The Role of Spirochetes in Periodontal Disease
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THE ROLE OF SPIROCHETES IN PERIODONTAL DISEASE WJ. LOESCHE The University of Michigan, School of Dentistry, School of Medicine, Ann Arbor, Michigan 48109- 1078 Adv Dent Res 2(2):275-283, November, 1988 ABSTRACT he spirochetal accumulation in subgingival plaque appears to be a function of the clinical severity of Tperiodontal disease. It is not known how many different spirochetal species colonize the plaque, but based upon size alone, there are small, intermediate-sized, and large spirochetes. Four species of small spiro- chetes are cultivable, and of these, T. denticola has been shown to possess proteolytic and keratinolytic enzymes as well as factors or mechanisms which suppress lymphocyte blastogenesis and inhibit fibroblast and poly- morphonuclear leukocyte (PMNL) function. All of these attributes could contribute to periodontal tissue insult. Yet independent of these potential virulence mechanisms, the overgrowth of spirochetes can be clinically useful if simply interpreted as indicating the result of tissue damage. In this case, the spirochetes would be indicators of disease and could be easily monitored by microscopic examination of plaque, or possibly by the measurement of benzoyl-DL-arginine-2-naphthylamide (BANA) hydrolytic activity in the plaque. INTRODUCTION of the pathogenesis of periodontal disease remains suspect. The oral spirochetes are often the dominant bac- ACQUISITION OF ORAL SPIROCHETES terial types observed in subgingival plaque removed from diseased periodontal sites, and yet they are one Definitive information on the acquisition of the oral of the least-studied and -understood members of the spirochetes is lacking, forcing one to sketch in outline plaque flora. Their contributions to periodontal dis- form what is known and/or presumed to be known. ease cannot be properly assessed until we know what The human oral spirochetes appear to be distinct from species are present and what their metabolic and human genital and intestinal spirochetes, from ani- physiological characteristics are. This dearth of mal species, from overtly pathogenic species, and from knowledge relative to the oral spirochetes reflects the free living forms (Canale-Parola, 1977; Harwood and difficulty that investigators have in isolating them from Canale-Parola, 1984). This implies that the oral spi- the plaque, and in maintaining them in vitro, once rochetes are acquired from other humans via oral con- isolated. At the present time, we do not know how tact. We have detected spirochetes by dark-field many different species of spirochetes can be culti- microscopy in the dental plaque of about 40% of the vated; whether certain types such as the intermedi- 3- to 5-year-old children and in about 50% of the 6- ate-sized and large spirochetes can be cultivated; what to 12-year-old children whom we have examined. proportion of the total spirochetes is composed of the However, their numbers were less than 0.5% of the known cultivable species; or what factors produced flora, and they were uncultivable. Almost all 6- to 12- by the spirochetes are harmful for the periodontium. year-old Dutch and Tanzanian children examined had Until these basic questions can be answered, there is detectable spirochetes in their plaque, and their num- no way to evaluate the role of spirochetes in peri- bers and proportions were greater when the plaque odontal disease, and accordingly, our understanding samples were removed from sites of gingival bleeding (Mikx et al, 1986). Presented at the Sunstar Portside Symposium, November 14-15, These data suggest that most if not all individuals 1986, Kobe, Japan acquire some type of spirochete in their early life. This investigation was supported by grants DE 030211 and DE Since the oral spirochetes comprise at least four spe- 06030 from the National Institute of Dental Research. cies, and undoubtedly more (Moore et al., 1985), one 276 LOESCHE Adv Dent Res November 1988 assumes from the above frequency data that the ac- disrupt the plaque — so as both to facilitate colony quisition of any one of these spirochetes is a likely formation from single cells and to maximize the num- event once the teeth erupt. Once acquired, the spi- ber of colonies — usually lyse the spirochetes. This rochetes show a predilection for the subgingival was demonstrated by experiments in which subgin- plaque, presumably because in this ecosystem these gival plaque samples were gently disrupted by me- motile organisms are not at as great a risk of being chanical mixing (10 sec, Vortex), for a microscopic swept away by the saliva and masticatory forces. Also, count to be obtained, and then subjected to vigorous the lower oxygen tension present in the subgingival disruption by either sonication or homogenization with plaque, combined with the availability of pre-formed a Tekmar homogenizer, or by being mechanically nutrients derived from the host and cohabitant plaque mixed for 60 sec (Vortex) (Salvador et al.,1987) (Table bacteria, would