Oral Microbiol Immunol 1992: 7: 257-262

Associations between microbiai species in dentai root canal infections

G. Sundqvist Department of Endodontics, University of UmeS, Sweden

Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992: 7: 257-262. The existence of commensal or antagonistic relationships between microorganisms in the root canals of teeth with apical periodontitis was investigated. Samples were taken from 65 infected human root canals and were analysed aecording to species, frequency of occurrence and proportion of the total isolated flora. The most frequent species were Fusobacterium nucleatum. Prevotella intermedia. Peptostreptococcus micros. Peplostreptococcus anaerobius. Eubacterium alactolyticum, Eubacterium lentum and Wolinella reeta. An odds ratio system was used to calculate positive or negative associations between the isolated bacteria. Strong positive associations were found between F nucleatum and P. micros, Porphyromonas endodontaiis, Selenomonas sputigena and W recta. There was also a positive association between P. intermedia and P. micros. P. anaerobius and the eubacteria. In general, speeies of streptococci, Propionibacterium propionica, Capnoeytophaga oehracea and VeiUonella parvuta showed no or negative assoei- Key words: microbial association: endodontic pathogen; oral ecology ations with the other bacteria. The results are consistent with the concept of a special and selective environment occurring in the root canal that is due, in part, G, Sundqvist, Department of Endodontics, University of UmeS, S-901 87 UmeS, Sweden to the cooperative as well as antagonistic nature of the relationships between bacteria in the root canal. Accepted for publication December 31, 1991

All bacteria that normally inhabit the oral cavity theoretically have the capacity to invade the root canal space during and following pulp necrosis and to participate in the infection of the canal and, ultimately, to enter the periapical tissues. Relatively few species do so. The bacteria isolated from infected root canals are composed of a restricted group of the oral flora (5, 19, 20, 45, 52), Most of the species that have been found in infected root canals have also been identified in the periodontal poeket (31, 32), Although the root eanal flora is not as complex as that of the periodonta! pocket, they are similar in that both contain a special and limited assortment of the oral flora. This implies that selective pressures operate in which certain bacteria are able to thrive, whereas others cannot survive. Studies on the dynamics of root canal infections have shown that the relative proportions of anaerobic microorganisms and bacterial cells increase with time, and the facultatively anaerobic bacteria are outnumbered when the canals have been infected for 6 months or more (9). Oxygen and oxygen products are likely to play an important role as

ecological determinants (24, 25), The proportionate decrease of facultative bacteria and concomitant increase of anaerobic bacteria with time may be due to the consumption of oxygen and the development of a low reduction-oxidation potential, which favours the growth of anaerobic bacteria. The nutrient supply is also important for the establishment of the microbial community in the root canal. Nutrients required for the growth of very fastidious organisms are present in the tissue fluid, and may also be available when connective tissue eells disintegrate (24), Bacteria can also utilize produets from other bacteria (14, 24, 27), and it is likely that sueh an exchange of nutrients takes place in the root canal. It has been shown that microbial associations play an important role in regulating the composition of the subgingival plaque tlora (8, 17, 39), and analogous commensal or antagonistic relationships may also influence the root canal microbiota. The more protective environment and relative simplicity of the root canal flora facilitates the analysis of positive and negative associations between bacterial species. The

purpose of the present study was to characterize the root canal flora of teeth with periapical lesions according to species, frequency of occurrence and proportion of the flora and to analyse the associations between these bacteria. Material and methods Clinical material and specimen collection

Sixty-five teeth were included in the study, all of which had single roots containing necrotic pulps, intact pulp chamber walls and radiographic evidence of periapieal bone resorption. None of the patients was under antibiotic therapy. The procedures outlined by Moller (33) were used for disinfection of the teeth and rubber dam. If upon opening there was exudation from the eanal, the exudate was soaked up with charcoalled paper points. When no exudate was present, a small amount of sterile saline was introduced into the canal by means of a syringe, and the canal was enlarged until a medium paper point could be introduced to a level approximately 1 mm short of the radiographic apex. The fiuid in the canal was absorbed with charcoalled

258

Sundqvist

paper points and transferred to a tube with 5 ml of anaerobic peptone yeast extract glucose (PYG) broth (18), Precautions were taken to avoid oxygen contamination of the broth by means of a mobile anaerobe laboratory (11), Microbiologic cultivation and identification

