# Mixed Red-Complex Bacterial Infection in Periodontitis

Authors: N. Suzuki; M. Yoneda; T. Hirofuji
Journal: International Journal of Dentistry
 (2013)

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## Abstract

The red complex, which includesPorphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (formerly Bacteroides forsythus), are recognized as the most important pathogens in adult periodontal disease. These bacteria are usually found together in periodontal pockets, suggesting that they may cause destruction of the periodontal tissue in a cooperative manner. This article discusses the interspecies pathogenic interactions within the red complex.


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## Body

## 1. Introduction

Periodontal diseases are polymicrobial immune-inflammatory infectious diseases that can lead to the destruction of periodontal ligaments and adjacent supportive alveolar bone. The subgingival plaque contains more than 700 bacterial species, and some of these microorganisms have been shown to be responsible for initiation/progression of periodontal diseases [1, 2]. The red complex, which includes Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia(formerly Bacteroides forsythus), encompasses the most important pathogens in adult periodontal disease [3]. Additionally, Fusobacterium nucleatum, Prevotellaspecies, Eikenella corrodens, Peptostreptococcus micros, and Campylobacter rectusare increased in deep periodontal pockets and are implicated as possible periodontopathogens [1–4]. These bacteria are not usually found alone, but in combination in the periodontal pockets, suggesting that some bacteria may cause destruction of the periodontal tissue in a cooperative manner [5]. Studies using animal models have reported the synergistic pathogenicity of mixed infections with P. gingivalis-T. denticola, P. gingivalis-F. nucleatum, P. gingivalis-T. forsythia, P. gingivalis-Aggregatibacter actinomycetemcomitans, F. nucleatum-T. forsythia, and P. gingivalis-T. denticola-T. forsythia [6–11]. Furthermore, coaggregation, nutrient effects, and modulation of virulence factors by periodontopathogens or by interspecies interactions between periodontopathogenic and nonpathogenic organisms have been reported to contribute to oral microbial pathogenesis [12]. This paper focuses on interspecies pathogenic interactions within the red complex, in particular the combinations of P. gingivalis-T. forsythia, P. gingivalis-T. denticola, and P. gingivalis-T. forsythia-T. denticola. Potential therapies using normal inhabitants of the oral microbiota that have an antagonistic relationship with the red complex are discussed.


