0099-2399[90/1611-0534/$02.00/0 JOURNAL OF ENDODONTtCS Copyright 9 1990 by The American Association of Endodontists
Printed in U.S.A.
VOL. 16, NO. 11,NOVEMBER1990
Localization and Identification of Root Canal Bacteria in Clinically Asymptomatic Periapical Pathosis H. Fukushima, DDS, K. Yamamoto, DDS, K. Hirohata, DDS, H. Sagawa, DDS, K-P. Leung, PhD, and C. B. Walker, PhD
percussion pain, or exudation were extracted, observed by scanning electron microscopy, and examined bacteriologically.
Twenty-one teeth with clinically asymptomatic periapical pathosis (class 3) were extracted and the isolation, identification, and localization of bacteria in the root apex were examined. Mixtures involving several bacteria were isolated from more than 60% of the cases. Scanning electron microscopy revealed bacterial masses to be associated with the apical part of the root canal, but not with the area of apical foramen or on the surface of root apex. Our results indicate that the bacteria in class 3 cases may be derived from organisms which colonized before or during endodontic treatment, but not from anachoresis. The bacteria-positive cases of asymptomatic periapical pathosis have the potential to progress to symptomatic periapical pathosis.
M A T E R I A L S AND M E T H O D S Twenty-one teeth from 21 volunteers diagnosed as class 3 ( 6 ) - - n o spontaneous pain, percussion pain, and e x u d a t i o n - were extracted, examined bacteriologically, and the existence of bacteria were confirmed by scanning electron microscopy.
Sampling The involved tooth and surrounding gingiva were disinfected with 70% ethanol and tincture of iodine, and extracted under local anaesthesia. The extracted tooth was immediately inserted into 4.5 ml of reduced transport fluid (7) under a stream of anaerobic gas (10% H2, 10% CO2, balance N2), sealed tightly with a butyl gum stopper, and transferred to the laboratory. The tooth was cut at a point 5 m m from the root apex under anaerobic conditions and the apex portion was washed 10 times with reduced transport fluid. One-hundred microliters of each wash was smeared on a blood agar plate and incubated anaerobically for 4 days to make sure that no bacteria were present. After washings, the apex was frozen by liquid nitrogen and cut longitudinally. One part of separated apex was fragmented into small pieces by a dental swage, inserted into reduced transport fluid, mixed vigorously by a vortex mixer and serially diluted to the 10% One-hundred microliters of each 10-fold dilution was plated on blood agar plates in duplicate. One plate was incubated anaerobically for 7 days and another aerobically for 4 to 7 days. Another part of the apex was used for examination by scanning electron microscopy.
It is well known that periapical pathosis results from endogenous infections (1). Recent studies incorporating anaerobic sampling and culture techniques have reported the association of various anaerobic bacteria with periapical pathosis (2-5). Although the particular isolates differ from patient to patient, species of Bacteroides, Fusobacterium, Peptostreptococcus, Peptococcus, and Eubacterium are considered the primary causative agents of this disease. In order to clarify the mechanism of exacerbation from a chronic to an acute periapical pathosis, it is necessary to elucidate the microorganisms associated with the infection. Closed canals or obturated teeth are convenient to study, since microorganisms from these samples are subject to less variation. In a previous study of obturated teeth with periapical pathosis (6), we found a mixture of anaerobic bacteria such as Bacteroides, Peptococcus, and Eubacterium in the root canal was correlated with the presence of clinical symptoms, classes 1 and 2. Anaerobic and facultative bacteria were isolated from almost half of clinically asymptomatic cases (class 3). These bacteria-positive class 3 cases have the possibility of proceeding to clinically symptomatic cases. However, the mechanisms leading to symptomatic periapical pathosis is uncertain. In this study, obturated class 3 teeth characterized by a radiolucent area at the apex, but without spontaneous pain,
Identification of Bacteria Isolation and identification of bacteria were performed according to previously described methods (6) with some modifications. Approximately 100 colonies were subcultured from the anaerobic primary isolation plates. Colonies from aerobic incubation were also subcultured to determine their facultative or aerobic status.