The PYG broth tubes with the samples were introduced into a glove box with an atmosphere of 10% hydrogen and 5Vu carbon dioxide in nitrogen. The samples were dispersed and diluted as described earlier (47), In brief, samples were agitated in a mechanical mixer until the paper points disintegrated, then serially diluted in prereduced anaerobically sterilized dilution solution (18) and plated on blood agar plates (18), After 7 days of incubation in the anaerobic box and after 2 days on plates incubated aerobieally, the different types of colonies were recorded. The total number of bacteria in the samples and the frequencies of the different colony types was determined. The isolated strains were identified according to standard methods (4, 16, 18, 21, 38), Identifieation of gram-positive anaerobic small rods, which grew poorly in PYG broth unless supplemented with Tween 80, was confirmed by comparing the electrophoretic mobility of their soluble cellular proteins in a polyacrylamide gel with that of reference strains (34, 47), Calculation of relationships between bacteria in the sampies

The odds ratio as described by Hillman et al, (17) and Socransky et al, (39) was used to meaure the associations between the organisms in the samples. The odds of an event are defined as the ratio of the probability of the event to the probability of its complement. For example, the relationship between Prevotella intermedia and Peptoslreptococcus micros is calculated as follows. There were 35 root canals in which neither speeies was • found. Of the other canals, 14contained both species together, 8 had P. intermedia only and the remaining 8 contained P. micros only. The odds of detecting P. micros in the root canals with P. intermedia was 14/8 (1,75) and the odds of detecting P. micros in the absence of P. intermedia was 8/35 (0,23), The odds ratio here is the odds of detecting P. micros in the presence of P. intermedia divided by the odds of detecting P. micros in the absence of P. intermedia.

Table 1. Bacterial species isolated from the root canals of 65 nonvital teeth with periapical lesions Bacterial species

No, of % strains Incidence

'

Streptococcus sp.' S. angino.susS. inilis S. innlans S. sungiiis S. nwrbitlortim Enterococcii.s Jaccatis

I 11 10 2 2 2 1

Peptoslreplococcus sp.' P. anaerobiu.s P. micros P. niger P. prcYolii

10 20 22 1

15 31 34 _ _ 11 — -.

1 7 1 3 1 1

Eubacterium ataclotyticiim E. brachy E. lentum E. nodatuin E. timidum

22 6 20 4 7

Propionibaclcriwn sp.' P. propionictis^ Laclobacitlus sp,' L. calcnafornic L. minuHis

2 5 21 1 2

Fusobacterium sp,' F nticlcatum

19 31

Prcvoletta (Bacteroides) sp, (saccharolytic)' Porphyromonas (Bacteroides) sp, (asaccharolytic)' Prcvotctki buccac Prevotella denticola Baclcroidcs gracitis Prcvolctki locscticii Prcvolclla oratis Baclcroidcs urcotyliciis Porphyromonas cndodonlalis* Porphyromonas gingivati.'," Prcvotelta intermedia

2 23 6 4 1 4 5 1 6 3 22

Capnoeytophaga octiracca Selenomonas sputigena Vcittonetta parvutu Wolinella recta Wolinella curva Eiticnctla corrodens Enterobacler aggtomcrans

? 6 6 16 2 1 1

-• •

27 22 _ — _ _ 14 24 _ -

17 15 • -

I

Actinomyccs sp,' A. isractii A. incyerii A. naeshmdii A. odonlotyticiis A. viscosiis

% of total llora (mean, when present)

34 9 31 6 11 _ 8 32 29 48 _ 35 9 6 6 8 9 •

:

—

3«» • 11 9 9 25 —

40 _ — 26 9 7 10 7 _ 55 15 •

-

10 13 _ 11 2 10 5 g _ 13 26 64 27 52 18 --

' Not identified to species level, - Including strains identified as S. conslcttatus (5), S. iiitcnncdius (4) and S. millcri (2) according to Whiley et al. (49), ' Formerly Arachnia propionica (3), ••Formerly Bacleroidcs endodontatis. B. gingivatis (35)

