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1 Gazi University, Faculty of Dentistry, Department of Endodontics and Conservative Treatment, 82. Sokak 06510 Emek, Ankara, Turkey; 2 NIOM, Scandinavian Institute of Dental Materials, Haslum, Norway
* corresponding author, guvenk{at}gazi.edu.tr
Abstract Introduction Aggregation Substance Surface Adhesins Sex Pheromones Lipoteichoic Acid Extracellular Superoxide Production Gelatinase Hyaluronidase Cytolysin AS-48 Other Bacteriocins Conclusion Acknowledgments REFERENCES
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Key words. Enterococcus faecalis, virulence factors, endodontic infection, apical periodontitis
| Introduction |
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Enterococci can withstand harsh environmental conditions. As originally defined by Sherman (1937), enterococci can grow at 10°C and 45°C, at pH 9.6, in 6.5% NaCl broth, and survive at 60°C for 30 minutes. E. faecalis can adapt to adverse conditions: Following pre-exposure to sublethal stress conditions, E. faecalis becomes less sensitive to normally lethal levels of sodium dodecyl sulfate, bile salts, hyperosmolarity, heat, ethanol, hydrogen peroxide, acidity, and alkalinity; furthermore, ‘cross-protection’ is pronounced against diverse challenges (Flahaut et al., 1996a,b,c, 1997). Starving E. faecalis cells maintain their viability for extended periods and become resistant to UV irradiation, heat, sodium hypochlorite, hydrogen peroxide, ethanol, and acid (Giard et al., 1996; Hartke et al., 1998). E. faecalis, moreover, can enter the viable but non-cultivable (VBNC) state, a survival mechanism adopted by a group of bacteria when exposed to environmental stress, and resuscitate upon returning to favorable conditions (Lleò et al., 2001). The ability of E. faecalis to tolerate or adapt to harsh environmental conditions may act as an advantage over other species. It may explain its survival in root canal infections, where nutrients are scarce and there are limited means of escape from root canal medicaments.
In in vitro studies, E. faecalis has been shown to invade dentinal tubules (Akpata and Blechman, 1982; Haapasalo and Ørstavik, 1987; Ørstavik and Haapasalo, 1990; Love, 2001), whereas not all bacteria have this ability (Akpata and Blechman, 1982; Perez et al., 1993). In animal studies, where pure cultures of various bacteria were inoculated separately into root canals, E. faecalis, unlike others, was found to colonize the root canal in most cases and to survive without the support of other bacteria (Fabricius et al., 1982; Sobrinho et al., 1998). E. faecalis is resistant to the antimicrobial effects of calcium hydroxide (Byström et al., 1985; Haapasalo and Ørstavik, 1987; Ørstavik and Haapasalo, 1990; Distel et al., 2002), probably partly due to an effective proton pump mechanism which maintains optimal cytoplasmic pH levels (Evans et al., 2002). Besides, E. faecalis, intrinsically or via acquisition, may be resistant to a wide range of antibiotics (Leclercq, 1997; Hunt, 1998), which, if used, may shift the microbial flora in favor of E. faecalis.
We undertook a literature search for the virulence factors of E. faecalis, which may relate to colonization of the host, competition with other bacteria, resistance against defense mechanisms of the host, and production of pathological changes directly through production of toxins or indirectly through induction of inflammation. The factors most extensively studied are: aggregation substance, surface adhesins, sex pheromones, lipoteichoic acid, extracellular superoxide, gelatinase, hyaluronidase, and cytolysin (hemolysin). Although not strictly acting as virulence factors, AS-48 and other bacteriocins are mentioned because of their possible contribution to the dominance of E. faecalis in persistent endodontic infections. From the data available, a model for the pathogenicity of E. faecalis in endodontic infections has been developed (Fig.
), where the elements of virulence factors and means of ecological advantage for this organism have been integrated.
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| Aggregation Substance |
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In addition to its adhesive function during the bacterial conjugation process, AS mediates adhesion of E. faecalis to a variety of eukaryotic cells in vitro, including renal tubular cells (Kreft et al., 1992) and intestinal epithelial cells (Olmsted et al., 1994). The sequence analysis of the AS indicates two RGD motifs (arginine, glycine and aspartic acid, amino acids) that are thought to facilitate adherence of the bacterium to the host cell via integrins, a family of eukaryotic cell-surface receptors (Galli et al., 1990). However, analysis of recent data suggests that a non-RGD-dependent pathway of Asc10 (a group of AS proteins) mediated cell internalization into enterocytes (Waters et al., 2003).
