Clinical Research

Identification of Cultivable Microorganisms from Primary Endodontic Infections with Exposed and Unexposed Pulp Space
Frederick C. S. Chu, BDS, MSc, C. S. Peter Tsang, BDS, PhD, Tak W. Chow, BDS, MSc, PhD, Lakshman P. Samaranayake, BDS, DDS
Abstract
This study was aimed at comparing the cultivable microorganisms in canals with periapical radiolucencies with exposed and unexposed pulp space. Microbiological samples were taken and analyzed from 45 canals with exposed pulp space, and 43 canals with unexposed pulp space. The canal contents were analyzed by aerobic/anaerobic culture, and conventional identification techniques. There were 211 isolates of bacteria belonging to 28 genera and 55 species recovered from exposed canals. In the unexposed group, 185 isolates of bacteria were recovered, of which 54 species of 28 genera were identified. Among the four most common genera, Prevotella was significantly more common in the exposed group (51/211 in the exposed group versus 30/185 in the unexposed group) (p 0.049), while there were no differences in prevalence of Actinomyces, Peptostreptococcus, and Campylobacter between two groups of canals. In addition, Fusobacterium nucleatum and Propionibacterium acne were significantly more common in the unexposed canals (p 0.047 and p 0.0051, respectively). Similarity in bacterial species in these two groups suggests that pulp space exposure may not be a significant factor in determining the type of bacteria present in infected canals.

From the Faculty of Dentistry, The University of Hong Kong, Hong Kong, China. This investigation was supported by the Committee on Research and Conference Grants of the University of Hong Kong. Address reprint requests to Dr. Frederick C. S. Chu, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong; E-mail address: cschu@hkucc.hku.hk. Copyright 2005 by the American Association of Endodontists

hen the dental pulp is exposed because of caries, tooth wear, iatrogenic tooth preparations, or traumatic fractures/cracks, oral bacteria can invade and cause pulp inflammation. Metabolic and other toxic products of these bacteria are thought to be responsible for such inflammatory reactions and subsequent disintegration and infection of pulp space. If infection in the canal persists, chronic inflammation of the periapical area ensues leading to bone loss (1). While more than 300 bacterial species are considered to be indigenous to the oral cavity, only certain groups of bacteria are isolated from infected human root canal systems using culture techniques (2). These include Actinomyces, Fusobacterium, Lactobacillus, Prevotella, Peptostreptococcus, and Streptococcus. In addition to culture techniques, the use of molecular technologies have led to more specific identification of not only known pathogens such as Peptostreptococcus micros (3) but also novel species like Tannerella forsythensis (formerly Bacteroides) (4) and Dialister pneumonsintes (5) in different forms of primary endodontic infections. Reorganization and taxonomic changes of bacteria associated with endodontic infections guided by phylogenetic data were also brought about by the molecular methods (6). Although molecular method like Polymerase Chain Reaction (PCR) is very sensitive, vitality of microorganisms identified remains unclear. A study comparing the uses of culture, PCR and DNA-DNA hybridization for identification of Fusobacterium nucleatum showed that the differences were small (7). Interestingly, bacteria can be isolated from pulpless canals with unexposed pulp space (8 11). Communication between the pulp and the periodontium through lateral canals is believed to be a possible pathway for the entry of bacteria to the root canals with necrotic pulp. In one study, identical genotypes of Porphyromonas gingivalis and Prevotella nigrescens were recovered from both the unexposed canals and the adjacent subgingival plaque (12). Similar results were obtained in a subsequent study, but the authors were unable to suggest the route by which these organisms traversed the intact tooth from gingival/periodontal tissue to the root canal chamber (13). Recent studies have also shown that some organisms such as Enterococcus facaelis (14 15), Pseudomonas aeruginosa (16), and Candida albicans (17), may be more resistant to endodontic therapy and cause treatment failure. It is also unclear whether these organisms were the cause of primary infection and developed resistance during chemo-mechanical therapy, or whether they entered the pulp space because of microleakage around restorations or contamination during treatment. There is little information on the microbiology of primary endodontic infection, i.e. infected canals without treatment, in ethnic Chinese populations; and the effect of pulp space exposure on root canal microflora also remains unclear. The aims of the current study were first, to compare the bacterial and yeast flora of infected canals with clinically exposed and unexposed pulp space, and second, to determine the differences, if any, in the clinical presentations of the latter two types of primary endodontic infections. The definition of clinical exposure is observed communication between pulp space and oral cavity before or after removal of existing caries and restorations. Such communication should be detectable visually under magnification and confirmed by a sharp straight probe. This definition gives rise to three groups of canals: (a) teeth without exposure even after complete removal of caries and/or restorations; (b) teeth with exposure before excavation, and (c) teeth with exposure