1,75/0.23 = 7,60, A posifive association existed when the odds ratio was above 2, i,e, the odds of detecting one organism was twice as likely with another organism present as with it absent. An odds ratio below 0,5 was considered a negative assoeiation. The proportion of

the organisms in each sample was not considered in these calculations. Results

All root canals contained bacteria, A total of 353 bacterial strains were iso-

Endodontic mierobiat associations

No. ot isolated spocies

Fig. 1. Size of the periapical lesion and number of isolated bacterial species frotn the root canal. The size of each lesion was calculated on radiographs by taking the average of the lesion's largest dimension and its extent in the direction perpendicular to the largest dimension.

lated. The mean number of strains in the canals was 5,4 (tnedian 6), The majority of the strains could be classified to species level (Table I), The most frequently isolated species was Fusobacterium nueleatum. which was present in 48% of the root canals. Other frequently isolated species were P. intermedia, P. micros, Peptostreptococcus anaerobius, Eubacterium alactolyticum, Eubacterium lentum and Wolinella recta. There was a weak correlation between the number of isolated species from the root canals and the size of the periapical lesions (Fig, 1), The number of bacterial species in the samples correlated weakly with the number of bacterial cells (Fig, 2), Forty-two strains that could not be

259

classified to speeies level were anaer- sp,, anaerobic lactobacillus sp, (4,94) obic, nonmotile, nonsaccharolytic and the unidentified fusobacteria (2,68), small, gram-negative, nonpigmenting but was negatively associated with P. rods not stimulated by formate and fu- intermedia. Eubacteria were positively marate. Nineteen of these produced a associated with the Peptostreptoeoecus small amount of butyric acid and were sp. and P. intermedia. hi general, species of streptococci, identified as Fu.sobacterium sp, and the other 23 strains, which produced minor Propionibacterium propionicus, Capnoamounts of acetic and/or succinic acid, cvtoptiaga oehracea and Veiltonelta parwere considered as Prevotetla (Baeter- vuta showed no or negative associations oides) sp. Twenty-one strains were small with the other bacteria. The odds ratios grarn-positive, anaerobie rods that grew for Actinomyces israelii was less than poorly in PYG broth unless it was 2,0 against all other species except P. supplemented with Tween 80, These propionicus (2,25), P. propionieus was strains are similar to strains categorized also positively associated with other Aeas Lactobacillus D2 and DIO by Moore tinotnyees sp. All other odds ratios for et al, (32) (L, V, H, Moore, personal this organism were less than 2,0, cointnunication). Possible associations between bac- Discussion terial taxa were determined by calculating the odds ratio among the most com- The relatively low number of species in monly isolated species (Table 2), In root canals indicates that special and some instances, where there was a selective mechanisms operate in which litniited number of isolates of some spe- certain bacteria are rnore capable of surcies, they were excluded from the calcu- viving and multiplying than others. The lation of odds ratios or were joined into purpose of this study was to identify groups (Streptococcus sp,, Actinomyces and clarify some of the relationships besp, and Eubacterium sp,), F nueleatum. tween individual species present in the which was the most prevalent species, infected root canal. There was a conwas positively associated (odds ratio spicuous dominance of anaerobic bacabove 2,0) with P micros, Porphyromon- teria, which made up 90% of the isolates as endodontaiis. Selenomonas sputigena from the canals. There were strong posiand Wolinella sp. There was a positive tive associations between soine species, association between P. intermedia and P. most noticably between P. intermedia micros. P anaerobius and the eubacteria, and P. anaerobius, P. intermedia and P. P. endodontaiis, which is specifically as- mieros as well as between E. lentum and sociated with endodontic infections two other bacteria - E. alactolyticum (50), was positively correlated with F. and P. anaerobius. In some instances nulceatum, E. alactolyticum, Wolinetta clear negative associations emerged.