AS was also found to mediate binding to extracellular matrix (ECM) proteins, including collagen type I. E. faecalis strain OG1X(pAM721), constitutively expressing AS, binds to collagen type I more than two times higher than the AS-negative strain OG1X(pAM944) (Rodzinski et al., 2001). Binding to collagen type I by bacteria may be of particular importance with respect to endodontic infections, since this is the main organic component of the dentin (Linde and Goldberg, 1993). A novel representative of the AS family, Asa 373, differing in its protein structure from classic AS, was reported to exhibit some moderately conserved amino acid motifs when its database sequence was compared with those of some other bacterial adhesins. The greatest similarity was with the amino acid sequence of Ag I/II polypeptides of oral streptococci (Muscholl-Silberhorn, 1999). The Ag I/II family of polypeptides has been found to mediate collagen recognition in some oral streptococci, and this has been associated with their ability to invade dentin tubules (Love et al., 1997). However, there is no direct evidence to support a role for Asa 373 to mediate binding to collagen.
AS has been reported to promote direct, opsonin-independent binding of E. faecalis to human neutrophils via a complement receptor-mediated mechanism (Vanek et al., 1999). As a consequence of this special type of binding, E. faecalis-bearing AS was shown to be resistant to killing by human neutrophils, despite marked phagocytosis and neutrophil activation (Rakita et al., 1999). Furthermore, both PMN-mediated extracellular superoxide production and phagosomal oxidant production against the AS-expressing strains were higher than those against the control strains lacking AS (Rakita et al., 1999). This oxidative burst by the neutrophils may be a possible contribution to tissue damage in case of infection with cells of E. faecalis expressing AS (Fig.
). AS was also reported to promote opsonin-independent adherence to and phagocytosis of E. faecalis by human macrophages as well, facilitating intracellular survival time in macrophages. However, AS suppresses the respiratory burst triggered by macrophages, as indicated by reduced concentrations of superoxide anion (Süßmuth et al., 2000). The responses to the AS-expressing E. faecalis by human neutrophils and macrophages, therefore, appear to vary; however, it can be concluded that AS serves as a protective factor in favor of the bacterium against the host defense mechanisms.
Superantigens are molecules produced by bacteria, viruses, parasites, and yeasts which can induce inflammation through stimulation of T-lymphocytes, followed by massive release of inflammatory cytokines, resulting in tissue damage (Jappe, 2000). AS, in combination with BS, was reported to possess superantigen activity (Schlievert et al., 1998). Cell extracts of AS- and BS-positive E. faecalis were found to induce T-cell proliferation, with subsequent release of tumor necrosis factor beta (TNF-ß) and gamma interferon (IFN-
), and to activate macrophages to release tumor necrosis factor alpha (TNF-
) (Fig.). The stimulation of lymphocyte proliferation and the production of the cytokines were comparable with those occurring following stimulation with the established staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1), as the positive control (Schlievert et al., 1998). The cytokines TNF- and TNF-ß have been implicated in bone resorption (Stashenko, 1998), while IFN- has been considered as an irreplaceable factor in host defense against infection and, at the same time, as an inflammatory mediator (Billiau, 1996). IFN- is well-known to potentiate respiratory burst responsiveness of macrophages to stimulants, resulting in increased production of hydrogen peroxide and superoxide anion. IFN- also stimulates the production of the cytotoxic agent nitric oxide (NO) by a variety of cells, including macrophages and neutrophils (Fig.), and may cause undesirable cell and tissue damage.
Results from animal studies concerning the role of AS in E. faecalis pathogenesis vary. In studies involving rabbits, AS promoted endocarditis (Chow et al., 1993; Schlievert et al., 1998), whereas this was not the case in a rat endocarditis model (Berti et al., 1998), and AS did not affect the severity of the disease in a rabbit endophthalmitis model (Jett et al., 1998). The issue of promotion of endocarditis by E. faecalis may be viewed in the general context of an interaction between a bacterial adhesin and a host target. The extracellular matrix of all mammalian tissues consists of glycoproteins (e.g., collagen, laminin, fibronectin) and proteoglycans that can be exploited by micro-organisms for colonization and initiation of infection (Westerlund and Korhonen, 1993). The ability of a bacterium to adhere to collagen has been shown to play an important role in the pathogenesis of endocarditis (Hienz et al., 1996). Since dentinal tissues (Linde and Goldberg, 1993) share common ECM proteins with the heart tissue (Bashey et al., 1992), a role for AS in endocarditis may also have relevance for endodontic infections. In epidemiologic studies, AS has frequently been detected in clinical isolates (Ike et al., 1987; Elsner et al., 2000) but is rarely found among fecal isolates from healthy volunteers (Coque et al., 1995), suggesting a possible role for AS in human enterococcal infections.
