0099-2399/91/1708-0380/$03.00/0 JOURNAL OF ENDODONTICS Copyright 9 1991 by The American Association of Endodontists
Printed in U.S.A.
VOL. 17, NO. 8, AUGUST1991
Bacteria in the Apical 5 mm of Infected Root Canals J. Craig Baumgartner, DDS, PhD, and William A. Falkler, Jr., MS, PhD
des/Porphyromonas (BPBP) were isolated and described before 1970 because of inadequate anaerobic culturing methods. Porphyromonas is a newly established genus that includes species previously identified as asaccharolytic black-pigmented Bacteroides. Included in the new genus are Porphyromonas asaccharolyticus, Porphyromonas gingivalis, and Porphyromonas endodontalis (3). Saccharolytic BPBP which remain in the genus Bacteroides include Bacteroides inter-
Ten freshly extracted teeth which had carious pulpal exposures and periapical lesions contiguous with the root apex were placed inside an anaerobic chamber and the apical 5 mm of the root canals cultured. In addition to anaerobic incubation, duplicate cultures were incubated aerobically. Fifty strains of bacteria from the 10 root canals were isolated and identified. The most prominent bacteria cultured from the 10 root canals were Actinomyces, Lactobacillus, black-pigmented Bacteroides, Peptostreptococcus, nonpigmented Bacteroides, VeilIonella, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus mutans. Of the 50 bacterial isolates, 34 (68%) were strict anaerobes. This study demonstrates the presence of predominately anaerobic bacteria in the apical 5 mm of infected root canals in teeth with carious pulpal exposures and periapical lesions.
medius, Bacteroides melaninogenicus, Bacteroides denticola, Bacteroides corporis, and Bacteroides loescheii. A significant breakthrough in culturing occurred in the early 1970's with the development of obligate anaerobic techniques for the isolation and incubation of microorganisms. The advent of anaerobic techniques demonstrated that the predominant bacteria of infected root canal systems were strict anaerobes. In addition to culturing sensitivity, the incidence of anaerobes varies with age, sample site, and disease state of the subject being cultured. Anaerobic infections occur where there is necrotic tissue, a compromised blood supply, and by succession following infections by aerobes and facultative microorganisms that lower the oxidation-reduction potential in the tissue. A recent study by Sundqvist et al. (4), using both aerobic and anaerobic incubation of culture samples from root canals of intact teeth with necrotic pulps and apical periodontitis, demonstrated a preponderance of anaerobic bacteria. The purpose of this study was to culture and identify the bacteria infecting the apical 5 mm of root canals in teeth having a coronal carious exposure and a periapical inflammatory lesion associated with the infected root canal
The most common pathway for microorganisms from the normal oral flora to the dental pulp is through an open cavity caused by dental caries in the crown of the tooth. Because of inadequate collateral circulation, a necrotic pulp is insulated from the usual clearing processes and immune mechanisms of the body and thus cannot heal. The root canal system becomes a "privileged sanctuary" for microorganisms, their by-products, and degradation products of both the microorganisms and the pulpal tissue (1). The microflora of infected root canals have been isolated and identified with varying results. The determination of which bacteria are present in an infected root canal depends on several factors. These factors include the method of sampling the root canal, the timing of the culture, the type of media (i.e. transport, selective, nonselective), the incubation conditions (i.e. aerobic, anaerobic), the use of microscopy (i.e. phase, darkfield), and the use of bacterial smears (i.e. Gram stain). Because culturing techniques were inadequate for the growth of strict anaerobes through the 1960's, the most common bacteria cultured from infected root canals were aerobic and facultative anaerobes such as a-hemolytic streptococci. In 1957, Brown and Rudolph (2) used phase contrast and darkfield microscopy and found that predominant bacteria in an infected root canal were often not cultivable using existing culturing methods. For example, very few strains of black-pigmented Bacteroi-