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Localization of Root Canal Bacteria
Vol. 16, No. 11, November 1990
Anaerobic bacteria and facultative Gram-positive rods were identified based on the Virginia Polytechnic Institute Anaerobe Laboratory Manual (8), Bergey's Manual of Systematic Bacteriology (9), and A Color Atlas of Anaerobic Bacteria (10). End product analysis was performed on all isolates. The presence of spore formers was detected by spore staining and incubation at 80~ for l0 min. Adonitol, amygdalin, arabinose, cellobiose, erythritol, esculin, fructose, galactose, glucose, glycerol, glycogen, inositol, inulin, lactose, maltose, mannitol, mannose, melezitose, melibiose, raffinose, rhamnose, ribose, sorbitol, sorbose, starch, sucrose, trehalose, and xylose were utilized for fermentation tests. Peptone-yeast extract-Fildes broth was used as a basal medium. Gas, H2S, lecithinase, lipase, catalase and indole production, growth at 15~ and on 20% bile, nitrate reduction, gelatin liquefaction, and hydrolysis of starch and esculin were performed as additional tests. Facultative Gram-positive cocci were identified on the basis of the methods described by Facklam (11, 12). Arabinose, glycerol, inulin, lactose, mannitol, sorbitol, and raffinose were tested for fermentation using semisolid fermentation medium (Eiken, Japan) as a basal medium. Color change from purple to yellow was regarded as positive reaction. Hydrolysis or arginine, esculin and hyppurate, reduction of litmus milk, production of insoluble glucan, growth in 40% bile, growth at 10~ and 450C and tolerance to heat at 60~ for 30 min were performed for characterization.
and then observed by a scanning electron microscope (X-560; Hitachi) at an accelerating voltage of 20 kV. RESULTS
Culture Findings Of 21 cases, 13 were culture positive (61.9%) as shown in Table 1; 11 cases yielded mixed cultures and the other 2 cases yielded pure cultures.
Proportional Distribution of Isolated Bacteria In one case of positive culture, too many colonies were present on the dilution plates for individual colonies to be isolated. Proportional distribution of bacterial isolated from 12 cases is given in Table 2. Anaerobic bacteria were isolated from all except one case, and facultatives were isolated from eight cases. Anaerobic bacteria were prevalent in most cases, and constituted >70% of the isolates from eight cases. Anaerobic Gram-positive cocci were found in seven cases and anaerobic Gram-negative rod in six cases. Of the facultatives isolated, Gram-positive cocci were prevalent and were the predominant organisms isolated from three cases.
Identification
Scanning Electron Microscopy For scanning electron microscopic study, part of the tooth apex was washed with phosphate-buffered saline (pH 7.2) three times and fixed with 1.2% glutaric aldehyde and 2% osmic acid. The sample was dehydrated by ethanol, replaced by isoamyl acetate, gold-coated by ion coater (IB-55; Eiko), TABLE 1, Bacteriological findings of root apex of teeth with asymptomatic periapical pathosis No. of Cases
%
13 11 2 8
61.9 52.4 9.5 38.1
Positive culture Mixed Pure Negative culture
The results of identification of the above-mentioned strains are summarized in Tables 3 to 5. In the Gram-positive rod taxa, eubacteria were isolated from six cases, followed by lactobacilli (five cases), propionibacteria (four cases), and actinomycetes (four cases). Propionibacterium acnes (four cases), Eubacterium aerofaciens, and Propionibacterium propionici (three cases, respectively) were identified as major isolates. Anaerobic Gram-positive cocci were isolated from seven cases. Peptostreptococci were the most prevalent and consisted ofP. productus, P. micros, and P. anaerobius (Table 3). Bacteroides species were most prevalent (six cases) of the Gram-negative rods, although species of this genus were different from case to case. Species of Fusobacterium (four cases), Selenomonas (two cases), Succinivibrio (one case), and
TABLE 2. Proportional distribution of bacteria isolated from root apex of teeth with asymptomatic periapical pathosis
Anaerobe G*(+) rods G(+) cocci G(-) rods G(-) cocci Facultative G(+) rods G(+) cocci No. of bacteria identified Colonial number * G, G r a m .
1
2
87.6 60.8 5.0 7.3 14.6 12.3 12.3
63.5 43.5 3.4 16.6
77.1 14.4 14.3 48.4
11.3 11.3
36.5 10.0 26.5
22.9 5.8 17.1
88.7 9.9 78.8
31 1.0 xl02
35 6.8 •
71 7.1 )<10
41 7.2 xl02
3
535
4
5 100 96.6 3.4
6 39.6 6.2 3.1 18.2 12.1 60.4
7 100 3.8 51.8 40.6 3.8
8 80.0 10.0 70.0
9
10
100 80.0 20.0
75.0 75.0
20.0 20.0
60.4 59 2.5 â&#x20AC;˘
33 6.0 xl02
54 2.0 xl03
20 2.1 xl0 a
5 5
11
12
100 100
25.0
100
25.0
100
32 3.2 xl0
5 5
32 3.2 xl03
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Fukushima et al.