Table 2. Odds ratios of associations among isolated species P. intermedia Streptococcus sp,' S. anginosus S. milis P. anaerobius P. micros Acliitomyccs sp.A. israelii Eubaclerium sp.' E. alaclotyticum E. lentum P. propionicu.s* F nucteatwn P. cndodonlatis P. inlermcdia C. octuacca S. sputigena V parvuta W recta/curva

0,46 0,59 2.23 9.08 7.60 2.96 1,53 3.56 3.96 2.00 0,46 1.66 0,36 •

'

_

•

0,36 0,98 1,88

E. alactolyticum

E nucteatwn

P. anaerobius

P. micros

0.46 0.97 0.43 5,26 1.18 0.29 0.29 2.52 _ 7,43 * 1.26 2,10 3,96 0.29 0,36 1,66 1,35

0.26 0.32 0,44 0,69 2.95 0.42 0.12 1,71 1,26 1,25 0,76 6.82 1.16 0.42 2,50 1.00 2.31

0.53 2,70 0.95 3,77 1.80 1.80 3.08 5.26 1.16 * 0,69 1.13 9.08 * 2.10 0.42 0,82

3.23 0.59 2.23 3.77 2.96 0.29 3.56 1.18 5.25 0.46 2.95 0.97 7.60 * 2.10 0.97 3.64

W recta/curva 1.83

0.46 1.14 0.82 3.^ 2.15 0.40 3.90 1.35 2.81 1.83 2.31 2.93 1,88 0.40 2.93 1.34 -

Boldface: positive associations with an average odds ratio > 2.0. * not present in samples with the actual species, ' Including 5. mutans, S. sanguis, S. morbittorum. E. faecalis. - Including Aclinoinvccs except A. israelii. ' Including E. braehv. E. nodatum. E. timidum. •* Fonnerly Araclmia propionica

260

Sundqvist

whereas bacteria that primarily obtain bacterial populations may depend on a energy by fermenting carbohydrates food chain in which the tnetabolistn of may be restricted in the closed environ- one species supplies essential nutrients tnent of the root canal by lack of avail- for the growth of other metnbers of the able nutrients. Disintegrated pulp tissue population (14, 23, 26-28), W recta is and the tissue fiuids constitute essential an example of an oral bacteria that apsources of nutrients in the root canal. pears to derive a source of etiergy frorn Important information with relevance the co-inhabiting microbial species. Hyfor the dynamics of root canal infections drogen and formate produced by other can be obtained from the recent studies bacteria stimulate the growth of H^ of ter Steeg et al, (41-43), They studied recta (13, 14, 48). The positive associNo. ot isoiatod specios Fig. 2. Number of isolated species from the the succession of species during enrich- ations observed in root canals between root canal and number of bacterial cells in ment growth of subgingival plaque or- the Wolinella sp, and the peptostreptocganisms in serum. Three phases could occi, Actinomyces sp,, Eubacterium sp. the sample be distinguished during growth; first, and F nucleattim (Table 2) tiiay partly the low content of carbohydrates in be dependent on the production of forThe flora of the infected root canal, serum was consumed by rapidly grow- mate by these bacteria (13, 27), Conalthough relatively sirnple, resembles ing saccharolytic bacteria, leading to versely, many of the isolated bacteria that of the periodontal pocket (31, 32) lactic and formic acid production. In the produce substances that may be toxic and in this context invites comparison. secotid phase, proteins were hydrolysed, for other members of the microciota in Some species were more prevalent in some amino acid fermentation took the root canal, A build up of metabolic the root canals than in the periodontal place and the remaining carbohydrates products such as fatty acids, sulfur cotnpockets, P. anaerobius, E. alactolyticum were metabolized. The carbohydrates pounds and ammonia (10, 34, 37, 44) and P. endodontaiis are examples of such were split off from the serum glyco- may significantly regulate the root canal organisms. On the other hand, sus- proteins. Growth during this phase was microfiora. pected periodontal pathogens as Actino- dominated by P. intermedia, V. parvula, Species of streptococci and C oehrabacillus actinomycetemcomitans and Eubacterium sp. and F. nucleatttm. In a cea have been found to be negatively Bacteroides forsythus were not found in ftnal phase, progressive protein degra- associated with suspected periodontal the canal samples and Porphyromonas dation and extensive amino acid fer- pathogens in subgingivai plaque gingivalis oecurred sparsely, van Wink- mentation occurred. The predominant samples (17, 39), In the root canal elhoff et al, (51) have previously shown species during this phase were P. mieros, streptococci, C oehracea. K parvula, A. that there is cross-inhibition within the F. nucleatum and Eubacterium sp. In an- israelii and P. propionicus (Table 2) group of black-pigrnenting Bacteroides other study P. micros was found to showed negative associations with rnost such that P endodontaiis inhibited the dominate serum cultures originating other bacteria. With regard to C oehragrowth of P. intertnedia. We found a frorn subgingival microbiotas (34), The cea and V parvula, it is likely that the negative assoeiation between these spe- ecological niche of P. micros may be nutritional demands of these bacteria eies and they were only present together related to its wide range of peptidase are the main reason for their tiegative in one root canal. Strong positive as- activities, lnaking ainino acids and pep- associations. Growth of C oehracea is soeiations were present between some of tides available from serum glyco- dependent on carbon dioxide and favthe species. Most of these associations proteins (41), P. intermedia, P. endodon- oured by carbohydrates (22, 40), but not have also been demonstrated in subgin- taiis and P. gingivalis are also able to favoured by low oxygen tension (25), gival plaque. Thus, Socransky et al, (39) degrade a number of glycoproteins in However, such conditions may be presalso found positive associatiotis be- serum (2, 46), The amino acids and pep- ent in the root canal during the initial tween F nucleatum and W recta and tides can be metabolized by these bac- phase of the infection, since the isolated between P. intermedia and P. mieros and teria (12, 15, 36), but can also be utilized strains of C oehracea were from Eubacterium sp. In eontrast, they found by other bacteria such as F. nueleatum samples containing one or a few strains, no association between F nucleatitm and and Eubaeterium sp,, which have little which came frorn teeth with small periP. micros, and in the subgingival or no proteolytic activity in serum (12, apical lesions. Vparvula is not favoured samples P. intermedia was negatively as- 41), by the conditions in the root canal, since sociated with Aetinomyces species. The results of these studies provides it depends on the metabolic intermediDistinct correlations between individ- good evidence for the notion that the ates lactate and pyruvate, which are ual bacteria in the special etivironment associations between Peptostreptococ- forrned frotn carbohydrate metabolisrn of both the root eanal and the perio- cus sp,, Eubacterium sp,, Bacteroides sp, by other bacteria (6, 29), Interestingly, dontal pocket are evident. Many factors and F nucleatum in endodontic infec- both C. oehracea and K parvula have to can infiuence the growth and coloniza- tions are based on the nutritional de- date been associated only with inactive tion at these sites. The availability of mands of these bacteria and, in particu- periodontal lesions (7, 8), However, the nutrients, the low oxygen tension in root lar, the availability of serum-like sub- species F. nucleatum, P. intertnedia, W. canals with necrotic pulps and bacterial strates in the root canal. In addition, a reeta and P. mieros, which were posiinteractions are important ecological wide range of nutritional interrelation- tively associated and prevaletit in the determinants. Conditions probably ships are recognized among oral bac- root canals, are associated with deep exist in the root canal that permit the teria and these may also infiuence the periodontal pockets and active lesions growth of anaerobic bacteria capable of associations between certain species (1, (7, 8, 32), fermenting amino acids and peptides. 13, 14, 24, 27, 30), Growth of tnixed The results of this study confirtn the