MATERIALS AND METHODS Patient Selection and Specimen Collection The Human Volunteers Research Committee at the University of Maryland at Baltimore gave approval to a protocol describing the collection and use of teeth for this investigation. The specimens were collected from 10 patients seen in the Emergency and Oral Surgery Clinics at the University of Maryland Dental School. Pain was the chief complaint and the reason all of the patients in this study presented for dental treatment. Immediately following extraction, each tooth with the attached periapical inflammatory lesion was placed directly into a sterile 50-ml centrifuge tube (Corning, Waxford, PA) containing 20 ml of enriched RPMI 1640 tissue culture medium (Flow Laboratories, Inc., McLean, VA) and refrigerated at 4~ Within 30 min, a #15 sterile scalpel (Martin
380
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Co., Arthur N. Thomas Dist., Philadelphia, PA) was used to remove the soft tissue lesions from the roots and the teeth were placed in an anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, MI) where all further culturing procedures were accomplished. The anaerobic atmosphere was maintained at 37~ and consisted of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide (Airco Products, Inc., Allentown, PA). Cultivation and Identification of Bacteria The surface of the root to be cultured was rinsed with sterile isotonic saline and dried with sterile gauze (Johnson & Johnson, East Winsor, N J). The root was secured with sterile locking pliers (Vice Grip, DeWitt, NE) while the apical 5 mm of root was cut free from the rest of the tooth with a coping saw containing a sterile blade. Before culturing the root canal, a sterile paper point (Johnson & Johnson) moistened with reduced transport fluid (RTF) (5) was used to sample the cut surface of the root for surface contamination. The paper point was placed in 1 ml of RTF in a 4-ml vial (Wheaton, Millville, N J) and vortexed (Genie; Thomas Scientific, Swedesboro, N J) for 30 s at level i0. One hundred microliters of the RTF was spread in duplicate onto nonselective Columbia Blood Agar (BBL, Cockeysville, MD) with 0.0005% hemin (Sigma, St. Louis, MO) and 0.0001% menadione (Sigma) (CBAHM). Sterile stainless steel endodontic reamers (Union Broach, NY, NY) were used to remove pulpal debris and dentinal filings from the root canal in the apical portion of the root of the tooth. The pulpal debris and dentinal filings including any microorganisms were suspended in 1 ml of R T F i n a 4-ml vial and vortexed for 30 s. After dispersing the material, the suspension was diluted with RTF using a 10-fold serial dilution technique to 10-4. One hundred microliters of the dilutions from 10-1 through 10-4 was cultured in duplicate on CBAHM. One hundred microliters of the 10-~ dilution was cultured in duplicate on nonselective MM 10 medium and on trypticase soy agar (BBL, Cockeysville, MD) with kanamycin (Sigma) (6). The latter medium is selective for BPBP. In addition, 25 ul of RTF from dilutions 10-~ to 10-4 was spotted onto selective media for Fusobacterium nucleatum (Crystal Violet Erythromycin Agar) (6), Veillonella species (Veillonella Agar) (Difco, Detroit, MI), Lactobacillus species (Rogosa SL Agar; Difco), Actinomyces species (Forsyth Actinomyces Agar) (6), Streptococcus mutans (Mitis-Salivarius Bacitracin Sucrose Agar) (6), and Enterococcus (Streptococcus)faecalis (S. faecalis Medium; Difco). From each specimen, one plate each of CBAHM (10 -~ dilution plate), MMI0 (10 -~ dilution plate), Rogosa SL Agar (spot plate), S. faecalis Medium (spot plate), and Forsyth Actinomyces Agar (spot plate) was aerobically incubated (Bellco Glass Inc., Vineland, NJ) with 5% CO2 at 37~ All culture plates were incubated for at least 2 wk but examined for growth every 3 days. Representative colonies of different types were streaked for isolation on the appropriate medium and identified. BPBP were isolated either from CBAHM or from trypticase soy agar with Kanamycin. Identification of microorganisms was based on growth in the anaerobic chamber and growth in the aerobic incubator with 5% CO2, colonial pigmentation, colonial morphology, Gram stain, hemagglutination (sheep red blood cells), and biochemical tests. In addition, API 20A and An-IDENT (API Analytic
Bacteria in Root Canals
381