| Surface Adhesins |
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The efaA gene was identified with the use of an antiserum from a patient with E. faecalis endocarditis (Lowe et al., 1995). The amino acid sequence of the associated protein, EfaA, revealed 55 to 60% homology to a group of streptococcal proteins known as adhesins. Thus, it was hypothesized that EfaA might be functioning as an adhesin in endocarditis. Production of EfaA by strains of E. faecalis is common. In one study, the efaA gene was detected in all medical (blood, pus, urine, feces, hospital environment) and almost all food (milk, cheese, meat) isolates of E. faecalis (Eaton and Gasson, 2001). In an animal model, mutants with the efaA gene showed prolonged survival, compared with E. faecalis strains bearing no efaA gene, indicating a role for the efaA gene in disease (Singh et al., 1998a). Recent studies suggest EfaA as a solute binding-protein receptor for a manganese transport system in E. faecalis. While manganese is required for the growth and survival of most micro-organisms, EfaA is strongly expressed in a manganese-ion-depleted environment, probably for the regulation of the cytoplasmic homeostasis of the cation (Low et al., 2003). The relatively low availability of manganese in serum (Krachler et al., 1999) and in dentin (Battistone et al., 1967) may induce expression of EfaA in vivo.
Studies have also focused on factors associated with the binding of bacteria to extracellular matrix (ECM) proteins. Various strains of E. faecalis obtained from different clinical materials were found to agglutinate strongly with ECM proteins, including collagen type I and type IV, and this was attributed to the surface hydrophobicity of the cells (Zareba et al., 1997). The study by Xiao et al.(1998) indicated that a protein was involved in the binding of E. faecalis to ECM proteins following growth in a stressful condition, defined as growth at 46°C. The so-called ‘conditional adherence’ of the bacterium to collagen, however, was impaired following pre-treatment with a protein-digesting enzyme or pre-incubation with soluble forms of collagen, or following digestion of the binding substrate with collagenase. The putative proteinaceous adhesin of E. faecalis was subsequently identified as Ace, a collagen-binding MSCRAMM (microbial surface component recognizing adhesive matrix molecules), which is structurally and functionally similar to the collagen-binding protein Cna of Staphylococcus aureus (Rich et al., 1999). It was recently shown that the disruption of the ace gene impaired the conditional binding of E. faecalis to the ECM proteins (Nallapareddy et al., 2000b). Identification of Ace-specific antibodies in sera obtained from patients with enterococcal infections, and especially from patients with E. faecalis endocarditis, indicated that Ace is commonly expressed in vivo during human infections by different strains, and not just at 46°C in vitro (Nallapareddy et al., 2000a). Recently, the influence of Ace on adhesion of the bacterium to dentin was tested (Hubble et al., 2003). As compared with the Ace-negative isogenic strain, Ace producing wild-type strain OG1RF adhered significantly more to dentinal surfaces when both strains were incubated at 46°C. In addition, serine protease was found to aid adhesion of the bacterium to dentin, probably by exposing binding sites for the adhesins or by modifying the adhesins. Interestingly, adhesion of the Ace-positive strain to dentin at 37°C was far superior to adhesion at 46°C. This may be an indication of a stronger adhesin working at 37°C or an unidentified factor present in dentin (or in the culture medium) which enhances the expression of Ace at 37°C.
Several investigators have demonstrated that serum may modulate bacterial surface antigen expression (Guzman et al., 1989, 1991; Lambert et al., 1990; Lowe et al., 1995). Guzman et al.(1989, 1991) suggested that adherence of E. faecalis to eukaryotic cells could be mediated by carbohydrate residues present on the bacterial cell surface. E. faecalis isolates from urinary tract infections (UTI) express D-galactose and L-fucose ligands when grown in serum, whereas the isolates normally did not express these ligands when they were grown in brain heart infusion broth (Guzman et al., 1991). Growth in serum raised the adherence of E. faecalis isolates from UTI and endocarditis to eukaryotic cells by at least 1•5- to three-fold, with the greatest (eight-fold) increase in adherence of UTI strain to heart cells (Guzman et al., 1989). A role for serum in invasion of dentinal tubules by E. faecalis was suggested by Love (2001). In the presence of human serum, dentin invasion and collagen adhesion by the other test species, Streptococcus gordonii DL1 and Streptococcus mutans NG8, was inhibited, while dentin invasion by E. faecalis JH2-2 was not inhibited, and binding to collagen was enhanced.
