Microbial role in periodontitis: Have we reached the top? Some unsung bacteria other than red complex

Abstract

The microbial etiology of periodontal disease has been the focus of researchers for a long time. The search for the pathogens of periodontal diseases has been underway for more than 100 years, and continues up today. Despite the increasing knowledge about oral microbiota, we are not able to implicate any one particular organism that can be considered as a candidate pathogen. In fact the term “candidate pathogen” has lost its steam with a myriad of microorganisms being incriminated from time to time. Most studies of the bacterial etiology of periodontitis have used either culture-based or targeted deoxyribonucleic acid approaches and so it is likely that pathogens remain undiscovered. The advent of 16S cloning and sequencing has facilitated identification of several uncultivable bacteria in the oral cavity. The concept that not one single organism, but several organisms contained in the biofilm orchestrating in a medley of the show appears to be more plausible. The present review highlights some lesser known bacteria associated with periodontal destruction.

INTRODUCTION

Periodontal disease is one of the most common afflictions faced by the human beings. A disease affecting more than 30% of the human race,[1] very few people escape its’ ravages. Gingival bleeding, spacing of teeth, suppuration, progressive loss of alveolar bone support and ultimate loss of tooth or more often many teeth are the hallmarks of periodontitis. Certainly of microbial etiology the exact etiology of periodontal disease is still an enigma. Multifactorial in origin, chronic and insidious in nature with a mixture of genetic and oral hygiene factors, periodontitis is still a complex phenomenon.

The microbial etiology of periodontal disease has been the focus of researchers for a long time. With about 400 species detected in the gingival sulcus and some of them viz. Porphyromonas gingivalis and Tannerella forsythia widely regarded as major pathogens and fulfill the criteria for candidate pathogen.[2]

In the initial era, microbial search for periodontal pathogens was confined to culture based methods and relied heavily on artificial media. The advent of deoxyribonucleic acid (DNA) based strategies has changed the scenario. Initially, the molecular based investigations were confined to detection of species, which were earlier identified by traditional culture techniques. This molecular based search while confirming the presence of already identified organisms the presence of several species, which were not detectable by prior culture based techniques remained unanswered. The novel and path-breaking ribosomal 16S cloning and sequencing has facilitated identification of several hitherto uncultivable bacteria in the oral cavity. Based on 16S cloning and sequencing of human subgingival flora, Paster et al. demonstrated that 40% bacterial species present to be novel species or phylotypes.[3]

The microbial role in periodontal pathogenesis although is increasingly realized, to our great chagrin nothing substantive has emerged. A play of words, semantics, it is all a charade. Although the presence of M. tuberculosis is an indication of tuberculosis and T. pallidum a positive diagnosis of syphilis, there is no single microorganism, which is attributable to chronic periodontitis (CP). About 20 years ago Socransky aptly summed up the microbial etiology of periodontal disease with a disarming statement “specific bacteria of right clonal type with essential genetic elements in numbers for that host with appropriate additional species in the right environment.”[4]

We next come to the issue that single microorganism may not be the culprit, but an orchestrated group of more than one might be the main offenders. In a classic paper in 1998 Socransky et al. used cluster analysis and community ordination methodologies. This elaborate and highly technological research on the subgingival microbiota resulted in organizing the organisms into several complexes indicated by various colors.[4] The colors varying from red to yellow have a different connotation. Red being the most pathogenic and yellow indicative of commensales. These researchers have been quizzed innumerable times about the rationale of choice of colors. Origin of the colors is supposed to be derived from VIBGYOR, they represent the colors seen in U.S. flag or they were named without any particular intent. 185 subjects, 13,261 subgingival samples and determination of 40 subgingival taxa was a phenomenal work and the work was a well-accepted in the research community because it “simplified” description of multiple organisms of questionable role into selected and few complexes with specific relationship to periodontal health and disease.[4]

The same group of researchers after a decade (2008) attempted to study the complexes in supragingival plaque. Adopting cluster analysis and community ordination they reported that complexes in the supragingival plaque were similar to subgingival plaque with few minor differences. The red complex observed in subgingival plaque was strengthened with the addition of Eubacterium nodatum and Trepenoma socranskii was loosely associated with this complex. Similar additions were observed in orange and yellow complex.[5]

With the plethora of the organism and their dubious role, Hajishengallis and Lamont in an invited review introduced the spirit of polymicrobial synergy and dysbiosis model of periodontal disease. The model revolves around certain species, termed “keystone pathogens,” to modulate host response in ways that impair immune surveillance and tip the balance from homeostasis to dysbiosis.[6]

Several unexplained situations need to be addressed: Red complex organism like P. gingivalis can be found in the absence of disease, precluding its role as an exogenous pathogen.[7] Periodontal microbiota is more heterogeneous than earlier believed, with 700 organism believed to exist, 200 present in any one individual and about 50 present at any one site.[8]

The doctrine that Gram-negative organism predominate is no longer tenable with a significant increase of Gram-positive organisms in deep diseased sites and found in greater abundance than in Gram-negative organisms in some studies, F. alocis is one such example.[9]

Unrecognized periodontal pathogens remain to be identified. Hence, the present review brings to focus some lesser known bacteria associated with periodontal destruction.