Journal of Endodontics
TABLE 3. Proportional distribution of anaerobic Gram-positive bacteria isolated from root apex of teeth with asymptomatic periapical pathosis 1
2
4.8
9.6 3.3
3
4
5
6
7
8
9
10
11
G*(+) rods
Eubacterium aerofaciens lentum alactolyticum contortum limosum moniliforme dolichum rectale Lactobacillus catenaforme minutus ruminis rogosae vitulinus sp. Propionibacterium acnes acidi-propionici freu. ss, freu/r sp. Actinomyces israelii viscosus meyeri G(+) cocci Peptostreptococcus productus micros anaerobius Peptococcus asaccharolyticus magnus Streptococcus intermedius morbillorum
42.4 3.1 52.5
3.8
24.1 2,5 100 1.7
2,5 7.3
4.2 1.4 75.0 3.3 10.0 2.8
9.7 4.8
9.6 12.9
5.7 2.9 2.9
3.3 3.3
2.9
80.0
2.5 2.5
2.5 2.5
2.8
3.3
14.8 11.4
30.0 5.0
37.0 2.9 20.0 35.0 3.1
9 G, G r a m .
t P. freudenreichii ss. freudenreichiL
Veillonella (three cases) species were identified in addition to Bacteroides as shown in Table 4. Facultative bacteria were isolated and identified from eight cases. Among them, lactobacilli, streptococci, and enterococci were prevalent (five, four, and four cases, respectively). Enterococcusfaecalis was isolated from three cases.
Scanning Electron Microscopic Findings Results of scanning electron microscopic observation of another chopped apex are given in Table 6. Bacteria were recognized between the end of filling material and the upper part of apical foramen in all of 13 cases which showed positive culture and two negative culture cases. Bacterial cells were also recognized at the surface of filling material in six of these cases. However, no bacteria was found at the apical foramen in any cases. Figure 1 shows an example of positive culture. Coccoids and filaments were clumping with each other only at the site of B.
DISCUSSION In our previous report (6), periapical pathosis was divided into three classes based on clinical symptoms, and the bacteria involved were identified. Microorganisms were demonstrated in cultures from root canals in all cases with severe clinical symptoms, especially percussion pain which often persists during endodontic procedures (classes 1 and 2). In the asymptomatic class 3, only 7 of 12 cases exhibited positive cultures. Colony-forming units of positive culture in this class were lower than those of classes 1 and 2. Oral streptococci and enteric bacteria were predominant in these cases. On the other hand, Bacteroides, Peptococcus, Peptostreptococcus, and Eubacterium species were frequently isolated from symptomatic cases, suggesting that these bacteria exert an important influence on exacerbation of chronic periapical lesions. In the present work, we have focused on the elucidation of the discrepancy between isolated bacteria from symptomatic and asymptomatic periapical pathosis, namely, to clarify the derivation of anaerobic bacteria such as Bacteroides, Peptococ-
Vol. 16, No. 11, November 1990
Localization of Root Canal Bacteria
TABLE 4. Proportional distribution of anaerobic Gram-negative bacteria isolated from root apex of teeth with asymptomatic periapical pathosis
1
2
3
5
6
7
TABLE 6. Scanning electron microscopic findings
Positive culture
Foramen -
+
-
+
-
3 4
+
+ +
-
14.1
5
-
+
-
2.9
6
-
+
-
2.9
7
-
+
-
6.0
38.7
2.9 2.5 5.7
6.0 3.1
2.5
1.9 Negative culture
17.0 6.4 2.9
G ( - ) cocci
Veillonella parvulla
Apical
Space
-
3.3
2.5
Empty
1
3.4 6.4
Filled Space
2
G*(-) rods
Bacteroides intermedius melaninogenicus asaccharolyticus capillosus eggerthfi furcosus ruminicola sp. Fusobacterium russfi mortiferum Selenomonas ruminantium Succinivibrio dextrinosotvens
12.1
14.5
3.8
537
8
+
+
-
9
+
+
-
10
+
+
11 12
-
+
-
+
+
-
13
+
+
-
14
-
+
-
15
-
+
-
16
-
-
-
17
-
-
-
18
-
-
-
19
-
-
-
20
-
-
-
21
-
-
-
* G, Gram.