| Sex Pheromones |
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Some of the E. faecalis sex pheromones and their inhibitory peptides were found to be chemotactic for human and rat neutrophils and also to induce superoxide production and lysosomal enzyme secretion (Ember and Hugli, 1989; Sannomiya et al., 1990) (Fig.). Studies have demonstrated a strong association between gingival crevicular fluid neutrophil lysosomal enzymes and chronic periodontal disease (Kunimatsu et al., 1995; Buchmann et al., 2002). A phagocytic lysosomal enzyme, arylsulfatase, has been detected in abundance in periapical lesions, whereas samples from healthy tissues showed little or no enzyme activity (Aqrabawi et al., 1993), and teeth with larger periapical lesions exhibit increased levels of the lysosomal enzyme beta glucoronidase (Kuo et al., 1998). Furthermore, neutrophil lysosomal enzymes may activate the complement system, which can contribute to bone resorption in the periapical tissues either by destruction of bone or by inhibition of new bone formation (Torabinejad et al., 1985).
| Lipoteichoic Acid |
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Through its lipidic moiety, the LTA molecule has been found to bind to a variety of eukaryotic cells, including platelets (Beachey et al., 1977), erythrocytes (Beachey et al., 1979a), lymphocytes (Beachey et al., 1979b), PMN leukocytes (Courtney et al., 1981), and epithelial cells (Beachey and Ofek, 1976). Erythrocytes bound by pneumococcal LTAs were found to be rendered susceptible to lysis both in vitro and in vivo when exposed to even their own complement system, pointing to the possibility of tissue damage triggered by LTA during bacterial infections (Hummell and Winkelstein, 1986). Other than binding to cells, LTAs from S. mutans strain BHT were found to exhibit high affinity for hydroxyapatite (Ciardi et al., 1977), a feature of LTA that could enhance Gram-positive colonization on dental surfaces. Also, LTA extracts from Enterococcus hirae ATCC 9790 bind to calcified matrix, as well as to cells of neonatal rat parietal and long bones (O’Grady et al., 1980). In a tissue culture study, LTA stimulated bone resorption (Hausmann et al., 1975). Corroborating data come from rat experiments where severe inflammatory lesions and bone resorption were induced in the periodontal tissues of the rats after repeated intragingival injection of LTA from S. mutans (Bab et al., 1979).
Apoptosis is essentially the programmed death of a cell without damage to adjacent cells. While this process occurs constantly in virtually all organs throughout life, it may also be involved in several diseases, including oral diseases and periradicular lesions (Satchell et al., 2003). LTA from streptococci has been found to cause apoptotic cell damage in cell culture (Wang et al., 2001) and in tissue culture studies (Schmidt et al., 2001). The effects of various components and particularly of LTA of E. faecalis in causing apoptosis in relevant cell lines (osteoblasts, osteoclasts, periodontal ligament fibroblasts, macrophages, and neutrophils) merit investigation, since it may bring new insight into the nature of periradicular lesions involving E. faecalis.
Lipoteichoic acids isolated from strains of E. faecalis or from other Gram-positive bacteria have been reported to stimulate leukocytes to release several mediators which are known to play a role in various phases of the inflammatory response. These include the release of TNF-, interleukin 1 beta (IL-1ß), interleukin 6 (IL-6) (Bhakdi et al., 1991), and interleukin 8 (IL-8) (Saetre et al., 2001) by cultured human monocytes and by human whole-blood leukocytes, respectively, the release of prostaglandin E2 (PGE2) by mouse peritoneal macrophages (Card et al., 1994), the release of lysosomal enzymes by rat peritoneal macrophages (Harrop et al., 1980), and the generation of superoxide anion by human monocytes (Levy et al., 1990) (Fig.).
These factors have all been detected in periapical samples, and each has a well-known tissue-damaging (TNF-, IL-1ß, IL-6, PGE2, lysosomal enzyme, superoxide anion) or leukocyte-attracting (IL-8) property.