While a lion's share of literature is devoted to red complex organisms, little has been spoken about other organisms, which also are reported to be associated with periodontal disease. The following are essentially associated with periodontitis: P. gingivalis, T. forsythia, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Prevotella melaninogenica, Fusobacterium nucleatum, Parvimonas micra, Eikenella corrodens, Prevotella nigrescens, Capnocytophaga gingivalis, Treponema denticola, Treponema socranskii, Eubacterium nodatum and Campylobacter rectus.[10]

The following organisms have also been implicated as periodontal pathogens: Porphyromonas endodontalis, Prevotella denticola, Filifactor alocis, Cryptobacterium curtum, Eubacterium saphenum, Mogibacterium timidum, Prevotella corporis, Prevotella disiens, Peptostreptococcus magnus, Slackia exigua, Treponema maltophilum, Treponema sp. Smibert-3, Treponema lecithinolyticum, Treponema putidum sp. nov., Enterococcus faecalis, Escherichia coli and Bartonella sp.[10] Nevertheless, the relevance of certain species as etiological agents of periodontal diseases remains controversial, even among microbiologists.

While it is not possible to dwell on each of the above organisms, few of them are mentioned here and the discerning reader is recommended to wade through a maze of information and draw necessary inferences accordingly.

Eubacterium nodatum

These species are all obligatory anaerobic, asaccharolytic and generally nonreactive and they grow poorly and slowly on media commonly used to isolate anaerobic bacteria. Three new species, E. nodatum, Eubacterium timidum, and Eubacterium brachy, were described, primarily from subgingival samples taken from patients with moderate and severe adult periodontitis. Except for the isolation of E. brachy from a pleuropulmonary infection, these species have not been reported from other infected body sites. The organism shows cellular and morphological properties of Actinomyces sp. Actinomyces israelii. Formation of molar tooth colonies and isolation from cases of lumpy jaw and from the genital tract of women in association with the use of an intrauterine contraceptive device are other similarities with A. israelii. These species are potential pathogens that are likely to be overlooked in infected clinical material without special attention periods of incubation and culture media.

Haffajee et al. reported higher mean counts, proportions and percentage of sites of P. gingivalis and T. forsythia as well as E. nodatum and T. denticola from subjects with periodontitis than from periodontally healthy subjects. The researchers reported a strong association of E. nodatum and T. denticola with periodontitis whether in the presence or absence of high levels of the consensus pathogens.[11] Not surprisingly the same research group while studying the subgingival samples found merit in including E. nodatum as a part of the red complex.[5]

Dialister pneumosintes

Originally named as Bacterium pneumosintes by Olitsky and Gates who isolated it from nasopharyngeal secretions of patients during the flu epidemic of 1918-1921 the organism was first placed in the genus Dialister. The organism had chequered etymological twists in its christening. Taxonomic revision in 1970 necessitated its restoration to the genus Bacteroides. In 1980 Shah and Collins proposed removing the organism from the genus Bacteroides and Moore and Moore transferred the species back to the genus Dialister.

As small, Gram-negative rod that grows with punctiform, circular, convex, clear, transparent, shiny, smooth colonies on blood agar. D. pneumosintes has been recovered from pus and body fluids and from human bite wounds. The organism has potential to inflict pathological damage in lung, brain, and dental root canals.

Slow to grow and requiring strict anaerobic condition, D. pneumosintes is non-fermentive and produces no catalase, indole or nitrate. Cells are 0.2-0.4 by 0.3-9.6 μm, arranged singly, in pairs, or in very short chains.

The involvement of D. pneumosintes in periodontal conditions was not recognized for a long time. Isolated in low proportions from children and young adults with gingivitis, few studies only reported the presence of D. pneumosintes in human periodontal disease and this could be due to difficulties in culture methods. Only in the recent past the importance of this organism as an important component of subgingival microbiota has been brought to limelight.

With refinements in molecular microbiology especially 16S ribosomal RNA (rRNA) polymerase chain reaction (PCR) identification method, Ghayoumi et al. determined the presence of D. pneumosintes from periodontal pockets and implicated it as “candidate pathogen.”[12]

Presence of this organism in 83% of patients with severe periodontitis and in 19% of patients with slight periodontitis lead to the suggestion of the organism being considered as “suspected periodontal pathogens.”