Endodontic mierobiat associations presetice of a characteristic tnicrobiota within the root canal. During the course of infection, strong inter-relationships develop between different microbial species and population shifts in the fiora are produced as a result of these interactions as well as the special etivironmetital and selective pressures in the root eanal. Acknowledgements

I thank Dr, Haroun Shah and Dr, David Figdor for valuable criticistn of the manuscript.

References 1, Carlsson J. Microbiology of plaque associated periodontal disease. In: Lindhe J, ed. Textbook of clinical periodontology. Copenhagen: Munksgaard, 1990: 129-152, 2, Carlsson J, Nilsson T, Sundqvist G, Effect of proteinases from black-pigmented Bacteroides on human plasma proteins. In: Lehner T, Cimasoni G, ed. The borderland between caries and periodontal disease III. Geneve: Medecine et Hygiene, 1986: 155-163. 3, Charfreitag O, Collins MD, Stackebrandt E, Reclassification of Arachnia propiontca as Propionibacterium propionicus comb, nov, Int J Syst Bacteriol 1988: 38: 354-357. 4, Cowan ST, ed. Cowan and Steel's manual for the identification of medical bacteria. 2nd edn. Cambridge: Cambridge University Press, 1974, 5, Dahlen G, Bergenholtz G, Fndodontic activity in teeth with necrotic pulps. J Dent Res 1980: 59: 1033 1040. 6, Delwiche EA, Pestka JJ, Tortorello ML. The Vcilloncllae: gram-negative cocci with a unique physiology. Ann Rev Microbiol 1985: 39: 175-193. 7, Dzink JL, Tantier ACR, Haffajee AD, Socransky SS. Gram negative species associated with active destructive periodontal lesions, J Clin Petiodontol 1985: 12: 648-659. 8, Dzink JL, Socransky SS, Haffajee AD. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases. J Clin Periodontol 1988: 15: 316-323, 9, Fabricius L, Dahlen G, Ohman AE, Mdller AjR. Predominant indigenous oral bacteria isolated from infected root canals after varied titnes of closure. Scand J Dent Res 1982: 90: 134 144. 10. Freter R, Brickner H, Botney M, Cleven D, Aranki A, Mechanisms that control bacterial populations in continous-flow culture models of large intestinal flora. Infect Immun 1983: 39: 676-685.