Change in vascular permeability is also an important phase in the course of inflammation, since extravasation of plasma, succeeded by diapedesis of circulating leukocytes, follows an increase in vascular permeability. LTA from S. aureus was shown to increase the vascular permeability in mice, probably through production of secondary mediators such as eicosanoids, platelet-activating factor, and histamine (Wada et al., 2000). A recent study indicates that streptococcal LTA up-regulates the expression of vascular endothelial growth factor (VEGF), a potent inducer of angiogenesis, vascular permeability, and edema, in macrophages and pulp cells (Telles et al., 2003). While an increase in vascular permeability is related to acute inflammation, angiogenesis is related more to chronic inflammation.
A proper autolytic activity appears to be necessary for the efficient killing of bacteria by cell-wall antibiotics. However, LTA has been found to inhibit the autolysis of isolated walls as well as intact cells of the former Streptococcus faecalis, now termed Enterococcus hirae ATCC 9790 (Cleveland et al., 1976). Compared with the parent strain, autolytic-defective mutants of E. hirae ATCC 9790 showed increased survival after exposure to cell-wall antibiotics. Moreover, they exhibited decreased rates of autolysis when treated with detergents, suspended in lytic buffers, or when grown in medium depleted of essential nutrients in the presence of increased levels of cellular LTA and lipids (Shungu et al., 1979). Thus, LTA appears to be associated with resistance against adverse conditions and may also be involved in resistance against root canal medicaments applied during endodontic treatment. Recently, LTA of E. faecalis was reported to be doubled in quantity during the viable but non-cultivable (VBNC) state, suggesting a role for LTA during this period (Signoretto et al., 2000).
D-alanylation of the cell-wall-associated LTA could be important in bringing about phenotypical advantages for the bacteria. A mutant strain of Streptococcus agalactiae, deficient in the D-alanine moiety on the LTA, was more susceptible to killing by macrophages and neutrophils than the wild-type strain, and exhibited decreased virulence in animal models (Poyart et al., 2003). Insertional inactivation of the gene dltD (responsible for expression of the protein that incorporates D-alanine into LTA) in Lactobacillus casei 102S resulted in enhanced antimicrobial activity of the disinfectants cetyltrimethylammonium bromide and chlorhexidine (Debabov et al., 2000). The latter is also used as an endodontic disinfectant.
Finally, LTA has been considered as a constituent of the binding substance of E. faecalis that acts as the receptor on the recipient cell for aggregation substance produced by the donor cell. This presumption stems from experiments where free LTA isolated from E. faecalis inhibited pheromone-induced cell clumping by acting as a competitive inhibitor of the cellular-binding substance (Ehrenfeld et al., 1986). Therefore, LTA can also be regarded as a molecule contributing to the virulence of E. faecalis through the facilitation of aggregate formation and plasmid transfer.
| Extracellular Superoxide Production |
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). In addition to production by host cells, bacteria can also produce superoxide anion. Production of superoxide by a clinical isolate of a Streptococcus D sp. strain was lytic for erythrocytes (Falcioni et al., 1981). Extracellular superoxide production has been reported to be a common trait in strains of E. faecalis. Among a total of 91 clinical and community isolates and type strains, 87 were found to produce detectable extracellular superoxide anion (Huycke et al., 1996). Isolates associated with bacteremia or endocarditis produced significantly higher extracellular superoxide than those from the stool of healthy subjects (Huycke et al., 1996). In a subcutaneous model, extracellular superoxide production was found to enhance the in vivo survival of E. faecalis in a mixed infection with Bacteroides fragilis (Huycke and Gilmore, 1997).
| Gelatinase |
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Gelatinase, as a member of the matrix metalloproteinase (MMP) family, can also be produced by a wide variety of mammalian cells, including inflammatory cells, epithelial cells, fibroblasts, osteoclasts, etc. Acting on substrates similar to those of the bacterial gelatinase, host gelatinase plays a role in normal physiological processes, such as regulation of formation and remodeling of tissues through its extracellular matrix-degrading functions. However, unregulated MMP activity has been implicated in certain pathological states, such as invasion of cancer cells, arthritis, and periodontitis. Gelatinase (Gelatinase A, MMP-2; and Gelatinase B, MMP-9) levels were elevated in oral rinses, crevicular fluid, and whole saliva samples (Mäkelä et al., 1994) and in gingival biopsy specimens (Soell et al., 2002) from periodontitis patients compared with those in healthy subjects. Inhibition of gelatinase decreases the rate of bone resorption in tissue culture experiments (Hill et al., 1994) and in experimental periodontal disease models (Ramamurthy et al., 2002). Recently, host gelatinase was reported to be higher in inflamed pulps and periapical lesions than in healthy tissues (Shin et al., 2002). Host gelatinase was also shown to have a significant effect in the degradation of dentin organic matrix (Tjäderhane et al., 1998). Certain peptides, generated as a consequence of fragmentation of collagen, attract monocytes (Postlethwaite and Kang, 1976), macrophages, and neutrophils (Riley et al., 1988; Laskin et al., 1994) to the site of breakdown (Fig.). Furthermore, the collagen peptides were found to stimulate the release of destructive reactive oxygen species, hydrogen peroxide and the superoxide anion, and also the lytic enzymes, elastase and gelatinase, by macrophages (Laskin et al., 1994) (Fig.). By analogy, collagen hydrolysis by the gelatinase of E. faecalis may therefore play an important role in the pathogenesis of periapical inflammation.