The role of D. pneumosintes in refractory periodontitis was investigated by Colombo et al.[13] and in a recent study Silva et al. reported higher counts in subgingival plaque of 156 chronic patients than 66 aggressive periodontitis (AgP) patients.[14]

Comparing subgingival biofilm and saliva samples from subjects with periodontitis and healthy patients, Ferraro found significant associations between the prevalence of D. pneumosintes and pocket depth, attachment loss and bleeding on probing. These findings buttress the association of D. pneumosintes with periodontitis.

Smokers harbored significantly higher numbers of D. pneumosintes associated with moderate and deep pockets.[15] Although tested positive in healthy subjects, the median values were higher in samples from periodontitis subjects.[16]

Filifactor alocis (A thread like organism inhabiting in a furrow)

Filum-thread; factor- a maker; alox - a furrow; referring to its isolation from a crevice of the gums.

Originally isolated in 1985 from human gingival crevice as Fusobacterium alocis, it was reclassified into genus Filifactor based on 16S rRNA and named as F. alocis.

It is a fastidious, Gram-positive, obligatory anaerobic rod possessing trypsin-like enzymatic activity similar to P. gingivalis and T. denticola. F. alocis has the ability to survive in periodontal pocket and share common virulence properties with Fusobacterium.

The fastidious nature of this organism has contributed to its low detection in culture based methods. The organism is associated to cause endodontic infection and periodontal destruction. This organism has been found in elevated numbers in AgP (77.8%) and CP (76.7%) compared with periodontally healthy individuals due to its potential to withstand oxidative stress and inflammatory microenvironment provided by periodontal pocket.[17]

Filifactor is attributed as the second most prevalent in CP and third most prevalent in generalized aggressive periodontitis and proposed to be an excellent marker organism for periodontal disease.[17] A study by Dahlén and Leonhardt in 2006 concluded that F. alocis should be added to the 12 species used for routine diagnostics of periodontitis-associated bacterial flora.[18]

T. lecithinolyticum (Lekithos-egg yolk-effecting the breakdown of egg yolk)

Diverse treponemal species are associated with periodontal disease and endodontic infections. One among them is T. lecithinolyticum belonging to group IV oral treponemes. It is a small saccharolytic spirochaete possessing phospholipase A and C activities.

T. lecithinolyticum activates matrix metalloproteinase-2 in human gingival fibroblasts and periodontal ligament cells and induce activation of osteoclast by a prostaglandin E2-dependent mechanism. Major surface protein and prcA-prtP gene are considered as virulence factors of this organism, which exhibit chymotrypsin like protease activity stimulating various inflammatory cytokines namely interleukin-1, 6, 8 and intercellular adhesion molecule-1.[19]

The prevalence of this organism is more pronounced in rapidly progressive periodontitis and CP compared to healthy individuals.[20] Hence, it is proposed to be a good indicator requiring the development of advanced probes for its microbial diagnoses.

Solobacterium moorei

Named in honor of an American microbiologist Moore. Identified in 2000 by Kageyama and Benno. S. moorei, previously known as “Bulleidia moorei” is a Gram-positive, non-spore forming, anaerobic bacillus originally isolated from human feces. In the last decade, S. moorei has been associated with halitosis and has been isolated from endodontic infections, periradicular lesions and subgingival plaques from patients with refractory periodontitis.

This bacterium was categorized on the basis of 16S rRNA sequence into a new genus named Solobacterium. S. moorei is currently the only member of the genus. In addition to ribosomal RNA sequence, S. moorei is also distinguished from closely related species in the genera Eubacterium, Lactobacillus, Propionibacterium and Bifidobacterium by metabolic end products.

Kazor et al. examined samples taken from the dorsal surface of the tongue in five normal subjects and in six subjects with halitosis. The bacteria in the tongue samples were identified by direct amplification of 16S rDNA genes followed by cloning, a technique known as broad range PCR. They found S. moorei in three of six subjects with halitosis and in one of five normal subjects.[21] Haraszthy et al. using both broad range PCR and bacterial culture, identified S. moorei on the dorsal surface of the tongue in 8 of 8 subjects with halitosis but in 0 of 5 normal subjects.[22] Recently Tanabe and Grenier reported that S. moorei can be a major source of malodorous compounds in halitosis by producing VSCs through a process involving the β-galactosidase activity of the bacterium and an exogenous source of proteases.[23]

Cryptobacterium curtum (Kryptos- hidden; curtum- shortened; a hidden rod-shaped bacterium)

