Periodontal diseases are a group of chronic inflammatory, polymicrobial infections, which result in gradual loss of tooth attachment to the bone and eventual loss of the tooth. The microbial etiology of periodontitis is defined by the sub gingival plaque biofilm in which resides an interdependent microbial community containing numerous species of bacteria. Many Gram-negative anaerobic bacilli such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, were considered as keystone pathogens and a pre-requisite for periodontal disease activity and progression. However, with the advent of open-ended molecular techniques such as 16s rRNA sequencing and cloning, organisms that were not considered pathogenic to periodontal infections are now emerging as possible contributors to the microbial etiology of periodontitis. Among these, the Gram-positive anaerobic bacteria are in the forefront. Over the past decade, various studies have showed the association of GPA with periodontal disease conditions. However, their absolute role in the same has not been clearly defined. This mini review aims at a comprehensive analysis of the literature available on the association of GPA (Gram Positive Anaerobes) with various periodontal disease conditions.

Keywords: chronic periodontitis, keystone pathogens, polymicrobial synergy dysbiosis, gram positive anerobes, diabetes mellitus, cardiovascular diseases

PSD Model, polymicrobial synergy dysbiosis model; GPA, gram positive anaerobes; DNA, deoxyribonucleic acid

Periodontitis is a polymicrobial infection leading to chronic inflammation of the supporting structures of the teeth that leads to pocket formation and breakdown of alveolar bone around the teeth, resulting in tooth loss.¹ Besides it’s local impact, periodontal infections are also associated with various far-reaching systemic effects, like diabetes mellitus and cardiovascular diseases.²

Advances in molecular research and the oral microbiome³ study have depicted the number of oral bacteria (cultivable and not) to a mind-boggling 700 species! The subgingival biofilm is the primary etiology of periodontal disease initiation and progression and for this reason periodontal microbiology has been the focus of research over centuries. It has gone through paradigm shifts of ideologies over the past century such as the non-specific/specific plaque hypotheses, the ecological plaque hypothesis and recently the PSD model. The “Non-Specific Plaque Hypothesis” proposed by Black and Miller in the late 1800s, believed that the total microflora could lead to disease. The “Specific Plaque Hypothesis”, which followed in 1976 concluded that only a few species of the total microflora are actively involved in disease taking into account differences in virulence among bacteria. The “Ecological Plaque Hypothesis” put forward by Marsh⁴ in 1994, emphasized that periodontal disease is the result of an imbalance in the microflora by ecological stress resulting in an enrichment of certain disease-related micro-organisms. “Keystone-Pathogen Hypothesis” in 2012⁵ proposes that certain low-abundance microbial pathogens can cause inflammatory disease by interfering with the host immune system and remodeling the microbiota.

The past few decades of periodontal microbial research have been spent primarily on certain keystone pathogens (mostly Gram-negative anaerobes), which were believed to be positively associated with periodontal disease. The currently accepted model of periodontal pathogenesis, however, believes that periodontitis is initiated by a synergistic and dysbiotic microbial community rather than by certain select keystone pathogens. Virulent pathogens such as Porphyromonas gingivalis, elevate the virulence of the entire biofilm community following interactive communication with accessory pathogens such as mitis group streptococci. This impairs the host immune surveillance and creates a dysbiotic environment eventually disrupting tissue homeostasis and causing destruction of periodontal tissues. This led to the currently believed model of periodontal pathogenesis, that is, polymicrobial synergy and dysbiosis or PSD model.⁶

This advancement in our understanding has made been possible, due to open-ended molecular techniques such as DNA-DNA checkerboard hybridization techniques and 16s rRNA cloning and the revolutionary oral microbiome study in 2010. Newer microorganisms which were previously associated with periodontal health have now been found to be contributing to the periodontal disease process.^7–11

Of these, Gram-positive anaerobes such as Filifactor alocis, Peptostreptococcus micros and Eubacterium nodatum which have been recently isolated from patients with periodontitis, are emerging to be considered as important contributors to the bacterial etiology of periodontitis. However, the literature evidence is controversial and inconsistent across various studies. This discrepancy could be explained by geographic variability,¹² or by difference in the depths of the pockets sampled,¹³ as well as the sample size and the DNA analytic bias.¹⁴

This review article aims to search all the available literature associating GPA and periodontal disease and draw a conclusion about the validity of GPA contributing significantly to periodontal pathology.