Products, Plainview NJ) rapid identification kits were used to identify the isolated microorganisms. A dissecting microscope (American Optical, Buffalo, NY) was used to enumerate colony-forming units on each culture plate. RESULTS The composition of the microorganisms isolated in this study and the number of bacteria in each sample are shown in Table 1. A total of 50 strains of bacteria were isolated from the apical 5 mm of the 10 root canals cultured in this study. The total number of strains of each species of bacteria isolated from the 10 root canals and the number of strains of each species associated with black-pigmented Bacteroides (five root canals) are shown in Table 2. The most commonly isolated microorganisms were Actinomyces species, Lactobacillus species, black-pigmented Bacteroides, Peptostreptococcus species,
Veillonella parvula, Bacteroides buccae, E. faecalis, Fusobacterium nucleatum, and S. mutans. The species of bacteria most often isolated from the same root canals as the BPBP isolated from 5 of the 10 root canals were Lactobacillus species, Peptostreptococcus species, B. buccae, E. faecalis, and V. parvula. Of the 50 microbial isolates, 34 (68%) isolates were strict anaerobes. All 10 (100%) of the root canals contained strict anaerobes and 2 root canals were infected exclusively with anaerobes. The number of colony-forming units on nonselective CBAHM from the five specimens without BPBP ranged from 5.6 x 104 to 4.3 x 106 with a median of 3.9 x 105 and the number of colony-forming units on nonselective CBAHM from the five specimens with black-pigmented Bacteroides ranged from 9.2 x 104 to 2.3 x 10 6 with a median of 4.4 x 105. The growth of bacteria from the cut surface of the roots was negligible. DISCUSSION This is the first time that both aerobic and strict anaerobic culturing techniques have been used to isolate and identify bacteria from the apical portion of root canals in teeth with coronal carious exposures of the pulp and with periapical lesions. Fifty strains of bacteria were isolated and identified from the apical 5 mm of the 10 infected root canals. The average of five isolates per infected root canal is consistent with polymicrobial root canal infections demonstrated by numerous investigators (4, 7-11). Sundqvist et al. (4) found a greater median number of microorganisms in the root canals of intact teeth infected with BPBP (2.8 x 105) than in root canals not infected with BPBP (3.0 x 103). In this study, there was not a great difference in the median number of colonyforming units enumerated between root canals infected with BPBP (4.4 x 105) and root canals not infected with BPBP (3.9 x 105). This finding may be related to the lower percentage of anaerobic bacteria detected in this study, differences in culturing methods, or the absence of members of the genus Porphyromonas. Six of the strains of Actinomyces, four of the seven strains of Lactobacillus, and the three strains of S. mutans were facultative. With the exception of facultative strains of S. mutans which are associated with caries, the species of bacteria that were cultured in this study are similar to those cultured by Sundqvist et al. (4) in the root canals of
382
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Journal of Endodontics
TABLE 1. Distribution of bacterial species isolated from the apical 5 mm of 10 root canals
Tooth Specimen Species 1 Actinomyces Actinomyces Actinomyces Actinomyces Actinomyces
sp.* sp. israelii viscosus* naeslundii
2
3
4
+
+
5
6
7
8
9
10
+ + + +
B. intermedius B. denticola B. buccae
+
+
+
+ +
+
+
+
+
Bifidobacterium sp. E. faecalis*
-t-
Eubacterium sp.
-I-
+
+
F. nucleatum Lactobacillus sp.* Lactobacillus sp.
+
Peptostreptococcus anaerobius Peptostreptococcus micros
+
Propionibacterium sp.
+
S. intermedius S. mutans*
+
+
+
-I-
+
+
+
+ +
+
+
+
+
-I-
+
Veillonella parvula No. of bacterial cells
+
+
+
+
+
+ +
5.6 x 104 9.2 x 104 3.9 x 105 4.3 • 106 4.4 • 105 3.8 x 105 1.3 • 108 8.8 • 105 2.3 • 108 1.9 • 106
* Facultative anaerobes.
TABLE 2. Bacterial species isolated from the apical 5 mm of 10 infected root canals
Total No. of Strains (10 canals)
No. of Strains with BlackPigmented Bacteroides (5 canals)
2 1 1 1 2
0 1 0 0 1
Black-pigmented Bacteroides B. intermedius B. denticola
5 1
1 1
Nonpigmented Bacteroides B. buccae Bifidobacterium sp. Eubacterium sp. Fusobacterium nucleatum Lactobacillus sp.* Lactobacillus sp. Peptostreptococcus anaerobius Peptostreptococcus micros Propionibacterium sp. E. faecalis* Streptococcus intermedius S. mutans* Veillonetta parvula
5 1 1 3 4 3 5 1 1 4 1 3 5
2 0 1 1 2 3 3 1 1 2 1 1 2
Species
Actinomyces Actinomyces Actinomyces Actinomyces Actinomyces
sp.* sp. israelii viscosus* naeslundii*
* Facuttative bacteria.