A zinc-containing metalloproteinase from Legionella pneumophila hydrolyzes similar substrates as the gelatinase of E. faecalis, and this enzyme has been associated with disease progression due to its cytotoxic and tissue-destructive potential and its inhibitory effects on phagocytes (Dowling et al., 1992).
Another condition where E. faecalis-derived gelatinase can produce pathological alterations may be seen in the study by Gold et al.(1975), where the gelatin-liquefying strain 2SaR induced caries in rats, whereas this was not the case with non-proteolytic strains.
Animal studies indicate increased lethality of a gelatinase-producing E. faecalis strain compared with the isogenic strain deficient in gelatinase production (Singh et al., 1998b).
In epidemiologic studies with human clinical isolates of E. faecalis (those isolated from hospitalized patients with infection at various sites), gelatinase production was detected in 45–68% of the isolates (Coque et al., 1995; Elsner et al., 2000; Kanemitsu et al., 2001), and the gelatinase activity was higher in clinical isolates than in fecal isolates from healthy volunteers (Coque et al., 1995).
| Hyaluronidase |
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Another role for hyaluronidase may be to supply nutrients for the bacteria, since the degradation products of its target substrates are disaccharides that can be transported and metabolized intracellularly by bacteria (Hynes and Walton, 2000). Hyaluronic acid as the substrate for hyaluronidase has also been detected in dentin (Jones and Leaver, 1974; Chardin et al., 1990). Streptococci, isolated from carious dentin, can grow in medium containing only hyaluronic acid, suggesting that the bacteria may derive the essential carbon for their growth through hydrolysis of the substrate (Toto et al., 1968). Production of hyaluronidase by streptococci and a strain of E. faecalis isolated from carious dentin could play a role in tissue destruction during the caries process (Parikh et al., 1965). Bacteria isolated from infected root canals associated with apical periodontitis also produce hyaluronidase, and the hyaluronidase activity appears to be related to the degree (acute and subacute) of clinical symptoms (Hashioka et al., 1994).
Hyaluronidase (‘the spreading factor’) is considered to facilitate the spread of bacteria as well as their toxins through host tissues. In addition to its own damaging effect, hyaluronidase may also pave the way for the deleterious effects of other bacterial toxins, thus increasing the magnitude of the damage. The presence of micro-organisms, including E. faecalis, in periapical lesions (Abou-Rass and Bogen, 1998; Sunde et al., 2002) may also be related to the activity of a degrading bacterial enzyme such as hyaluronidase. It may act to facilitate the migration of bacteria from the root canal into the periapical lesion. Interestingly, a large number of species reported in the aforementioned studies are capable of producing hyaluronidase. However, due to the lack of studies concerning the role of hyaluronidase in enterococcal virulence, the contribution of this factor to the apical periodontitis caused by enterococci remains hypothetical.
| Cytolysin |
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Among the target cells of cytolysin are the erythrocytes (Basinger and Jackson, 1968; Miyazaki et al., 1993), PMNs and macrophages (Miyazaki et al., 1993), and a broad range of Gram-positive, but not Gram-negative, organisms (Jackson, 1971; Jett and Gilmore, 1990). It has been hypothesized that if the bacteriocin effect of cytolysin of E. faecalis favors colonization of the Gram-negatives, there could be a shift to a bacterial flora usually associated with periodontal disease (Jett and Gilmore, 1990).