Classification system of bacteria discovered by Gram in 1884, allows a large proportion of clinically important bacteria to be classified as either Gram positive or negative. The following flow chart depicts the classification system of Gram +ve species in detail (Figure 1).

Figure 1 Classification system of Gram Positive species.

The potentially pathogenic role of bacteria, which were not considered as primary keystone pathogens in periodontal disease, has been implicated in periodontal literature time and again. Paul Keyes, way back in 1970, said - “I am convinced that although many clinicians and investigators do not exclude the role of bacteria in periodontal lesions, at this point interest in microorganisms often dissipates and attention shifts to other areas”.

Gram-positive anaerobes have been isolated way back in 1990s from periodontal biofilms.¹⁵ Kumar et al.⁸ also identified the dominance of Gram positive anaerobic species in periodontally diseased sites compared to healthy individuals.⁸ In another study supporting this evidence, Haffajee et al.¹⁶ examined supragingival biofilm samples to understand the nature of the microbial complexes that exist in supra-gingival plaque. An interesting observation was that Eubacterium nodatum, a Gram-positive anaerobe was found both in the mature and the long-term redevelopment biofilms along with the red complex species, P. gingivalis, T. forsythia, and T. denticola usually observed in subgingival plaque. Not surprisingly the same research group while studying the subgingival samples found merit in including E. nodatum as a part of the red complex.

Eubacterium nodatum is an obligate anaerobe, filamentous or club-shaped asaccharolytic, Gram-positive rod. They grow slowly in culture and share cultural, biochemical, or morphological characteristics with other well-known species of anaerobic bacteria. They elaborate virulence factors such as esterases, acid phosphatases and aminopeptidases and have been isolated from a significant proportion of the subgingival microbiota of chronic periodontitis ranging from 10.8 to 54%.¹⁷ Three species, E. nodatum, Eubacterium timidum, and Eubacterium brachy, have been described, primarily from subgingival samples taken from patients with moderate and severe periodontitis.¹⁸

Parvimonas micra is a species of the orange microbial complex put forward by Socransky et al.¹³ The presence of P. micra has been positively associated with periodontitis over the past 2 decades. Previously known as Peptostreptococcus micros, this Gram-positive, micro- aerophiliccoccus is usually associated with polymicrobial infections such as intracranial abscesses, sinus infections, and periodontitis. Peptostreptococcus sp. are colonisers of the oral cavity, vagina, skin, GI tract and urinary tract. They are also found to cause systemic infections such as abscesses, necrotizing tissue infections, and infections of GIT and urinary tract in immunocompromised individuals. P. micra possesses several virulence factors that contribute to its pathogenic potential. The cell wall of P. micra has been shown to induce a potent inflammatory response in macrophages.^19,20 They elaborate proteases that enable it to penetrate the basement membrane.²¹ It also makes a carbohydrate- mediated co-aggregation with Fusobacterium nucleatum and Porphyromonas gingivalis.¹⁹ These data suggest that peptostreptococci may play a role in preventing wound healing in chronic disease and may be important in the physical structure of a disease-associated biofilm.

Filifactor alocis is a fastidious, Gram-positive, obligatory anaerobic rod possessing trypsin-like enzymatic activity similar to P. gingivalis and T. denticola. It has the ability to survive in the periodontal pocket and shares common virulence properties with Fusobacterium. Filifactor alocis (ATCC 35896T) was first isolated in 1985 from the human gingival crevice as Fusobacterium alocis and later reclassified as Filifactor alocis. Oxidative stress resistance- Sialidase activity exhibited by F. alocis results in release of sialic acids that scavenge oxidative stress in the periodontal pocket.²² 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 Aggressive Periodontitis (77.8%) and Chronic Periodontitis (76.7%) compared with periodontally healthy individuals due to its potential to withstand oxidative stress and inflammatory microenvironment provided by periodontal pocket.¹⁷