TABLE 5. Proportional distribution of facultatives isolated from root apex of teeth with asymptomatic periapical pathosis 1
2
3
4
6
8
3.1
5.0
10
12
G*(+) rods
Lactobacillus brevis buchneri fermentum jensenfi lactis casei ss. rhamosus Actinomyces naeslundii Arachnia propionica Propionibacterium granulosum
2.5
9.9
7.3 2.5 3.1 6.4 5.0 10.0
2.9 2.9
G ( + ) cocci
Streptococcus anginosus -constellatus MG -intermedius sanguis I sanguis II sp. (viridans) sp. Enterococcus faecalis sp.
16.1
76.0
6.4
1.4 6.0 21.2 5.7 1.4
6.0 F~G 1. S c a n n i n g e l e c t r o n m i c r o g r a p h o f t h e r o o t a p e x in a c l a s s 3
11.4
25.0 100 27.2
case. P a r t s A t o C a r e e n l a r g e m e n t s o f A , B, a n d C in part D. N o b a c t e r i a w e r e f o u n d at apical f o r a m e n (A). C o c c o i d s a n d f i l a m e n t s w e r e c l u m p i n g w i t h e a c h o t h e r o n l y b e t w e e n t h e filling material a n d
* G, Gram.
cus, and Eubacterium isolated from symptomatic periapical pathosis. The routes that bacteria may follow to invade to the periapical region, are as follows: (a) progression of deep dental caries via pulp to the periapical region; (b) penetration through dentinal tubes to pulp due to cutting during dental therapy; (c) penetration to pulp as a result of tooth fracture; (d) invasion by bacteria in deep periodontal pockets via accessory
t h e u p p e r p a r t o f t h e apical f o r a m e n (A).
canals to pulp; (e) expansion of periapical pathosis to a neighboring tooth: and (f) anachoresis via the blood stream (14). Since asymptomatic, obturated teeth with a radiolucency at the apex were selected for this experiment, all of the root canals were filled with filling material. The root canals and periapical regions were not exposed to oral microbial flora, as is evident in the observations from scanning electron microscopy. Therefore, it is considered that the existence of bacteria
538
Fukushima et al.
in this area is dependent upon anachoresis or on the persistence of bacteria that had invaded before or during endodontic treatment. In this experiment, bacteria were recovered from the chopped apex of more than 60% of the samples. This value is higher than previous data from class 3 cases (6). In addition, the proportional distribution of isolated bacteria was quite different from those isolated from root canals of class 3 cases. In this study, anaerobic bacteria predominated in 9 of the 12 cases yielding positive culture. The average number of bacterial species present in each sample of this study was also different from previous data of class 3. The possibility of contamination during extraction was ruled out, because no microorganisms were recognized at the site of the apical foramen or at the surface of root apex. Therefore, these differences may be due to the sampling procedure, i.e. sampling from the root canal may not reflect the whole bacterial composition at the apex of a class 3 lesion. On the other hand, sampling from root canals in symptomatic periapical infections is not difficult because exudates containing pus come out of the periapical area through the root canals after opening. In the previous study (6), the average number of bacterial species present in each sample of symptomatic periapical pathosis was 3.8 (class 1) and 3.3 (class 2), respectively. These figures are quite similar to the results of Sabiston et al. (16), Aderhold et al. (17), and Lewis et al. (18), indicating that specific bacteria among mixture of various bacterial species may grow when the exacerbation of chronic to acute periapical pathosis occur. Species of Bacteroides, Peptococcus, Peptostreptococcus, and Eubacterium were identified as major constituents. These genera are same as those found in symptomatic periapical pathosis (6). The association of these anaerobic bacteria with acute periapical pathosis has been definitively established. Sundqvist (4), Griffee et al. (13), and Haapasalo (19) have indicated that there is a significant association between the clinical symptoms and the presence of certain Bacteroides species, van Winkelhoffet al. (20) have demonstrated the specificity of B. endodontalis, new black-pigmented Bacteroides, for the root canal infection. Further identification of isolated black-pigmented Bacteroides in this study using DNA-DNA dot hybridization (data not shown), however, did not identify this bacterium in our cultures. The scanning electron microscopic study revealed bacterial masses to be associated with the apical part of the root canal, but not with the area of apical foramen or on the surface of root apex. Our data suggest that the derivation of bacteria in these cases might be due to persistence of bacteria which colonized before or during endodontic treatment, but not due to anachoresis, since most of the bacteria-positive cases were mixed infections. Therefore, it appears that at least 60% of the class 3 teeth we examined have the potential to progress to symptomatic periapical pathosis, when the environmental factors surrounding microorganisms are changed or when host defense mechanisms are lowered.