11, Fulghum RS. Mobile anaerobe laboratory. Appl Microbiol 1971: 3: 101-105. 12, Gharbia SE, Shah HN. Utilization of aspartate, glutamate, and their corresponding peptides by Fusobacterium nucleatum subspecies and Porphyromonas gingivatis. Curr Microbiol 1991: 22: 159-163, 13, Gillespie J, Holt SC. Growth studies of Wotinctta recta, a gram-negative periodontopathogen. Oral Microbiol Immunol 1987: 2: 105-111. 14, Grenier D, Mayrand D. Nutritional relationships between oral bacteria. Infect Immun 1986: 53: 616 620. 15, Grenier D, Mayrand D, McBride BC, Further studies on the degradation of immunoglobulins by black-pigmented Bacteroides. Oral Microbiol Immunol 1989: 12: 12-18. 16, Hill GH, Ayers OA, Kohan AP. Characteristics and sites of infection of Eubactcritnn nodatuni Eubacteriwn timidum. Eubaeterium brachy, and other asaccharolytic eubacteria. J Clin Microbiol 1987: 25: 1540-1545. 17, Hillman JD, Socransky SS, Shivers M. The relationships between streptococcal species and periodontopathic bacteria in human dental plaque. Arch Oral Biol 1985:30: 791-795. 18, Holdeman LV, Cato EP, Moore WEC. Anaerobe laboratory manual. 4th edn. Blacksburg, VA: Virginia Polytechnic Institute Anaerobe Laboratory, 1977, 19, Kantz WE, Henry CA, Isolation and classification of anaerobic bacteria from intact pulp chambers of non-vital teeth in man. Arch Oral Biol 1974: 19: 91-96. 20, Keudell K, Conte M, Fujimoto L, Ernest M, Berry HG. Microorganistns isolated from pulp chambers. J Endod 1976: 2: 146-148, 21, Krieg NR, Holt JG, ed. Bergey's manual of systematic bacteriology, Baltimore: Williams & Wilkins, 1984, 22, Leadbetter ER, Holt SC, Socransky SS, Capnoeytophaga: new genus of gramnegative gliding bacteria. I, General characteristics, taxonomic considerations and significance. Arch Microbiol 1979: 122: 9-16, 23, Lev M, Keudell KC, Milford AF. Succinate as a growth factor for Bacteroides inctaninogcnicus. J Bacteriol 1971: 108: 175-178. 24, Loesche WJ. Importatice of nutrition in gingival crevice microbial ecology. Periodontics 1968: 6: 245-249. 25, Loesche WJ, Gusberti F, Mettraux G, Higgins T, Syed S. Relationship between oxygen tension and subgingival bacterial fiora in untreated human periodontal pockets. Infect Immun 1983: 42: 659-667. 26, Matsh PD, Host defenses and tnicrobial homeostasis: role of microbial interactions. J Dent Res 1989: 68: 1567-1575. 27, Mayrand D, Virulence protnotion by mixed bacterial infections. In: Bayer-