contribute to their establishment in 2). this site (Loesche, 1976; Loesche and Laughon, 1982). Spirochetes averaged about 55% of the microscopic count but were less than 1% of the viable count. Only ISOLATION small spirochetes were isolated, and these were es- sentially T. denticola and a few T. socranskii isolates. Sonication, which gave the highest viable recoveries There are four species of oral spirochetes — Tre- for plaque bacteria, yielded the lowest percentage re- ponema denticola, T. socranskii, T. vincentii, and T. pec- covery of spirochetes. Homogenization yielded the tinovorum — which have been well-characterized, and highest percentage recovery of spirochetes and a re- American Type Culture strains are available (Table spectable recovery of other bacteria. Vortex mixing, 1). Two other species, T. macrodentium and T. oralis, which is a very common method of dispersing plaque have been described in the older literature (Socransky when paper points are used for plaque collection, et al., 1969), but since type cultures are nonexistent, yielded the lowest recovery of plaque bacteria, but their relationship to the above species, and hence their was comparable with the homogenizer in the per- uniqueness, has not been established. T. denticola and centage recovery of spirochetes. T. vincentii are asaccharolytic species, whereas T. so- These data indicate that the routine dispersal pro- cranskii ferments glucose and other sugars, and T. cedures used in cultural studies severely discriminate pectinovorum has a requirement for pectin (Smibert against the recovery of spirochetes as they lyse the and Burmeister, 1983). No one has reported on the spirochetes, i.e., spirochetes cannot be found by mi- proportions of any single species such as T. denticola, croscopic examination of the dispersed plaque. How- T. socranskii etc., in plaque associated with health or ever, it is possible that some of the spirochetes might disease until 1988 (Simonson et al, 1988). Rather, when have survived dispersion, but did not grow because spirochetes were isolated from the plaque, the data the medium was lacking in essential nutrients. This were always qualitative, i.e., frequency of isolation was not the case for T. denticola, T. socranskii, T, vin- (Smibert et al., 1984), number of morphotypes iso- centii, and T. pectinovorum, since our medium could lated (Cheng and Chan, 1983; Fiehn and Wester- support their growth. Thus, the low or non-recovery gaard, 1984), and so forth. This is because of the of these species reflected either that they were pres- difficulty investigators have in the quantitative recov- ent in appreciable numbers in the plaque and were ery of spirochetes from the plaque. lysed by the dispersal procedures, or that they were Spirochetes are delicate organisms relative to the present only in low numbers in the plaque. other bacterial types that are found in dental plaque. Most intermediate-sized and large spirochetes have This affects their isolation, since procedures used to probably never been cultivated. This could reflect the TABLE 1 TAXONOMIC CHARACTERISTICS OF ORAL SPIROCHETES Species Size No. of Axial Carbohydrate % Fibrils Fermentation G + C T. denticola small 2-4-2, 5-10-5 no 37-38 T. vincentii intermediate 5-10-5 no 44 T. socranskii small 1-2-1 yes 51 T. pectinovorum small 2-4-2 pectin 39 T. oralis small 1-2-1 no ? T. macrodentium small 1-2-1 yes ? Based upon data which appeared in Loesche and Laughon (1982), Smibert and Burmeister (1983), and Smibert et al. (1984). Vol. 2 No. 2 THE ROLE OF SP1R0CHETES IN PERIODONTAL DISEASE 277 TABLE 2 16 citations) and average 50% of the flora in early- EFFECT OF DISPERSAL PROCEDURES ON RECOVERY onset periodontitis (EOP) (Lindhe et al, 1980; Loesche OF SPIROCHETES FROM SUBGINGIVAL PLAQUE et al, 1985). Only in localized juvenile periodontitis SAMPLES (n = 15) (LJP) are high proportions of spirochetes an incon- sistent observation (Loesche et al, 1985; Newman and Dispersal Morphotype Socransky, 1977; Slots, 1976). In none of these micro- Procedure small intermediate large Total scopic studies were data on the presence of any par- Spirochetes as % of Microscopic Count ticular species provided, although the proportions of small, intermediate-sized and large spirochetes were Gentle Mechanical 31% 15% 8% 54% often noted. Spirochetes as % of Viable Count Cultural studies indicate that T. denticola is signif- Harsh Mechanical 0.8% no growth no growth 0.8 icantly associated with diseased sites (Moore et al, Homogenization 0.9% " " 0.9 1985). Simonson et al (1988) have used monoclonal antibodies to T. denticola to show that the absolute Sonication 0.1% " " 0.1 and relative numbers of this species increased signif- a91% of isolates were T. denticola, 5% were T. socranskii, and icantly in plaque removed from diseased sites com- 4% were unspeciated. pared with plaque removed from non-diseased sites. Adapted from Salvador et ah, 1987. We also have data obtained with monoclonal anti- bodies which associate T. denticola with clinical dis- ease (Lopatin, Bretz, and Loesche, in preparation).