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after excavation. Groups (b) and (c) are both regarded as the exposed canals because these groups had extensive carious lesions and bacterial invasion into the pulp chamber. Dental Products, Ontario, Canada) under copious sterile saline coolant. Care was taken not to leave any caries or restorative material in front of the preparation of the access cavity above the pulp chamber, and a straight probe was used to examine if any pulp space exposure was already present. Any exposure detected at this stage was sealed with a cotton pellet moistened with Oraseal (Ultradent) before the access cavity above the pulp chamber was disinfected with 4% chlorhexidine and 10% iodine. To check the sterility, a sterile, dry cotton pellet was used to swab the wall of the access cavity above the pulp chamber and that was then transferred to a vial containing 3 ml of sterile Reduced Transport Fluid (RTF) for culture (19). This sterility check is to ensure that the paper point sampling of the intracanal contents would not be contaminated by the access cavities, which could be carious or contaminated by microleakage around restorations. Chlorhexidine gel was used for disinfection instead of hydrogen peroxide solution proposed by Mo ller (20) to avoid the flow of disinfectant into the pulp chamber and canals. The combined use of chlorhexidine and iodine was proven to be an efficient skin disinfection regime for catheter insertion (21) and collection of periapical abscess (22). After estimating the canal length with the preoperative periapical radiograph, a size 15 K-flexofile (Dentsply, Milford, DE) was placed gently into the root canal to determine its patency. For multi-rooted teeth, only the root with periapical lesion was sampled. If periapical lesions were present around all the roots, samples were taken from the widest root only. Three fine, sterilized paper points (Absorbent paper points, Diadent Group International, Burnaby, B.C., Canada), were sequentially placed to reach the estimated length of the root determined with the preoperative radiograph, for 15 to 20 s. If the canal was dry, then a small amount of saline was used to wet the canal before the paper points were inserted. The retrieved paper points were immediately placed in 0.5 ml of Reduced Transport Fluid (RTF) in a Bijoux container (Bibby Sterilin, Stone, England) and transported to the Oral Bio-Science Laboratory within the same premises, for microbiological analysis.

Materials and Methods

There were 100 consecutive patients attending the Primary Care Clinic of the Prince Philip Dental Hospital, University of Hong Kong, with periapical radiolucencies associated with infected canals were invited to participate. Patients with uncontrolled systemic diseases or requiring antibiotic cover were excluded. A total of 103 teeth were sampled, and 88 of them were included in this study. Fifteen samples were discussed because of contamination of operation field or prolonged delay in delivery to laboratory. Forty-five teeth (21 single-rooted and 24 multirooted) with exposed pulp space, and 43 teeth (31 single-rooted and 12 multi-rooted) with unexposed pulp space were included in the study. Patients of the exposed group comprised 16 males and 29 females, and their age ranged from 16 to 49 yr (mean age 34.4 12.3 yr). The unexposed group comprised 13 males and 29 females with ages between 16 and 71 yr (mean age 47.3 21.8 yr). Each infected canal was examined to determine if it is associated with swelling, pain and tenderness to percussion. The criteria and frequencies of clinical findings are presented in Table 1. The study was approved by the Ethics Committee of the Faculty of Dentistry, The University of Hong Kong. Informed consent was obtained from all individuals before the study, which was an integral component of the management protocol.