A study by Dahlen & Leonhardt¹⁸ concluded that F. alocis should be added to the 12 species used for routine diagnostics of periodontitis-associated bacterial flora. This is one of the marker organisms and is considered an important periodontal pathogen. The organism is now identified to be significant to the pathogenic structure of biofilms associated with periodontal inflammation.^23–25 In comparison with the other traditional periodontal pathogens, the high incidence of F. alocis in the periodontal pocket compared with its absence in healthy individuals or those who are periodontitis- resistant has highlighted its importance in the infectious disease process.^17,23,24 Streptococcus sanguinis are Gram-positive cocci are non-motile and non-spore forming Pili of S. sanguinis bind to salivary α-amylase, and contribute to the formation of biofilm on saliva-coated surfaces. They initiate aggregation of other oral bacteria and maturation of dental plaque. Sortase A (SrtA) of S. sanguinis have an influence on the expression of various cell surface virulence factors.²⁶

E. Streptococcus parasanguinis are Gram-positive, non-motile, non-spore forming cocci. They are facultative anaerobes. The long peritrichous fimbriae of S. parasanguinis are critical for the formation of biofilms on solid surfaces.²⁷ It is one of the major early colonizers of dental surfaces in the human oral cavity. Fim A protein is a potential virulence factor (Table 1).

As evident from the above literature review, a number of Gram-positive anaerobes such as Filifactor alocis, Parvimonas micra, Eubacterium nodatum and Streptococcus parasanguinis are now considered as potential periodontal pathogens.^50–52 But these findings are not consistent across all studies. The possible contributory factors to this variability are study design, the population studied and the methods of detection of microorganisms. Also, we need to keep in mind while interpreting the results of association studies, the “causal versus casual” concept. Analysis of the associations based on the Hill’s criteria of causality can give us an insight into this aspect.

The paradigm shift in the understanding of periodontal pathogenesis is attributed to the introduction of novel theories about the ecological events associated with periodontal destruction. The Polymicrobial Synergy and Dysbiosis model makes us question the individual role of these Gram-positive anaerobes implicated in periodontal pathogenesis.^53–55 This has to be confirmed by future studies. Further, whether they fulfill all of Koch’s postulates in being an infectious organism is yet to be studied.

This mini review clearly highlights that the etiology of periodontitis is more complex than a previous model associating Gram-positive bacteria with health and implicating Gram-negative bacteria as the causative agents in disease. Whether Gram Positive Anaerobes contribute independently to the periodontal pathogenesis is to be decided by further uniformly designed studies and future systematic reviews.^56–59

None.

The author declares no conflict of interest.

