several steps are required before its efficient activation (102). Recently it was discovered that toll-like receptors (TLRs) play a crucial role in recognition of invading pathogens (102, 112). There are currently 10 known TLRs, each of which recognizes a different spectrum of pathogen-associated molecular patterns (PAMPs), e.g. TLR2 recognizes bacterial peptidoglycan and lipoproteins, TLR3 recognizes double-stranded RNA, TLR4 lipopolysaccharide, TLR5 flagellin and flagellated bacteria, TLR9 procaryotic DNA (113). TLRs are known to be expressed in a number of tissues and by a variety of cell types including monocytes, neutrophils, endothelial cells, fibroblasts, osteoblasts and dendritic cells (110). Signaling through TLRs leads to a set of innate immune response - production of proinflammatory cytokines and upregulation of costimulatory molecules, and ultimately also induction of adaptive immunity (113, 114). The molecular basis of TLR-dependent signal transduction is an extremely active area of investigation, as these findings might explain different innate immune responses to various pathogens.
One of the recent exciting discoveries was that TLRs are critical molecules in adaptive immune response as well. They are required for the upregulation of co-stimulatory molecules such as CD80/86 and major histocompatibility complex (MHC) on dendritic cells (DCs) (113). TLRs can also regulate T-cell differentiation status by producing proinflammatory cytokines such as IL-12.
Genetic variations or polymorphisms associated with TLRs might explain to some extent the species-specific response and thereby different susceptibility of host to infections (113, 115).

The primary function of the innate immune system is to provide a rapid response to bacterial pathogens. Bacterial products are chemotactic for neutrophils, activate the plasma proteinase cascade systems, trigger mast cells to release biogenic amines, and stimulate inflammatory cells and resident tissue cells to form cytokines (IL-1, tumor necrosis factor), platelet activating factor and prostanoids (e.g. prostaglandins, leukotrienes) (116-119). Polymorphonuclear leukocytes (PMNs) seem to play a central role in the pathogenesis of periodontitis (123). Specific adhesion molecules like ICAM-1, ELAM-1 promote the movement of PMNs from blood vessels into the connective tissue and sulcus, where they phagocytose the bacteria (120). The defects in vitality and function of PMNs are modifying factors for disease pattern or severity (121).

Most of the tissue destruction results from direct effect of the bacteria, together with the resulting inflammatory and immunological host responses. Reactive oxygen products from inflammatory cells injure tissue cells, and proteases from both inflammatory cells and resident tissue cells degrade components of the extracellular tissue matrix (122, 123). In the periodontal tissues prostanoids, cytokines and thrombin directly or indirectly induce degradation of the extracellular matrix, activate osteoclasts and initiate bone resorption (116, 124). An involvement of TLR4 in bone resorption was demonstrated with TLR4-deficient mice. It was observed that bone loss was significantly less in TLR4-deficient mice than in wild-type controls (125). This decrease was correlated with reduced expression of the bone resorptive cytokines IL-1 and IL-1 as well as the proinflammatory cytokine IL-12. An immunohistochemical investigation of gingival tissue of periodontitis patients showed the association of the expression of the TLR4 with severe periodontal disease (126). It becomes apparent that TLR levels influence the magnitude of inflammatory responses, underscoring the need to clarify the molecular mechanisms modulating TLR expression (113).
Only now are we beginning to appreciate the complexities of the evolutionary conserved innate immune system, and the essential role it plays in maintaining homeostasis. The disruption of an intact innate immune system is detrimental to the health of the host in either a localized or a systemic manner.
The adaptive immunity is based on specific antigen-antibody reaction, as well as specific T-cell recognition. Antigens are presented by Langerhans cells to lymph tissue, where the B-cells will be converted to plasma cells to produce antibodies. The reaction is supported by T-cells. The antibodies induce aggregation of bacteria, inhibit adhesion of bacteria to epithelium, lead to antibody-complement-mediated bactericidal activity or through opsonisation to phagocytosis by neutrophils and macrophages (127). Persons able to provide an effective antibody reaction are supposed to be more resistant to periodontitis than those with a quantitatively or qualitatively ineffective response. It has been shown that the production of IgG2 predominates over IgG1 concentration by patients with early-onset periodontitis (128). This suggests that functionally less-effective IgG2 antibodies play an important role in susceptibility and dimension of periodontal destruction in those patients. Some authors have stressed the importance of testing the titer of antibodies to putative pathogens and the avidity of antibodies in determining the status of periodontal disease (129).

The bacteria play an important role in the etiology of periodontitis, but the response of the host is the decisive factor for the susceptibility of periodontitis. Risk factors like smoking, diabetes, stress modify to a large extent the susceptibility of the host and progression of the disease (130, 131). It turns out that smoking has the highest impact on the course of the disease, modulated by all the other factors (132). Regardless of the different microbial profiles identified in smokers and non-smokers in the majority of the investigations it is unclear whether the increased presence of certain microorganisms is the cause or the consequence of a more severe disease condition. However, conflicting results have been reported about the influence of smoking on the subgingival microflora of periodontitis patients (133). It can be concluded that smoking and stress influence host-related factors, thereby modifying the microflora to be more pathogenic. Bergsröm et al. (134) proposed to regard destructive periodontal disease as a systemic disease in the same way as heart disease or lung disease. In smokers the periodontal disease is initiated and driven by smoking, where the elevated morbidity does not depend on particular microflora (134).