intact teeth. Sundqvist et al. (4) isolated 25 strains of BPBP from 22 of 72 (30%) cultured root canals. Of the 25 strains of BPBP isolated by Sundqvist et al. (4), B. intermedius and P. endodontalis were the most numerous. P. gingivalis, B. loescheii, and B. denticola were also isolated by Sundqvist et al. (4). In this study six strains of BPBP were isolated from 5 of the 10 (50%) root canals. Of the six strains isolated, five strains were B. intermedius and one strain was B. denticola. No strain of bacteria from the asaccharolytic genus of Porphyromonas was isolated. Sundqvist et al. (4) cultured intact teeth and found that 91.4% of the microorganisms were strict anaerobes whereas in this study we cultured teeth with carious exposures and found that 68% of the bacteria were strict anaerobes. Direct access of saliva and oral microbes via a carious exposure to the pulp cavity provides an environment conducive to the growth of facultative microorganisms. Considering the presence o f a carious exposure, the relatively high percentage (68%) of anaerobic bacteria isolated from the apical 5 m m of these root canals seems to suggest a selective process for anaerobes from the oral flora. This process may be similar to the selection of microorganisms that seems to take place in the gingival sulcus during the development of different types of periodontal diseases (12). Of the more than 350 species of bacteria estimated to reside in the human oral cavity, periodontal research suggests that only 15 to 20 species are associated with periodontal diseases (12). The results of studies culturing infected root canals in intact teeth and the results of this study suggest that only a limited number of species of bacteria are consistently selected out of the total oral flora for growth in an infected root canal. As bacteria penetrate apically
Vol. 17, No. 8, August 1991
in necrotic root canals, anaerobic succession apparently occurs because of the compromised blood supply, the lower oxidation-reduction potential in the necrotic tissue, and the establishment of synergistic relationships with other bacteria. A study by Fabricius et al. (13) using monkeys demonstrated that the ratio of anaerobes to facultative bacteria increased with time following the sealing of the tooth after previous exposure of the pulp cavity to oral flora. Similarly to periodontal diseases, Koch's postulates have not been fulfilled for infections of endodontic origin because the infections are polymicrobial. However, BPBP have been implicated with symptomatic infections of endodontic origin by several investigators (4, 7, 8, l l, 14-16). Other bacteria implicated as possible endodontopathogens include Peptostreptococcus species (17), Peptococcus species (11) F. nucleaturn (18), Actinomyces species (19), and B. buccae (20). Future investigations should attempt to better define virulence factors and the ecological interrelationships required to achieve pathogenicity among bacteria isolated from infections of endodontic origin. The informed consent of all human subjects who participated in the experimental investigation reported or described in this article was obtained after the nature of the procedure and possible discomforts and risks had been fully explained. The opinions and assertions herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. This study was part of a dissertation submitted in partial fulfillment of the requirements for a PhD degree at the University of Maryland Dental School, Baltimore, MD. The authors express their thanks to the American Association of Endodontists Endowment and Memorial Foundation for funding the purchase of the culture media used in this study. Dr. Baumgartner is chief, Microbiology Branch, United States Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, DC. Dr. Falkler is professor and chairman, Microbiology Department, University of Maryland Dental School, Baltimore, MD. Address requests for reprints to Dr. J. Craig Baumgartner, 10376 Currycomb Ct., Columbia, MD 21044.
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383