1. Page RC, Offenbacher S, Schroeder HE, et al. Advances in the pathogenesis of periodontitis: Summary of developments, clinical implications and future directions. Periodontol 2000. 1997;14:216–248.
2. Maurizio T, Kenneth SK. Special issue: Periodontitis and Systemic Diseases – Proceedings of a workshop jointly held by the European Federation of Periodontology and American Academy of Periodontology. J Periodontol. 2013;84(4–s):S1–S214.
3. Dewhirst FE, Chen T, Izard J, et al, The Human Oral Microbiome. J Bacteriol. 2010;192(19):5002–5017.
4. Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res. 1994;8(2):263–271.
5. Hajishengallis G, Liang S, Payne MA, et al. Low–abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe. 2011;10(5):497–506.
6. Hajishengallis G, Lamont RJ. Beyond the red complex and into more complexity: the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease etiology. Mol Oral Microbiol. 2012;27(6):409–419.
7. Laksmana T, Kittichotirat W, Huang Y, et al. Metagenomic analysis of subgingival microbiota following non–surgical periodontal therapy:a pilot study. Open Dent J. 2012;6:255–261.
8. Kumar PS, Griffen AL, Moeschberger ML, et al. Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J Clin Microbiol. 2005;43(8):3944–3955.
9. Griffen AL, Beall CJ, Campbell JH, et al. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing. ISME J. 2012;6(6):1176–1185.
10. Liu B, Faller LL, Klitgord N, et al. Deep sequencing of the oral microbiome reveals signatures of periodontal disease. PLoS One. 2012;7(6):e37919.
11. Wang J, Qi J, Zhao H, et al. Metagenomic sequencing reveals microbiota and its functional potential associated with periodontal disease. Sci Rep. 2013;3:1843.
12. Nasidze I, Li J, Quinque D, et al. Global diversity in the human salivary microbiome. Genome Res. 2009;19(4):636–643.
13. Socransky SS, Haffajee AD, Cugini MA, et al. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25(2):134–144.
14. Von Wintzingerode F, Gobel UB, Stackebrandt E. Determination of microbial diversity in environmental samples: pitfalls of PCR–based rRNA analysis. FEMS Microbiol Rev. 1997;21(3):213–219.
15. Rams TE, Feik D, Listgarten MA, et al. Peptostreptococcus micros in human periodontitis. Oral Microbiol Immunol. 1992;7(1):1–64.
16. Haffajee AD, Socransky SS, Patel MR, et al. Microbial complexes in supragingival plaque. Oral Microbiol Immunol. 2008;23(3):196–205.
17. Wade WG. The Role of Eubacterium Species in Periodontal Disease and Other Oral Infections. Microbial ecology in health and disease. 1996;9(6):367–370.
18. Dahlen G, Leonhardt A. A new checkerboard panel for testing bacterial markers in periodontal disease. Oral Microbiol Immunol. 2006;21(1):6–11.
19. Hill GB, Ayers OM, Kohan AP. Characteristics and Sites of Infection of Eubacterium nodatum, Eubacterium timidum, Eubacteriumbrachy, and Other Asaccharolytic Eubacteria. J Clin Microbiol. 1987;25(7):1540–1545.
20. Kremer BH, van Steenbergen TJ. Peptostreptococcusmicros coaggregates with Fusobacterium nucleatum and non–encapsulatedPorphyromonas gingivalis. FEMS Microbiol Lett. 2000;182:57–62.
21. Tanabe S, Bodet C, Grenier D. Peptostreptococcus micros cell wall elicits a pro–inflammatory response in human macrophages. J Endotoxin RCS. 2007;13(4):219–226.
22. Grenier D, Bouclin R. Contribution of proteases and plasmin–acquired activity in migration of Peptostreptococcus micros through a reconstituted basement membrane. Oral Microbiol Immunol. 2006;21(5):319–325.
23. Kumar PS, Griffen AL, Barton JA, et al. New bacterial species associated with chronic periodontitis. J Dent Res. 2003;82(5):338–344.
24. Kumar PS, Leys EJ, Bryk JM, et al. Changes in periodontal health status are associated with bacterial community shifts as assessed by quantitative 16S cloning and sequencing. J Clin Microbiol. 2006;44(10):3665–3673.
25. Schlafer S, Riep B, Griffen AL, et al. Filifactor alocis – involvement in periodontal. BMC Microbiol. 2010;10:66.
26. Yamaguchi M, Terao Y, Ogawa T, et al. Role of Streptococcus sanguinis sortase A in bacterial colonization. Microbes and Infection. 2006;8(12–13):2791–2796.
27. Froeliger EH, Fives Taylor P. Streptococcus parasanguisfimbria–associated adhesin fap1 is required for biofilm formation. Infect Immun. 2001;69(4):2512–2519.
28. Tsai CY, Tang CY, Tan TS, et al. Subgingival microbiota in individuals with severe chronic periodontitis. J Microbiol Immunol Infect. 2016;S1684–1182(16):30037–30038.
29. Feres M, Bernal M, Matarazzo F, et al. Subgingival bacterial recolonization after scaling and root planing in smokers with chronic periodontitis. Aust Dent J. 2015;60(2):225–232.
30. Ana Paula VC, Clarissa BM. Periodontal–disease–associated biofilm: A reservoir for pathogens of medical importance. Microb Pathog. 2016;94:27–34.