Clinical studies seeking evidence for etiological role of bacteria

A major limitation of many microbiological studies has resulted from the selection of appropriate patients and controls. Favored subjects most often chosen are those with the most advanced cases of periodontitis. Analysis of the complex microflora of these samples from cross-sectional studies did not reveal whether the investigated microorganisms initiated the disease or whether they colonized later. The presence of suspected pathogens may result from, rather than cause the disease.
There are multiple forms of destructive periodontal disease that are difficult to define clinically. Combining subjects that represent two or more disease types into a single group diminishes the likelihood of discriminating the pathogens from other species.

Prevalence studies

A positive correlation between bacterial numbers and severity of gingivitis or periodontitis and amount of bone loss has been demonstrated in cross-sectional studies (135). A higher prevalence and increased proportions of P. gingivalis, T. forsythensis, P. intermedia, Fusobacterium spp., Campylobacter and Treponema spp. were detected in periodontitis patients as compared to periodontally healthy subjects (30, 32, 44).

Progression of disease

An important piece of evidence in defining periodontal pathogens comes from longitudinal studies examining the subgingival microflora in active sites undergoing attachment loss (27, 33, 39, 135, 136). Several resident putative periodontal pathogens have been reported to be responsible for the progression of attachment loss. Haffajee et al. (137) followed longitudinally the changes in pockets that subsequently lost attachment. Significantly higher levels of P. gingivalis, C. rectus and significantly lower levels of C. ochracea were found in active subjects prior to breakdown.
By studying the microbiota of active destructive periodontal lesions and inactive sites, species such as T. forsythensis, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, Str. intermedius, P. micros, A. actinomycetemcomitans were found frequently in high numbers, suggesting that they may represent causative agents. In inactive sites S. mitis, S. sanguis, Actinomyces spp., C. ochracea and V. parvula were elevated (33, 39, 49, 138 -142).
Liljenberg et al. however, compared periodontitis patients with progressive and non-progressive disease in a cross-sectional study and found no differences in the subgingival microbiota between groups (143). Furthermore, even patients with progressive disease did not show differences between progressive and non-progressive sites (144).

Risk factor studies

Risk assessment studies were used to confirm etiological agents in periodontal diseases. A periodontal site in a carrier-state with bacterial pathogens was considered to be a future risk indication of periodontal breakdown. It has been observed that subgingival colonization with T. forsythensis, P. gingivalis and A. actinomycetemcomitans was associated with a risk for attachment and severe bone loss (3, 145 -147). Similarly an increase in levels of bacteria was associated with an increased risk of attachment loss, however, different threshold levels were reported for different bacterial species. Additionally, several combinations of species were associated with an increased risk for disease progression (48). However, some authors refer to periodontal pathogens as minor risk indicators due to the fact that the odds ratios between the presence of these specific bacteria individually and periodontitis are not high enough to classify them as risk factors (148).

Treatment studies

Successful therapy is aimed at diminishing the level of pathogens, supporting the colonization with beneficial species and leading to an attachment level gain (55, 149-151). The prerequisite for an efficient therapy is an excellent oral hygiene. Scaling and root planing is considered the standard therapy of periodontitis. Combined with a regular maintenance program the supra- and subgingival debridement has been shown to be effective in most cases of periodontal therapy (152 -154).
Surgical intervention in the form of modified Widman flap surgery or apically repositioned flap may be needed if non-surgical therapy was not effective and deep pockets still persist. It provides better access to the roots for the debridement. Comparison between the surgical and non-surgical therapy demonstrated higher attachment gain in deep pockets after the surgery (149).
The mechanical debridement not only decreases plaque mass but also radically changes the composition of the subgingival microbiota (139, 149, 152). The disruption of biofilm is effective in altering the biofilm's composition so that the putative pathogens are eliminated or reduced to nonpathogenic levels, and bacteria associated with health are positively selected. The qualitative shift may be mediated not only through the direct effect of mechanical debridement but also indirectly through an altered immune response (155).
Patients with aggressive or refractory periodontitis often need an adjunctive systemic antibiotic therapy. Refractory periodontitis is characterized by ongoing deterioration of periodontal sites and associated with a continued presence of T. forsythensis, P. gingivalis, C. rectus, P. intermedia, P. micros, spirochetes, enterococci (147, 156, 157). Predictable results have been achieved with the administration of a metronidazole / amoxicillin combination (158, 159). The strictly anaerobic gram-negative species and A. actinomycetemcomitans are the main targets of this antibiotic combination.
Although some success has been reported due to antibiotic therapy, several limitations have become evident. Most of these limitations are due to the fact that periodontal infections result from the formation of biofilm. Therefore, disrupting the biofilm mechanically is still the basis for successful periodontal treatment (160).
The clinical stability of periodontal status means a dynamic balance between the presence of opportunistic bacteria and immune response. The maintenance therapy is thus aimed at keeping the bacterial colonization under control.