References 1. Naidorf IJ. Inflammation and infection of pulp and periapical tissues. Oral Surg 1972;34:486-96. 2. Brown LR. Rudolph CE. Isolation and identification of microorganisms from unexposed canals of pulp involved teeth. Oral Surg 1957;10:1094-8. 3. Shah HN, Collins MD. Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacterio11988;38:128-31. 4. Sundqvist G, Johansson E, Sj6gren U. Prevalence of black-pigmented Bacteroides species in root canal infections. J Endodon 1989;15:13-9. 5. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbio11972;24:638-44. 6. Baumgartner JC. Microbiological and immunological investigation of periapical pathosis [PhD Dissertation]. Baltimore: University of Maryland, 1990. 7. 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. 8. Haapasalo M, Ranta H, Rantah K, Shah H. Black-pigmented Bacteroides spp. in human apical periodontitis. Infect Immun 1986;53:149-53. 9. Kantz WE, Henry CA. Isolation and classification of anaerobic bacteria from intact pulp chambers of non-vital teeth in man. Arch Oral Bio11974;19:916. 10. Bergenholtz G. Microorganisms from necrotic pulp of traumatized teeth. Odontol Revy 1974;25:347-58. 11. 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. 12. Peros W, Savitt E. The microbiology of periodontal disease. Clin Microbiol Newslett 1989;11:49-51. 13. Fabricius L, Dahlen G, Ohman AE, M611er,&JR. Predominant indigenous oral bacteria isolated from infected root canals after varied times of closure. Scand J Dent Res 1982;90:134-44. 14. Sundqvist GK. Bacteriological studies of necrotic dental pulps [PhD Dissertation]. Umea, Sweden: University of Umea, 1976. 15. Van Winkelhoff AJ, Carlee AW, de Graaff J. Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses. Infect Immun 1985;49:494-7. 16. Van Winkelhoff AJ, Van Steenbergen TJM, de Graaff J. The role of black-pigmented Bacteroides in human oral infections. J Clin Periodontol 1988;15:145-55. 17. Sundqvist GK, Eckerbom MI, Larsson ,&P, Sjogren UT. Capacity of anaerobic bacteria from necrotic dental pulps to induce purulent infections. Infect Immun 1979;25:685-93. 18. Heimdahl A, Von Konow L, Satoh T, Nord CE. Clinical appearance of orafacial infections of odontogenic origin in relation to microbiological findings. J Clin Microbio11985;22:299-302. 19. Happonen RP. Periapical actinomycosis: a follow-up study of 16 surgically treated cases. Endod Dent Traumato11986;2:205-9. 20. Haapasalo M Bacteroides buccae and related taxa in necrotic root canal infections. J Clin Microbio11986;24:940-4.
Printed in U.S.A.
VOL. 17, NO. 8, AUGUST1991
Bacteria in the Apical 5 mm of Infected Root Canals J. Craig Baumgartner, DDS, PhD, and William A. Falkler, Jr., MS, PhD
des/Porphyromonas (BPBP) were isolated and described before 1970 because of inadequate anaerobic culturing methods. Porphyromonas is a newly established genus that includes species previously identified as asaccharolytic black-pigmented Bacteroides. Included in the new genus are Porphyromonas asaccharolyticus, Porphyromonas gingivalis, and Porphyromonas endodontalis (3). Saccharolytic BPBP which remain in the genus Bacteroides include Bacteroides inter-
Ten freshly extracted teeth which had carious pulpal exposures and periapical lesions contiguous with the root apex were placed inside an anaerobic chamber and the apical 5 mm of the root canals cultured. In addition to anaerobic incubation, duplicate cultures were incubated aerobically. Fifty strains of bacteria from the 10 root canals were isolated and identified. The most prominent bacteria cultured from the 10 root canals were Actinomyces, Lactobacillus, black-pigmented Bacteroides, Peptostreptococcus, nonpigmented Bacteroides, VeilIonella, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus mutans. Of the 50 bacterial isolates, 34 (68%) were strict anaerobes. This study demonstrates the presence of predominately anaerobic bacteria in the apical 5 mm of infected root canals in teeth with carious pulpal exposures and periapical lesions.
medius, Bacteroides melaninogenicus, Bacteroides denticola, Bacteroides corporis, and Bacteroides loescheii. A significant breakthrough in culturing occurred in the early 1970's with the development of obligate anaerobic techniques for the isolation and incubation of microorganisms. The advent of anaerobic techniques demonstrated that the predominant bacteria of infected root canal systems were strict anaerobes. In addition to culturing sensitivity, the incidence of anaerobes varies with age, sample site, and disease state of the subject being cultured. Anaerobic infections occur where there is necrotic tissue, a compromised blood supply, and by succession following infections by aerobes and facultative microorganisms that lower the oxidation-reduction potential in the tissue. A recent study by Sundqvist et al. (4), using both aerobic and anaerobic incubation of culture samples from root canals of intact teeth with necrotic pulps and apical periodontitis, demonstrated a preponderance of anaerobic bacteria. The purpose of this study was to culture and identify the bacteria infecting the apical 5 mm of root canals in teeth having a coronal carious exposure and a periapical inflammatory lesion associated with the infected root canal