Cells are short Gram-positive rods. Occasionally Gram-variable are in stationary phase. Obligatory anaerobic, non-motile and non-sporing. Catalase negative and asaccharolytic. Individual cells occur singly or in masses. C. curtum is characterized as opportunistic pathogen with a typical occurrence in the oral cavity, involved in dental and oral infections such as periodontitis, inflammation and abscess. Nakazawa et al. in their study proposed novel Eubacterium-like isolates, from the periodontal pocket of an adult patient with periodontal disease and necrotic dental pulp and suggested that it should be classified in a new genus and species C. curtum.[24]

Mitsuokella dentalis

Named after Mitsuoka, a Japanese bacteriologist who first described the organism. M. dentalis is a nonmotile, nonspore forming, Gram-negative rods approximately 0.7 μm wide by 1-2 μm long. Flynn et al. reported that M. dentalis is a constituent of the pathogenic microbiota in human periodontitis.[25]

Porphyromonas endodontalis

P. endodontalis is an asaccharolytic, black-pigmented, Gram-negative anaerobic bacterium. Originally known as Bacteroides endodontalis, P. endodontalis is highly sensitive to oxygen and is therefore difficult to cultivate from clinical samples. P. endodontalis is primarily found in infections that originate in the pulp, but has also been isolated from the tonsillar area, the dorsum of the tongue and the periodontal pockets of patients with periodontal and pulp lesions. Tran et al. were the first to report the detection of this species in periodontal pockets, although it was present at a low concentration.[26]

Lombardo et al. showed a high prevalence of P. endodontalis in addition to P. gingivalis and T. forsythia, in diseased periodontal sites when compared to healthy sites, with a statistically significant reduction after periodontal therapy.[27]

Peptostreptococcus micros

P micros is a Gram-positive anaerobic coccus, which is primarily found in human oral cavity. Also called as Parvimonas micra it exists in three morphologies smooth, rough and smooth variant of the rough type. The organism is considered to be a commensal of the oral cavity, as it comprises of <3% of the subgingival flora in periodontally healthy subjects. However, it has been isolated more frequently and in higher numbers in patients with periodontitis and has been shown to be associated with periodontal destruction, particularly in sites from disease active patients. P. micros is also a known pathogen in other mixed anaerobic infections of oral cavity including endodontic abscesses, peritonsillar infections and other mixed infections throughout the body.

Rams et al. in a cross-sectional study involving 907 people reported prevalence of P. micros in 58-63% of periodontitis subjects. In culture-positive patients, P. micros averaged 12-15% of total viable counts and it was concluded to be potential pathogen in adult periodontitis.[28]

Fusobacterium nucleatum

F. nucleatum belongs to the Bacteroidaceae family and is a dominant microorganism within the periodontium. It is a Gram-negative anaerobic species of the phylum Fusobacteria, numerically dominant in dental plaque biofilms, and important in biofilm ecology and human infectious diseases.

F. nucleatum which refers to group of three subspecies (nucleatum, vincentii and polymorphum) is a central species in physical interactions between Gram-positive and Gram-negative species that are likely to be important in biofilm colonization and contributes to the reducing conditions necessary for the emergence of oxygen-intolerant anaerobes. It is considered as an intermediate colonizer bridging the attachment of commensals that colonize the tooth and epithelial surface with true pathogens.

It also promotes physio-chemical changes in the gingival sulcus, allowing pathogenic successors to establish and proliferate. It was also suggested that F. nucleatum facilitates invasion of host cells by P. gingivalis.[29]

F. nucleatum is demonstrated to be significant marker for destructive periodontal disease in adult subjects and is identified more in active sites compared to inactive sites, and associated with higher pocket sulfide levels in CP subjects.[30]

CONCLUSION

The paper attempts to highlight the hitherto unappreciated bacteria in pathogenesis. It may not have a direct clinical significance like, say, regeneration or root coverage, but provokes curiosity and inquiry. Focusing on these bacteria will encourage research and invite future deliberations and would be of help to postgraduate students and teachers in periodontology. The oral microbiota is one of the best characterized microbiomes that colonize the human body. Despite the increasing knowledge about oral microbiota, we are not able to zero on any one particular organism that can be christened as a “candidate pathogen”. In fact the term “candidate pathogen” has lost its steam with an array of microorganisms being incriminated from time to time. The concept that not one single organism but several organisms contained in the biofilm orchestrating in a medley of show with host response throwing its weight appears to be more plausible. Periodontitis although unusual to occur in the absence of microbial components, we are nowhere near the elusive principle actor and only have sporadic, but inconclusive evidence. The role of supporting actors and bit players need to be reappraised. The room with a view as far as microbial populace in periodontitis is concerned needs a fresh stance on the condition of “temporality.”