Journal of Endodontics
CONCLUSIONS Sixty percent of extracted tooth apices with asymptomatic periapical pathosis (class 3) contained mixtures of bacteria between the filling material and the upper part of the apical foramen. These organisms predominantly consisted of the species of Bacteroides, Peptococcus, Peptostreptococcus, and Eubacterium which have also been in symptomatic cases (classes 1 and 2). Therefore, it is concluded that bacteria persisting in the apex of a class 3 lesion may have endodontopathic potential to progress to class 1 and 2 lesions. Dr. Fukushima is affiliated with the Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL and Department of Bacteriology, Osaka Dental University, Osaka, Japan. Drs. Yamamoto, Hirohata, and Sagawa are affiliated with the Department of Bacteriology, Osaka Dental University, Osaka, Japan. Drs. Leung and Walker are affiliated with the Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL.
References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg 1965;20:340-9. 2. Wittgow WC, Sabiston CB Jr. Microorganisms from pulpal chambers of intact teeth with necrotic pulps. J Endodon 1975;1:168-71. 3. Keudell K, Conte M, Fujimoto L, Ernest M, Berry HG. Microorganisms isolated from pulp chambers. J Endodon 1976;2:146-8. 4. Sundqvist GK. Bacteriological studies of necrotic dental pulps [PhD Thesis]. Umea University Odontol Dissertation no. 7, 1976:1-94. 5. Williams BL, McCann GF, Schoenknecht FD. Bacteriology of dental abscesses of endodontic origin. J Clin Microbio11983;18:770-4. 6. Yoshida M, Fukushima H, Yamamoto K, Ogawa K, Toda T, Sagawa H. Correlation between clinical symptoms and microorganisms isolated from root canals of teeth with periapical pathosis. J Endodon 1987;13:24-8. 7. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbio11972;24:638-44. 8. Holdeman LV, Cato EP, Moore WEC. Anaerobe laboratory manual. 4th ed. Blacksburg, Virginia: Virginia Polytechnique Institute and State University, 1977. 9. Holdeman LV, Kelley RW, Moore WEC. Anaerobic gram-negative straight, curved and helical rods. In: Krieg NR, Holt JG, eds. Bergey's manual of systematic bacteriology. 1st ed. Baltimore: Williams & Wilkins, 1984:60262. 10. Mitsuoka T. A color atlas of anaerobic bacteria. 1st ed. Tokyo, Japan: Sohbunsha, 1980. 11. Facklam RR. Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl Microbiol 1972;23:1131-9. 12. Facklam RR. Physiological differentiation of viridansstreptococci. J Clin Microbiol 1977;5:184-201. 13. Griffee MB, Patterson SS, Miller CH, Kafrawy AH, Newton CW. The relationship of Bacteroides melaninogenicus to symptoms associated with pulpal necrosis. Oral Surg 1980;50:457-61. 14. Blechman H. Infection of the pulp and periapical tissues. In: Nolte WA, ed. Oral microbiology. 3rd ed. St. Louis: CV Mosby, 1977:535-58. 15. Sundqvist GK, Eckerbom MI, Larsson AP, Sjogren UT. Capacity of anaerobic bacteria from necrotic dental pulps to induce purulent infections. Infect Immun 1979;25:685-93. 16. Sabiston CB, Grigsby WR, Segerstorm N. Bacteriology of pyogenic infections of dental origin. Oral Surg 1976;41:430-5. 17. Aderhold L, Knothe H, Frenkel G. The bacteriology of dentogenous pyogenic infections. Oral Surg 1981 ;52:583-7. 18. Lewis MAO, MacFarlane TW, McGowan DA. Quantitative bacteriology of acute dento-alveolar abscesses. J Med Microbiol 1986;21:101-4. 19. Haapasalo M. Bacteroides buccae and related taxa in necrotic root canal infections. J Clin Microbio11986;24:940-4. 20. van Winkelhoff AJ, Martijn TJ, van Steebergen TJM, Kippuw N, de Graaf J. Further characterization of Bacteroides endodontalis, an asaccharolytic black-pigmented Bacteroides species from the oral cavity. J Clin Microbiol 1985;22:75-9.