The most common pathway for microorganisms from the normal oral flora to the dental pulp is through an open cavity caused by dental caries in the crown of the tooth. Because of inadequate collateral circulation, a necrotic pulp is insulated from the usual clearing processes and immune mechanisms of the body and thus cannot heal. The root canal system becomes a "privileged sanctuary" for microorganisms, their by-products, and degradation products of both the microorganisms and the pulpal tissue (1). The microflora of infected root canals have been isolated and identified with varying results. The determination of which bacteria are present in an infected root canal depends on several factors. These factors include the method of sampling the root canal, the timing of the culture, the type of media (i.e. transport, selective, nonselective), the incubation conditions (i.e. aerobic, anaerobic), the use of microscopy (i.e. phase, darkfield), and the use of bacterial smears (i.e. Gram stain). Because culturing techniques were inadequate for the growth of strict anaerobes through the 1960's, the most common bacteria cultured from infected root canals were aerobic and facultative anaerobes such as a-hemolytic streptococci. In 1957, Brown and Rudolph (2) used phase contrast and darkfield microscopy and found that predominant bacteria in an infected root canal were often not cultivable using existing culturing methods. For example, very few strains of black-pigmented Bacteroi-
MATERIALS AND METHODS Patient Selection and Specimen Collection The Human Volunteers Research Committee at the University of Maryland at Baltimore gave approval to a protocol describing the collection and use of teeth for this investigation. The specimens were collected from 10 patients seen in the Emergency and Oral Surgery Clinics at the University of Maryland Dental School. Pain was the chief complaint and the reason all of the patients in this study presented for dental treatment. Immediately following extraction, each tooth with the attached periapical inflammatory lesion was placed directly into a sterile 50-ml centrifuge tube (Corning, Waxford, PA) containing 20 ml of enriched RPMI 1640 tissue culture medium (Flow Laboratories, Inc., McLean, VA) and refrigerated at 4~ Within 30 min, a #15 sterile scalpel (Martin
380
Vol. 17, No. 8, August 1991
Co., Arthur N. Thomas Dist., Philadelphia, PA) was used to remove the soft tissue lesions from the roots and the teeth were placed in an anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, MI) where all further culturing procedures were accomplished. The anaerobic atmosphere was maintained at 37~ and consisted of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide (Airco Products, Inc., Allentown, PA). Cultivation and Identification of Bacteria The surface of the root to be cultured was rinsed with sterile isotonic saline and dried with sterile gauze (Johnson & Johnson, East Winsor, N J). The root was secured with sterile locking pliers (Vice Grip, DeWitt, NE) while the apical 5 mm of root was cut free from the rest of the tooth with a coping saw containing a sterile blade. Before culturing the root canal, a sterile paper point (Johnson & Johnson) moistened with reduced transport fluid (RTF) (5) was used to sample the cut surface of the root for surface contamination. The paper point was placed in 1 ml of RTF in a 4-ml vial (Wheaton, Millville, N J) and vortexed (Genie; Thomas Scientific, Swedesboro, N J) for 30 s at level i0. One hundred microliters of the RTF was spread in duplicate onto nonselective Columbia Blood Agar (BBL, Cockeysville, MD) with 0.0005% hemin (Sigma, St. Louis, MO) and 0.0001% menadione (Sigma) (CBAHM). Sterile stainless steel endodontic reamers (Union Broach, NY, NY) were used to remove pulpal debris and dentinal filings from the root canal in the apical portion of the root of the tooth. The pulpal debris and dentinal filings including any microorganisms were suspended in 1 ml of R T F i n a 4-ml vial and vortexed for 30 s. After dispersing the material, the suspension was diluted with RTF using a 10-fold serial dilution technique to 10-4. One hundred microliters of the dilutions from 10-1 through 10-4 was cultured in duplicate on CBAHM. One hundred microliters of the 10-~ dilution was cultured in duplicate on nonselective MM 10 medium and on trypticase soy agar (BBL, Cockeysville, MD) with kanamycin (Sigma) (6). The latter medium is selective for BPBP. In addition, 25 ul of RTF from dilutions 10-~ to 10-4 was spotted onto selective media for Fusobacterium nucleatum (Crystal Violet Erythromycin Agar) (6), Veillonella species (Veillonella Agar) (Difco, Detroit, MI), Lactobacillus species (Rogosa SL Agar; Difco), Actinomyces species (Forsyth Actinomyces Agar) (6), Streptococcus mutans (Mitis-Salivarius Bacitracin Sucrose Agar) (6), and Enterococcus (Streptococcus)faecalis (S. faecalis Medium; Difco). From each specimen, one plate each of CBAHM (10 -~ dilution plate), MMI0 (10 -~ dilution