261

Symposium VIll. The pathogenesis of bacterial infections. Berlin: Springer-Verlag 1985: 281-291. 28. Mayrand D, McBride BC. Ecological relationships of bacteria involved in a simple mixed anaerobic infection. Infect Itnmun 1980: 27: 44-50, 29. Mays TD, Holdernan LV, Moore WEC, Rogosa M, Johnson JL. Taxonotny of the genus Vcittonctia Prevot. Int J Syst Bacteriol 1982: 32: 28-36. 30. Mikx FMH, van Campen GJ. The microbial comtnunity on the teeth, with special reference to neerotising ulcerative gingivitis in dogs. Eur J Chemother Antibiodcs 1982: 2: 19-26. 31. Moore WEC, Holdeman LV, Smibert RM, Hash DE, Burmeister JA, Ranney RR, Bactetiology of severe periodontitis in young adult humans. Infect Immun 1982: 38: 1137-1148. 32. Moore WEC, Holdeman LV, Cato EP et al. Comparative bacteriology of juvenile periodontitis. Infect Itntnun 1985: 48: 507-519. 33. Moller AjR. Microbiological examination of root canals and periapical tissues of human teeth. Thesis. Odontol Tidskr 1966: 74: 1-380. 34. Persson S, Ediund M-B, Claesson R, Carlsson J. The formation of hydrogen sulfide and methyl mercaptan by oral bacteria. Oral Microbiol Immunol 1990: 5: 195 201, 35. Shah HN, Collins MD, Proposal for reclassification oi Bacteroides asacctiarolytictis. Bacteroides gingiva/is. and Bacteroides endodontatis in a new genus, PorpJivromonas. Int J Syst Bacteriol 1988: 38: 128-131. 36. Shah HN, Williams RAD. Utilization of glucose and amino acids by Bacteroides intcrmcdius and Baclcroidcs gingivatis. Curr Microbiol 1987: 15: 241-246. 37. Singer RE, Buckner BA, Butyrate and propionate: itnportant compotients of toxic dental plaque extracts. Infect Immun 1981: 32: 458-463, 38. Sneath PHA, Mair NS, Shape ME, Hoh JG, ed. Bergey's tnanual of systematic bacteriology. Baltitiiore: Williams & Wilkins, 1986. 39. Socransky SS, Haffajee AD, Dzink JL, Hillman JD. Associations between microbial speeies in subgingival plaque samples. Oral Microbiol Immunol 1988: 3: 1-7. 40. Soeransky SS, Holt SC, Leadbetter ER, Tanner ACR, Savitt E, Hammond BF. Capnocylophaga: new genus of gramnegative gliding bacteria. III. Physiological characterization. Arch Microbiol 1979: 122: 29-33. 41. Steeg PF ter, van der Hoeven JS. Development of periodontal microflora on human serutn. Microbial Ecol Health Dis 1989: 2: I-IO. 42. Steeg PF ter, van der Hoeven JS, de Jong MH, van Munster PJJ, Jansen MJH, Enrichment of subgingival microflora on

262

43,

44,

45,

46,

Sundqvist human serum leading to accumulation of Bacteroides species, Pcptostreptoeocci and Fusobacteria. Antonie van Leeuwenhoek 1987: 53: 261-271. Steeg PF ter, van der Hoeven JS, de Jong MH, van Munster PJJ, Jansen MJH, Modelling the gingival poeket by enrichment of subgingival microflora in human serum in chetnostats. Microbial Ecol Health Dis 1988: 1: 73-84, Steenbergen TJM van, van der Mispel LMS, de Graaff J, Effects of amtnonia and volatile fatty aeids produced by oral bacteria on tissue culture cells, J Dent Res 1986: 65: 909-912, Sundqvist G. Bacteriological studies of necrotic dental pulps. Thesis. University of Umea, Umea, Sweden, 1976: 1-94. Sundqvist G, Carlsson J, Herrmann B, Tarnvik A. Degradation of human im-

munoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides. J Med Microbiol 1985: 19: 85-94, 47. Sundqvist G, Johansson E, Sjogren U. Prevaletice of black-pigmented Baeteroides species in root canal infections. J Endod 1989: 15: 13-19. 48. Tanner ACR, Badger S, Lai C-H, Listgarten MA, Visconti RA, Socransky SS. Wolinella gen, nov,, Wolinetta succinogcncs (Vibrio succinogenes Wolin et al,) comb, nov,, and description of Bacteroides gracilis sp. nov,, Wolinctla recta sp, nov,, Campylobacler concisus sp, nov,, and Eit