## 2.P. gingivalis and T. forsythia

P. gingivalispossesses many virulence factors, such as fimbriae, lipopolysaccharides, and proteases [13–15]. The arg-gingipain (Rgp) and lys-gingipain (Kgp) cysteine proteinases are important for the virulence of P. gingivalisas they elicit dysfunction of inflammatory and immune responses and can degrade various connective tissue proteins [16, 17]. Rgp is encoded by two separate genes (rgpAand rgpB), whereas Kgp is encoded by a single gene (kgp) [18]. In a murine abscess model, rgpAand rgpBdouble and kgpsingle-mutants induced smaller abscesses than did the wild type, and the rgpA-rgpB-kgptriple (gingipain-null) mutant showed negligible lesion formation [19]. These findings indicate that gingipains play an important role in abscess formation in mice.Compared to the abscess formation induced by monoinfection with each bacterium in the abovedescribed murine model, mixed infection with wild-typeP. gingivalisATCC 33277 and T. forsythiaATCC 43037 showed a synergistic effect on abscess formation [19]. On the other hand, mixed infection with P. gingivalismutants devoid of gingipain (rgpA rgpB, kgp, and rgpA rgpB kgp) with T. forsythiashowed only just an additive effect on abscess formation. These findings suggest that the gingipains of P. gingivalis play an important role in the pathological synergism between P. gingivalisand T. forsythia. A combination of T. forsythiastrains isolated from periodontitis patients and P. gingivalis also showed synergistic pathogenesis in a rabbit abscess model [20]. The researchers found 100% abscess formation in rabbits with mixed infection using P. gingivalisand T. forsythia but 0% in rabbits with monoinfection with each bacterium. Recently, Verma et al. [21] injected P. gingivalisFDC 381 and T. forsythiaATCC 43037 into the oral cavity of rats and evaluated the synergistic effect of mixed infection on periodontal disease. Mixed colonization by P. gingivalisand T. forsythiain the rat oral cavity was confirmed by polymerase chain reaction (PCR). The induction of moderate periodontal inflammation and pronounced apical migration of junctional epithelium, generation of a specific immunoglobulin G (IgG) antibody response, and stimulation of both Th1- and Th2-like immune responses as reflected by the serum IgG subclass profiles were evident. Mixed infection with P. gingivalisand T. forsythiaresulted in a significant increase in interproximal alveolar bone resorption compared to that in rats infected with a single bacterial strain and controls, but there was no synergy between the bacteria [21]. The researchers indicated that the virulence of P. gingivalisand T. forsythiamixed infection resulted from the immune-inflammatory responses and the lack of humoral immune protection during periodontitis in rats.Humoral immune responses were induced byP. gingivalisin the murine abscess model [22]. Mice that were first infected with a wild-type strain and subsequently reinfected with the same wild-type strain showed significantly smaller lesions than control mice that were mock-infected with medium only and then re-infected with the wild-type strain. Yoneda et al. [23] assessed the serum IgG antibody response in a murine abscess model after mixed infection with various levels of P. gingivalisATCC 33277 or T. forsythiaATCC 43037 whole bacteria cell antigens. After mixed infection, IgG antibody responses to P. gingivalisincreased in proportion to the level of T. forsythiainjected. In contrast, IgG antibody responses to T. forsythiadid not correlate with the level of P. gingivalisinjected. Reasons for this difference may be that P. gingivalisand T. forsythiainduced different antibody responses after mixed infection in mice; alternatively, these bacteria may exhibit different interactions in terms of growth at the injected sites.T. forsythia, which is a fastidious anaerobic Gram-negative rod, is frequently isolated together with P. gingivalis, especially from the active state of periodontitis [24–27]. It is well known that the growth of T. forsythiais accelerated on blood agar when cocultivated with P. gingivalisor F. nucleatum, suggesting that a form of symbiosis occurs with respect to nutrition [28]. Furthermore, the growth-promoting factors appear to be proteinaceous in nature. Sonicated cell extracts of T. forsythiaATCC 43037 stimulate growth of P. gingivalisATCC 33277 in nutrition-decreased medium in a dose-dependent manner [29], whereas the cell extract of T. forsythiahad no stimulatory effect on the growth of the rgpA rgpB kgptriple (gingipain-null) mutant of P. gingivalis. These results suggest that gingipains of P. gingivalisplay an important role in the digestion or uptake of the growth-promoting factor derived from T. forsythia. The growth-promoting interaction between P. gingivalisand T. forsythia may be partly related to synergistic virulence in a murine abscess model.T. forsythiais a member of the polymicrobial flora that invades buccal epithelial cells taken directly from the mouth [30]. Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses and for forming reservoirs important in recurrent infections. T. forsythiapossesses some putative virulence factors, such as a trypsin-like protease, a sialidase, hemagglutinin, components of the bacterial S-layer, and a cell surface-associated and secreted protein (BspA) [31]. BspA has been recognized as a virulence factor important for alveolar bone loss in mice [31]. Inagaki et al. [32] investigated the epithelial cell adherence and invasion abilities of T. forsythiaand reported that these are dependent on BspA. Additionally, they found that P. gingivalis FDC 381 or its outer membrane vesicles enhance the attachment and invasion of T. forsythiaATCC 43037 to epithelial cells.