plate), Rogosa SL Agar (spot plate), S. faecalis Medium (spot plate), and Forsyth Actinomyces Agar (spot plate) was aerobically incubated (Bellco Glass Inc., Vineland, NJ) with 5% CO2 at 37~ All culture plates were incubated for at least 2 wk but examined for growth every 3 days. Representative colonies of different types were streaked for isolation on the appropriate medium and identified. BPBP were isolated either from CBAHM or from trypticase soy agar with Kanamycin. Identification of microorganisms was based on growth in the anaerobic chamber and growth in the aerobic incubator with 5% CO2, colonial pigmentation, colonial morphology, Gram stain, hemagglutination (sheep red blood cells), and biochemical tests. In addition, API 20A and An-IDENT (API Analytic
Bacteria in Root Canals
381
Products, Plainview NJ) rapid identification kits were used to identify the isolated microorganisms. A dissecting microscope (American Optical, Buffalo, NY) was used to enumerate colony-forming units on each culture plate. RESULTS The composition of the microorganisms isolated in this study and the number of bacteria in each sample are shown in Table 1. A total of 50 strains of bacteria were isolated from the apical 5 mm of the 10 root canals cultured in this study. The total number of strains of each species of bacteria isolated from the 10 root canals and the number of strains of each species associated with black-pigmented Bacteroides (five root canals) are shown in Table 2. The most commonly isolated microorganisms were Actinomyces species, Lactobacillus species, black-pigmented Bacteroides, Peptostreptococcus species,
Veillonella parvula, Bacteroides buccae, E. faecalis, Fusobacterium nucleatum, and S. mutans. The species of bacteria most often isolated from the same root canals as the BPBP isolated from 5 of the 10 root canals were Lactobacillus species, Peptostreptococcus species, B. buccae, E. faecalis, and V. parvula. Of the 50 microbial isolates, 34 (68%) isolates were strict anaerobes. All 10 (100%) of the root canals contained strict anaerobes and 2 root canals were infected exclusively with anaerobes. The number of colony-forming units on nonselective CBAHM from the five specimens without BPBP ranged from 5.6 x 104 to 4.3 x 106 with a median of 3.9 x 105 and the number of colony-forming units on nonselective CBAHM from the five specimens with black-pigmented Bacteroides ranged from 9.2 x 104 to 2.3 x 10 6 with a median of 4.4 x 105. The growth of bacteria from the cut surface of the roots was negligible. DISCUSSION This is the first time that both aerobic and strict anaerobic culturing techniques have been used to isolate and identify bacteria from the apical portion of root canals in teeth with coronal carious exposures of the pulp and with periapical lesions. Fifty strains of bacteria were isolated and identified from the apical 5 mm of the 10 infected root canals. The average of five isolates per infected root canal is consistent with polymicrobial root canal infections demonstrated by numerous investigators (4, 7-11). Sundqvist et al. (4) found a greater median number of microorganisms in the root canals of intact teeth infected with BPBP (2.8 x 105) than in root canals not infected with BPBP (3.0 x 103). In this study, there was not a great difference in the median number of colonyforming units enumerated between root canals infected with BPBP (4.4 x 105) and root canals not infected with BPBP (3.9 x 105). This finding may be related to the lower percentage of anaerobic bacteria detected in this study, differences in culturing methods, or the absence of members of the genus Porphyromonas. Six of the strains of Actinomyces, four of the seven strains of Lactobacillus, and the three strains of S. mutans were facultative. With the exception of facultative strains of S. mutans which are associated with caries, the species of bacteria that were cultured in this study are similar to those cultured by Sundqvist et al. (4) in the root canals of
382
Baumgartner and Falkler
Journal of Endodontics
TABLE 1. Distribution of bacterial species isolated from the apical 5 mm of 10 root canals
Tooth Specimen Species 1 Actinomyces Actinomyces Actinomyces Actinomyces Actinomyces
sp.* sp. israelii viscosus* naeslundii
2
3
4
+
+
5
6
7
8
9
10
+ + + +
B. intermedius B. denticola B. buccae
+
+
+
+ +
+
+
+
+
Bifidobacterium sp. E. faecalis*
-t-
Eubacterium sp.
-I-
+
+
F. nucleatum Lactobacillus sp.* Lactobacillus sp.
+
Peptostreptococcus anaerobius Peptostreptococcus micros
+
Propionibacterium sp.
+
S. intermedius S. mutans*
+
+
+
-I-
+
+
+
+ +
+
+
+
+
-I-
+
Veillonella parvula No. of bacterial cells
+
+
+
+
+
+ +
5.6 x 104 9.2 x 104 3.9 x 105 4.3 • 106 4.4 • 105 3.8 x 105 1.3 • 108 8.8 • 105 2.3 • 108 1.9 • 106
* Facultative anaerobes.