Recent studies investigated the influence of environmental factors on the expression of cytolysin genes. In one study, a quorum-sensing mechanism for production of cytolysin was identified (Haas et al., 2002). According to this study, the products of two regulatory genes, cylR1 and cylR2, work together to repress the transcription of the cytolysin structural genes. As soon as the level of one of the cytolysin subunits, CylLS'' (the fully mature form), reaches an extracellular threshold, de-repression occurs and cytolysin expression is induced. Another study suggests that the genes cylLL and cylLS, encoding the structural subunits of cytolysin, are regulated in response to changing oxygen conditions, and increased amounts of cytolysin are produced under anaerobic conditions (Day et al., 2003). From an endodontic point of view, this finding is important, in that cells of E. faecalis may encounter anaerobic conditions in the root canal following depletion of oxygen by aerobes. Anaerobic conditions may also prevail in the layers of bacterial biofilms in the root canal, and E. faecalis has the capacity to produce biofilms (Distel et al., 2002).
Epidemiological investigations partly support a role for cytolysin in disease occurrence. Ike et al.(1987) reported that approximately 60% of E. faecalis clinical isolates were hemolytic, in contrast to only 17% of E. faecalis isolates derived from fecal specimens from healthy individuals. In another study, cylA occurred more frequently among bacteremia isolates than in isolates from cases of endocarditis or from stools from healthy subjects (Huycke and Gilmore, 1995). In contrast, the study by Coque et al.(1995) did not reveal any difference in cytolysin incidence among E. faecalis isolates from endocarditis, bacteremia, or stool from healthy subjects. In another study, where only 16% of E. faecalis clinical isolates produced cytolysin, the role of this protein as a main virulence factor was concluded to be small (Elsner et al., 2000). However, results from a recent study suggested that silent cyl genes from clinical isolates of E. faecalis may give a negative phenotypic profile (no hemolytic activity on blood agar plates), but environmental factors, such as those found in the infection site, may activate the genes (Eaton and Gasson, 2001).
Data from animal models (Ike et al., 1984; Jett et al., 1992, 1995; Chow et al., 1993; Singh et al., 1998b) and a nematode model (Garsin et al., 2001) suggest cytolysin to be an important virulence factor. In a rabbit endophthalmitis experiment, antibiotic treatment against E. faecalis together with corticosteroid therapy was effective in cases of non-cytolytic strains in preventing visual loss as a consequence of tissue damage, whereas this therapy was useless in the case of infection with the cytolytic strain, suggesting a pathogenic role for cytolysin in endophthalmitis (Jett et al., 1995).
| AS-48 |
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| Other Bacteriocins |
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| Conclusion |
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, and a model of the endodontic disease related to the virulence factors is presented in the Fig.
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). It may be that the most interesting among these surface adhesins is ‘Ace’, which is expressed by the bacterium under disease conditions and particularly under stress (Rich et al., 1999). Bacteria face a variety of stressful conditions in the root canal, such as nutrient deficiency, toxins of other bacteria, and endodontic medicaments. These conditions may modulate the adhesin expression of the bacterium. In addition, leakage of serum into the root canal can induce the expression of AS and other carbohydrate moieties, thereby increasing the adhesiveness of the bacterium. Adhesion to dentin and penetration along dentinal tubules by E. faecalis may serve as a means of protection from endodontic medicaments. An example is calcium hydroxide. When calcium hydroxide is placed in the root canal, the pH decreases sharply toward deeper dentinal zones (Tronstad et al., 1981; Nerwich et al., 1993). Thus, bacteria that have penetrated more deeply into the dentinal tubules and established footholds peripheral to the main root canal are at an advantage. Another mechanism by which E. faecalis survives may be through LTA, which has been associated with resistance of the bacterium against a variety of lethal conditions (Shungu et al., 1979). Since E. faecalis suppresses the growth of other bacteria with its cytolysin, AS-48, and other bacteriocins, the activity of these toxins against Gram-positive and -negative bacteria can explain, in part, the low number of other species in persistent endodontic infections where E. faecalis is dominant. The latter factors are not believed to be pathogenic in humans. However, along with cytolysin, they facilitate the dominance of E. faecalis in a mixed infection and serve as means to obtain ecological advantages which can result in disease in man.
The root canal is hardly a nutrient-rich medium, but E. faecalis may derive the energy it needs from the hyaluronan present in the dentin through degradation by hyaluronidase. E. faecalis may also feed on serum components present in the fluid in the dentinal tubules. Moreover, an inadequate apical seal of root fillings may allow serum to flow into the root canal. Therefore, it seems that, even in a well-debrided and coronally well-sealed root canal, remaining or arriving cells of E. faecalis may still grow and utilize local sources of energy and nutrients.