31. Matthew RM, Philip MP, Haikady N, et al. The subgingival microbiome of clinically healthy current and never smokers. ISME J. 2015;9:268–272.
32. Al hebshi NN, Al Alimi A, Taiyeb Ali T, et al. Quantitative analysis of classical and new putative periodontal pathogens in subgingival biofilm:a case– control study. J Periodont Res. 2015;50(3):320–329.
33. Moon JH1, Lee JH, Lee JY. Subgingival microbiome in smokers and non–smokers in Korean chronic periodontitis Patients. Mol Oral Microbiol. 2015;30(3):227–241.
34. Lourenço TG, Heller D, Silva Boghossian CM, et al. Microbial signature profiles of periodontally healthy and diseased patients. J Clin Periodontol. 2014;41(11):1027–1036.
35. Pérez Chaparro PJ, Gonçalves C, Figueiredo LC, et al. Newly identified pathogens associated with periodontitis: a systematic review. J Dent Res. 2014;93(9):846–858.
36. Fine DH, Markowitz K, Fairlie K, et al. A Consortium of Aggregatibacter actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis is present in sites prior to bone loss in a longitudinal study of localized aggressive periodontitis. J Clin Microbiol. 2013;51(9):2850–2861.
37. Aruni AW, Roy F, Sandberg L, et al. Proteome variation among Filifactor alocis strains. Proteomics. 2012;12(12):3343 3364.
38. Griffen AL, Beall CJ, Campbell JH, et al. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing. ISME J. 2012;6(6):1176–1185.
39. Colombo AP, Bennet S, Cotton SL, et al. Impact of periodontal therapy on the subgingival microbiota of severe periodontitis: comparison between good responders and “refractory” subjects by the human oral microbe identification microarray (HOMIM). J Periodontol. 2012;83(10):1279–1287.
40. Shaddox LM, Huang H, Lin T, et al. Microbiological characterization in children with aggressive periodontitis. J Dent Res. 2012;91(10):927–933.
41. Moffatt CE, Whitmore SE, Griffen AL, et al. Filifactor alocis interactions with gingival epithelial cells. Mol Oral Microbiol December. 2011;26(6):365–373.
42. Aruni AW, Roy F, Fletcher HM. Filifactor alocis has virulence attributes that can enhance its persistence under oxidative stress conditions and mediate invasion of epithelial cells by porphyromonas gingivalis. Infect Immun. 2011;79(10):3872–3886.
43. Abusleme L, Dupuy AK, Dutzan N, et al. The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation. ISME J. 2013;7(5):1016–1025.
44. Urbán E, Terhes G, Radnai M, et al. Detection of periodontopathogenic bacteria in pregnant women by traditional anaerobic culture method and by a commercial molecular genetic method. Anaerobe. 2010;16(3):283–288.
45. Shchipkova AY, Nagaraja HN, Kumar PS. Subgingival microbial profiles of smokers with periodontitis. J Dent Res. 2010;89(11):1247–1253.
46. Shchipkova AY, Nagaraja HN, Kumar PS. Subgingival Microbial Profiles of Smokers with Periodontitis. J Dent Res. 2010;89(11):1247–1253.
47.  Booth V, Downes J, Van den Berg J, et al. Gram–positive anaerobic bacilli in human periodontal disease. J Periodontol Res. 2004;39(4):213–220.  
48. Van Winkelhoff AJ, Bosch Tijhof CJ, Winkel EG, et al. Smoking affects the subgingival microflora in periodontitis. J Periodontol. 2001;72(5):666–671.
49. Tanner A, Maiden MF, Macuch PJ, et al. Microbiota of health, gingivitis, and initial periodontitis. J Clin Periodontol. 1998;25(2):85–98.
50. Nezar NAH, Hussein MS. Subgingival periodontal pathogens associated with chronic periodontitis in Yemenis. BMC Oral Health. 2014;14:13.
51. Nonnenmacher C, Dalpke A, Mutters R, et al. Quantitative detection of periodontopathogens by real–time PCR. J Microbiol Methods. 2004;59(1):117–125.
52. Mahajan A, Singh B, Kashyap D, et al. Interspecies communication and periodontal disease. Scientific World Journal. 2013;2013:765434.
53. Murphy EC, Frick IM. Gram–positive anaerobic cocci – commensals and opportunistic Pathogens. FEMS Microbiol Rev. 2013;37(4):520–553.
54. Wu HJ, Wang AH, Jennings MP. Jennings discovery of virulence factors of pathogenic bacteria. Curr Opin Chem Biol. 2008;12(1):1–9.
55. Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology. 2003;149(Pt 2):279–294.
56. Aruni W, Chioma O, Fletcher HM. Filifactor alocis:the newly discovered kid on the block with special talents. J Dent Res. 2014;93(8):725–732.
57. Boles BR, Thoendel M, Singh PK. Self–generated diversity produces ‘‘insurance effects’’ in biofilm communities. PNAS. 2004;101(47):16630–16635.
58. Roberts FA, Darveau RP. Microbial protection and virulence in periodontal tissue as a function of polymicrobial communities:symbiosis and Dysbiosis. Periodontol 2000. 2015;69(1):1–27.
59. Marsh PD, Devine DA. How is the development of dental biofilms influenced by the host? J Clin Periodontol. 2011;38(Suppl 11):28–35.