TABLE 2. Bacterial species isolated from the apical 5 mm of 10 infected root canals
Total No. of Strains (10 canals)
No. of Strains with BlackPigmented Bacteroides (5 canals)
2 1 1 1 2
0 1 0 0 1
Black-pigmented Bacteroides B. intermedius B. denticola
5 1
1 1
Nonpigmented Bacteroides B. buccae Bifidobacterium sp. Eubacterium sp. Fusobacterium nucleatum Lactobacillus sp.* Lactobacillus sp. Peptostreptococcus anaerobius Peptostreptococcus micros Propionibacterium sp. E. faecalis* Streptococcus intermedius S. mutans* Veillonetta parvula
5 1 1 3 4 3 5 1 1 4 1 3 5
2 0 1 1 2 3 3 1 1 2 1 1 2
Species
Actinomyces Actinomyces Actinomyces Actinomyces Actinomyces
sp.* sp. israelii viscosus* naeslundii*
* Facuttative bacteria.
intact teeth. Sundqvist et al. (4) isolated 25 strains of BPBP from 22 of 72 (30%) cultured root canals. Of the 25 strains of BPBP isolated by Sundqvist et al. (4), B. intermedius and P. endodontalis were the most numerous. P. gingivalis, B. loescheii, and B. denticola were also isolated by Sundqvist et al. (4). In this study six strains of BPBP were isolated from 5 of the 10 (50%) root canals. Of the six strains isolated, five strains were B. intermedius and one strain was B. denticola. No strain of bacteria from the asaccharolytic genus of Porphyromonas was isolated. Sundqvist et al. (4) cultured intact teeth and found that 91.4% of the microorganisms were strict anaerobes whereas in this study we cultured teeth with carious exposures and found that 68% of the bacteria were strict anaerobes. Direct access of saliva and oral microbes via a carious exposure to the pulp cavity provides an environment conducive to the growth of facultative microorganisms. Considering the presence o f a carious exposure, the relatively high percentage (68%) of anaerobic bacteria isolated from the apical 5 m m of these root canals seems to suggest a selective process for anaerobes from the oral flora. This process may be similar to the selection of microorganisms that seems to take place in the gingival sulcus during the development of different types of periodontal diseases (12). Of the more than 350 species of bacteria estimated to reside in the human oral cavity, periodontal research suggests that only 15 to 20 species are associated with periodontal diseases (12). The results of studies culturing infected root canals in intact teeth and the results of this study suggest that only a limited number of species of bacteria are consistently selected out of the total oral flora for growth in an infected root canal. As bacteria penetrate apically
Vol. 17, No. 8, August 1991
in necrotic root canals, anaerobic succession apparently occurs because of the compromised blood supply, the lower oxidation-reduction potential in the necrotic tissue, and the establishment of synergistic relationships with other bacteria. A study by Fabricius et al. (13) using monkeys demonstrated that the ratio of anaerobes to facultative bacteria increased with time following the sealing of the tooth after previous exposure of the pulp cavity to oral flora. Similarly to periodontal diseases, Koch's postulates have not been fulfilled for infections of endodontic origin because the infections are polymicrobial. However, BPBP have been implicated with symptomatic infections of endodontic origin by several investigators (4, 7, 8, l l, 14-16). Other bacteria implicated as possible endodontopathogens include Peptostreptococcus species (17), Peptococcus species (11) F. nucleaturn (18), Actinomyces species (19), and B. buccae (20). Future investigations should attempt to better define virulence factors and the ecological interrelationships required to achieve pathogenicity among bacteria isolated from infections of endodontic origin. The informed consent of all human subjects who participated in the experimental investigation reported or described in this article was obtained after the nature of the procedure and possible discomforts and risks had been fully explained. The opinions and assertions herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. This study was part of a dissertation submitted in partial fulfillment of the requirements for a PhD degree at the University of Maryland Dental School, Baltimore, MD. The authors express their thanks to the American Association of Endodontists Endowment and Memorial Foundation for funding the purchase of the culture media used in this study. Dr. Baumgartner is chief, Microbiology Branch, United States Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, DC. Dr. Falkler is professor and chairman, Microbiology Department, University of Maryland Dental School, Baltimore, MD. Address requests for reprints to Dr. J. Craig Baumgartner, 10376 Currycomb Ct., Columbia, MD 21044.
Bacteria in Root Canals
383
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