Production of extracellular superoxide and release of the lytic enzymes gelatinase and hyaluronidase and the toxin cytolysin by E. faecalis can cause direct damage in the dentinal as well as in the periapical tissues (Fig.). In contrast, E. faecalis can also induce host-mediated tissue damage in the periradicular tissues. Since cells of E. faecalis in the dentinal tubules cannot be reached and eliminated by the cells of the host defense system, they may elicit a permanent provocative effect on these cells. PMN leukocytes, lymphocytes, monocytes, and macrophages are stimulated by a group of virulence factors of E. faecalis, which will contribute to the periradicular damage.
It has been proposed that, since strains of E. faecalis frequently harbor plasmids determining antibiotic resistance, cytolysin, and/or bacteriocin, they may represent a reservoir of genetic information available to other bacteria in the intestine (Clewell and Weaver, 1989). This applies to the root canal microbiota as well. While antibiotic resistance and other virulence traits can be disseminated by means of the sex-pheromone-responsive plasmid transfer among the strains of E. faecalis, gene transfer is also possible from E. faecalis to bacteria of other species or even of other genera through sex-pheromone-independent conjugation. So far, there is no information on whether multiple strains of E. faecalis simultaneously participate in endodontic infections in utilizing the sex-pheromone-related gene transfer. However, E. faecalis frequently colonizes the root canal together with bacteria of other species and/or genera, and it may use the latter pathway of gene transfer. This is associated mainly with the transfer of antibiotic resistance genes. Thus, bacteria resistant to multiple antibiotics can be generated within the root canals, where E. faecalis plays a pivotal role. It has been reported that micro-organisms from the root canal can be seeded into the bloodstream during endodontic treatment, and this has the potential to bring about serious systemic diseases such as endocarditis, brain abscesses, and septicemia, particularly for compromised patients (Debelian et al., 1994, 1995). Although this may be a rare clinical occurrence, there are reported cases related to endodontic infections and endodontic treatment (Henig et al., 1978; Lee, 1984; Green and Haisch, 1988). In this context, bacteria resistant to multiple antibiotics pose particular problems. Indeed, in marginal periodontitis refractory to conventional treatment, an increased prevalence of bacteria resistant to antibiotics may be found (Handal et al., 2003).
It cannot be excluded that bacteria may pass through the apical foramen to the periradicular lesion during the course of endodontic infection and elicit host responses. However, the focus of infection is the root canal and the dentinal tubules, which are inaccessible to the elements of the host defense system. Therefore, treatment or preventive procedures should mainly include local, rather than systemic, means. In addition to disinfectants, physical removal of cells of E. faecalis through debridement of the root canal remains essential, since remnants containing LTA may still sustain the inflammation.
The use of agents blocking the expression of virulence genes or modulating their products may find a role in future treatments of persistent endodontic infections with E. faecalis. For example, sensitization of the bacteria to root canal medicaments, which are otherwise ineffective, particularly through targeting the LTA synthesis or d-alanylation of the LTA chain, may be possible, but a better understanding of the regulation of the virulence genes is necessary.
Another possible preventive measure to avoid invasion of the dentinal tubules by E. faecalis may be through disruption of the dentinal collagen, the target for the adhesins. Enzymatic modulation is one possible way of altering the collagen. Similarly, proteinaceous bacterial adhesins may be targeted by protein-digesting agents such as trypsin. These methods have been tested in vitro and resulted in decreased adherence of E. faecalis to collagen-coated surfaces (Xiao et al., 1998).
In conclusion, this review has dealt with the virulence factors of E. faecalis that may enable the bacterium to establish an endodontic infection and maintain a periradicular inflammation. A model of endodontic disease related to these factors has been proposed. The pathogenesis of the periradicular lesions is definitely a very complex process that may involve a large number of host and microbial factors (Torabinejad et al., 1985; Stashenko, 1998; Takahashi, 1998). In the present context, only those aspects of the immune and inflammatory events likely to occur within the periradicular lesion in relation to the virulence factors of E. faecalis have been discussed. It has been established that the primary periradicular lesion is a consequence of a mixed microbial flora rather than solely of E. faecalis. However, in apical periodontitis that persists despite root canal treatment, E. faecalis is frequently the dominant, sometimes the only, pathogen, suggesting that this species alone has the potential to maintain root canal infection and periradicular lesion. A better understanding of the role of the virulence factors of E. faecalis in endodontic infections may help in the development of new strategies to prevent or to eliminate the infection by this species, thereby improving treatment results in endodontics.
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