## 3.P. gingivalis and T. denticola

T. denticola, a small oral spirochete, is frequently found withP. gingivalisin progressing periodontitis lesions [1, 33–35]. T. denticolais located within the surface layers of the subgingival plaque, whereas P. gingivalisis observed predominantly beneath the spirochete layer [33]; a symbiotic nutrient utilization relationship between these two periodontopathogens has been shown in vitro [36]. The growth-stimulating factors produced by P. gingivalisATCC 33277 and T. denticolaATCC 35405 have been identified as isobutyric acid and succinic acid, respectively [36].Biofilm formation is also an important step in the etiology of periodontal diseases.P. gingivalis, but not T. denticolastrains, formed biofilms in vitro[37]. Coculture of P. gingivalisFDC 381 and T. denticolaATCC 35405 induced synergistic biofilm formation and coaggregation. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as the primary colonizer followed by coaggregation with T. denticolato form a mixed biofilm [38]. The T. denticolaflagellar mutant and cytoplasmic filament mutant exhibit significantly reduced biofilm formation with P. gingivalis. Similarly, the P. gingivalisgingipain mutant and major fimbriae mutant exhibited significantly reduced biofilm formation with T. denticola[38]. Using two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalisfimbriae, Hashimoto et al. [39] determined that dentilisin, a chymotrypsin-like proteinase of T. denticola,  was the P. gingivalisfimbriae-binding protein. These results support the hypothesis that these two organisms assist each other’s survival in subgingival sulcus and explain why they are frequently isolated together from subgingival plaque.Synergistic virulence of mixedP. gingivalisand T. denticolainfection has been assessed in several animal lesion models. Using a murine abscess model, one group reported that high doses of P. gingivalisW50 (1-2×1010 cells per dose) together with T. denticolaATCC 35404 (1×109 or 1×1010 cells per dose) had no effect on the formation and size of the spreading lesion caused by P. gingivalis [40]. However, at low P. gingivalis doses (1-2×109 cells), addition of T. denticola(1×1010 cells) significantly enhanced the virulence of P. gingivaliscompared with monoinfection [40]. Investigation of the synergistic virulence of P. gingivalisand T. denticolausing a murine experimental model of periodontitis found that a 1 : 1 ratio of P. gingivalisW50 and T. denticolaATCC 35405 coinoculum at 5×108 or 1×109 total bacterial cells induced the same level of bone loss as four doses of 1×1010  P. gingivalis[41]. Coinoculation induced strong P. gingivalis-specific T cell proliferative and interferon-γ (IFN-γ) cytokine responses and induced a strong T. denticola-specific IFN-γ cytokine response. Another study using a rat model of periodontal disease reported that a mixed P. gingivalisFDC 381 (5×109 cells) and T. denticolaATCC 35404 (5×109 cells) infection produced significantly more interproximal and horizontal alveolar bone loss compared to monoinfections (1×1010 cells); however, there was no synergy between P. gingivalisand T. denticola[42]. Furthermore, colonization of these bacteria was observed in the rat oral cavity during 7 weeks of periodontal disease, resulting in the generation of a specific serum IgG antibody response that reflected oral infection and the induction of an inflammatory response consistent with the established characteristics of periodontitis. These results suggest that P. gingivalisand T. denticolaact synergistically (with no synergy between the bacteria) to stimulate the host immune response and to induce alveolar bone loss in a rat model of periodontitis [42].


## 4.P. gingivalis, T. forsythia, and T. denticola

Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells contribute to periodontal tissue destruction. Bodet et al. [43] investigated the inflammatory response of an in vitro macrophage/epithelial cell coculture model following mono- or mixed infections with whole bacterial cells of the red-complex and their lipopolysaccharide (LPS). Mono- or mixed infections of the coculture model induced the secretion of interleukin-1 beta (IL-1β), IL-6, IL-8, prostaglandin E2 (PGE2), and MMP-9. All LPS mono- or mixed infections induced an increase in chemokine, MMP-9, and PGE2 production. Compared to mono-infections with individual bacterial species, no synergistic effects on cytokine, PGE2, or MMP-9 production by the bacterial mixtures tested were observed. P. gingivalisand T. forsythiainduced the secretion of RANTES (regulated and normal T cell expressed and secreted), whereas T. denticolaalone or in combination with P. gingivalisresulted in a significant decrease in RANTES levels. RANTES degradation by mono- or mixed infections with red-complex bacteria resulted in massive proteolytic degradation of RANTES by T. denticola. The ability of periodontopathogens to degrade cytokines and chemokines in vivomay play an important role in their pathogenicity by disrupting the host inflammatory response.Recently, polymicrobial infection withP. gingivalis, T. denticola, and T. forsythiain a rat model of periodontal disease was investigated [44]. A 1×1010 cell mixture (1 mL) containing an equal number of cells of each bacterium was injected into the rat oral cavity. PCR of the bacterial DNA in the oral sample revealed that polymicrobial infection enhanced colonization by P. gingivalis, T. denticola, and T. forsythia compared to their levels in monomicrobial infections. Oral infection of rats with a polymicrobial consortium comprising P. gingivalis, T. denticola, and T. forsythiainduced significant increases in maxillary and mandibular alveolar bone resorption compared to those resulting from any of the monomicrobial infections (P<0.001). The levels of serum IgG against all of the bacteria in the polymicrobial infection were lower than the respective levels induced by monomicrobial infections. This suggested that the host response to the polymicrobial infection was altered, resulting in enhanced evasion of protective immune responses by the bacterial consortium.