Detection methods

A variety of methods have been developed and applied for the detection and identification of microorganisms. Bacterial culture has long been regarded as the "gold standard". However, culture-based techniques suffer the limitation that they are highly delicate and time-consuming, requiring experienced personnel and strict quality assurance procedures. Many organisms will not grow on currently available culture media. Several studies showed that culture-based analyses of complex microbiota do not reflect the true composition of the microbial population as often only these species which grow easily in vitro are cultured (161). Additionally, identification based upon phenotypic characterization has been found to be unreliable. One disadvantage of culture techniques is that only small numbers of samples can be studied. More rapid unbiased techniques are required for examining large numbers of samples and reflecting more reliably the real diversity of the flora. These techniques include immunofluorescence using monoclonal or polyclonal antibodies, hybridization using either whole-genomic DNA probes or oligonucleotide probes, and PCR amplification assays.
The 16S rRNA with its altering conserved and variable domains has been found to be the most reliable and stable molecule for identification, enumeration and phylogenetic classification of procaryotes (162, 163).
Molecular biologic methods have a higher sensitivity and specificity as compared to bacterial culture hence increasing the accuracy of the analysis (161, 164). They are especially valuable for the detection of slow-growing, fastidious or yet uncultured bacteria.
In the situation where the putative pathogens belong to an indigenous flora, the detection of minute amounts of bacteria is irrelevant. In epidemiological studies, however, estimation of accurate prevalence of these bacteria in different population groups allows the assessment of their possible association with the disease.
New techniques like transmission or scanning electron microscopy, fluorescence-in-situ hybridization help to visualize, identify, localize and enumerate the microorganisms in biological samples. The development of a real-time PCR-amplification assay allows reliable quantification of bacterial species and assessment of their proportions in a total flora (165).

Aggressive periodontitis Clinical diagnosis

It has become popular to speak about different periodontal disease entities, which may have different specific etiology. However, only necrotizing periodontal diseases and localized aggressive periodontitis (earlier LJP) are well-defined disease entities. Most periodontitis cases are difficult to classify clinically in the gradual range from gingivitis to more-or-less advanced or aggressive periodontitis. This makes statistical associations between the disease status and microflora problematic.
According to the new classification aggressive periodontitis is a specific form of periodontitis with distinct clinical and laboratory characteristics (166). These include:

Besides periodontitis subjects systemically healthy
Rapidly progressing periodontal destruction
Familiar aggregation

Often, but not always:

Disproportion between dental plaque and tissue destruction
High prevalence of A. actinomycetemcomitans or P. gingivalis
Abnormal function of neutrophils or monocytes
Hyperresponsive macrophage-phenotype with increased production of PGE2 and IL-1ß
The destructive process may cease spontaneously or greatly slow down

The localized form:

Begins in puberty
First molars and central incisors are affected
High level of antibodies against putative pathogens in serum

The generalized form (GAP):

Patients younger than 30 years
Generalized destruction of the dentition
At least 3 teeth are affected
Intermittent course of the disease
Low level of antibodies against putative pathogens in serum

Microorganisms associated with aggressive periodontitis

There have been few studies concerning the associated pocket microflora of generalized aggressive periodontitis, but the available data implicate P. gingivalis, T. forsythensis, A. actinomycetemcomitans, P. intermedia, E. corrodens, F. nucleatum, C. rectus, C. ochracea, V. parvula, spirochetes, Eubacterium spp., P. micros as important suspected pathogens. They have been found in higher proportions and more frequently in aggressive periodontitis patients (10, 13, 15, 17, 167 -169, 170). However, aggressive periodontitis is considered a distinct form of periodontitis, microbiological criteria are not regarded as primary features in defining the disease entity (52).

Control group (Elderly)

A well-documented epidemiological study, the so-called "New England Elders Dental Study", revealed a high prevalence of periodontal destruction among older adults (171). Moderate pockets with pocket depth (PD) 4-6 mm were found in 66% of the study sample, and severe periodontal pockets (PD>6 mm) were observed in 21% of subjects. Only 8% of that population had no pockets. The authors drew the conclusion that age was significantly associated with periodontal destruction within this elders' population.
The only study investigating subgingival microbiota of an elderly population and comparing it with healthy and periodontitis patients was performed by Haffajee et al. (34). The mean probing depth of the 35 elders was 2.6±0.4 mm and as few as 6% of the sites revealed PD 4-6 mm. No pocket >6mm was documented. Several subjects had periodontal treatment in the past. At the time of the study all the subjects were on regular maintenance (mean duration 14.2 years). These individuals exhibited minimal evidence of disease progression and tooth loss. Marked similarities in the subgingival microbiotas of the healthy and well-maintained elders was observed.