## 5. Future Directions of Periodontitis Therapy

Most therapeutic modalities for treatment of periodontitis aim to remove pathogens and kill all bacteria in the periodontal pocket. Understanding the effect of interbacterial interactions on the pathogenesis of periodontitis may facilitate development of novel treatment modalities, such as the inhibition of adherence using antagonists, passive immunization, replacement therapy, regulation of levels of nonpathogenic bacteria to modulate virulence, probiotics, and interference with signaling mechanisms [12]. The disruption of the harmonic relationship between the host and commensal microorganisms is considered to be an important factor for the development of oral pathologies. The microbiota composition of healthy and periodontitis patients differs [45–47]. Here, we discuss the significance of interbacterial antagonism for maintenance and recovery of a healthy oral microbiota. Antagonistic bacteria present a substantial barrier to colonization by exogenous and opportunistic endogenous pathogens. Moreover, antagonistic bacteria have the potential for probiotic action, which may protect against periodontitis.Studies have identified organisms antagonistic to periodontopathogens. With regard to the red complex,Staphylococcus aureusand Streptococcus mutansisolates inhibited the growth of T. denticolaand P. gingivalis[48]. S. aureus strains produced a bacteriocin-like inhibitory substance, whereas the inhibitory effect of S. mutanswas related to the production of lactic acid. S. sanguinis, S. cristatus, S. salivarius, S. mitis, Actinomyces naeslundii, and Haemophilus parainfluenzaeinhibited the adhesion of standard P. gingivalisstrains in vitro [49]. S. cristatusarginine deiminase repressed FimA, a major subunit protein of the long fimbriae, and inhibited biofilm formation by P. gingivalis[50].Other studies have identified organisms with probiotic potential against periodontopathogens. An analysis of the ability of clinical isolates from healthy and periodontitis patients to inhibit the growth of periodontopathogens showed that the number of isolates from healthy volunteers that inhibited eitherP. gingivalisor P. intermedia was significantly higher than that from diseased patients [51]. These isolated growth-inhibiting strains included some viridans group Streptococcus, Actinomyces, and Bifidobacterium strains. Compared to these isolates, commercial dietary probiotics showed stronger inhibition of the periodontopathogens. In a study that compared oral lactobacilli from patients with chronic periodontitis and periodontally healthy subjects, the most prevalent species in healthy subjects were Lactobacillus gasseriand L. fermentum, whereas the most prevalent species in subjects with periodontitis was L. plantarum[52]. Furthermore, the greatest antimicrobial activities were associated with L. paracasei, L. plantarum, L. rhamnosus, and L. salivarius. The international guidelines for the evaluation of probiotics confirm that these four organisms exhibit both high antimicrobial activity and high tolerance to environmental stress [53].Some oral bacteria possess the potential for antagonism towards periodontopathogens, which highlights the therapeutic potential of stimulation of oral health using beneficial effector strains. Most evidence indicates that probiotics in the gut do not populate the gastrointestinal microbiota permanently, and they disappear from feces soon after cessation of probiotic ingestion [54]. Similarly, altering the composition of established oral plaque microbial communities is difficult [55]. Probiotic bacteria used in the human oral cavity include Bifidobacteriumand Lactobacillusspecies, and most of them were not derived from the oral cavity [56]. L. reuteriand L. salivariuscolonized the oral cavity of patients; however, the study was of only 2 weeks duration [57–59]. Another study reported that oral administration of L. salivarius decreased the proportion of L. salivariusin saliva during the 4- and 8-week intervention periods, although the sampling and analysis methods differed [60]. Organisms antagonistic to periodontopathogens that are derived from typical representatives of the oral microbiota and have probiotic potential may overcome the weaknesses associated with exogenous probiotic bacteria.



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