Clinical Procedures As part of the standard initial oral examination, a panoramic radiograph is taken for every new patient at the hospital. An additional periapical radiograph was taken if a periapical radiolucency was suspected for any tooth. The pulp status was assessed with heat, cold, electric pulp tests, and only teeth with no response were included in the study. Any pre-existing exposure of the pulp space because of a fracture, a crack line, a defective restoration or caries was carefully ascertained under 2.5 magnification. After cleansing with pumice, the tooth selected was isolated under rubber dam and the opening of the pulp space exposure was sealed with a sterile cotton pellet moistened with a small amount of Oraseal (Ultradent, Salt Lake City, UT) to prevent the possible entry of the disinfectants into the exposed canal. The operating field was swabbed twice using 4% chlorhexidine gluconate (Hibiscrub, Zeneca, Cheshire, UK) (18). The disinfectant was allowed to remain on the tooth surface and the rubber dam for 3 min, and then it was washed away thoroughly using sterile saline. A 10% iodine tincture (Betadine, Seton Healthcare Group, Oldman, UK) was then used to scrub the field for 1 min, and it was inactivated with sterile 5% sodium thiosulphate. All instruments, including the handpiece, were sterilized in an autoclave immediately before use. After disinfection of the operating field, caries and previous restorations were removed using a high-speed tungsten carbide bur (Beavers

Culture and Identification of Isolates In the laboratory, the contents of the Bijoux container were dispersed on a vortex mixer for 30 s (Autovortex mixer SA2, Stuart Scientific, Surrey, UK), the RTF solution diluted to 1:10, 1:100, and 1:1000, and spiral-plated (Model DU, Spiral Systems, Cincinnati, OH) onto the following culture media: (1) Columbia blood agar base (CBABS) supplemented with 5% defibrinated horse blood, 0.0005% (5 mg/L) haemin (Sigma Chemical, St. Louis, MO) and 0.00005% (0.5 mg/L) menadione (E. Merck, Darmstadt, Germany) for assessment of total cultivable microflora (2), Mitis salivarius agar for total streptococcal count (Difco, Detroit, MI) (3), Mitis salivarius Bacitracin agar for Streptococcus mutans, and (4) Rogosa SL agar (Difco) for Lactobacillus spp. The plates were incubated at 37C for 5 to 10 days in an anaerobic chamber (Forma Scientific, Marietta, OH) under 80% N2, 10% H2, and 10% CO2 (Hong Kong Oxygen & Acetylene, Hong Kong). In

TABLE 1. Criteria and frequencies of clinical findings associated with infected canals with exposed and unexposed pulp space No. of canals Exposed n 45
Swelling (localized or diffuse enlargement of soft tissue adjacent to the infected canal) Pain (sharp or dull pain associated with the infected canal, which may be spontaneous or elicited by stimuli) Tenderness to percussion (presence of pain when gentle percussion is performed by tapping on the incisal or occlusal surface with the end of a mirror handle held perpendicular to the crown)
No statistically significant differences in sign and symptoms were found in these two groups of canals (P 0.05).

Unexposed n 43
22 22 3

20 17 8

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addition, the 1:10, 1:100, and 1:1000 dilutions were spiral-plated on MacConkey agar (Oxoid, Unipath, Basingstoke, Hampshire, England) and Sabouraud dextrose agar (Oxoid) for the isolation of coliforms and yeasts, respectively. The resultant anaerobic growth was quantified by evaluating the colony forming units (CFUs) on the CBABS medium using a stereomicroscope (Nikon SMZ-1B, Nikon, Japan), and the number of colonyforming units per milliliter (CFU/ml) for each dilution was calculated for each sample. The growth was semi-quantitatively determined as very heavy, heavy, moderate, sparse, and very sparse depending on the CFUs ranging from 100,000, 100,000, 10,000, 1,000, 100 colony forming units per 1.0 ml RTF, respectively (14). Ten well separated and evenly dispersed colonies with varying colonial morphology were randomly selected and subcultured on CBABS to obtain a pure growth for qualitative studies. Speciation was based on colony morphology, type of hemolysis, cell morphology, Gram-staining reaction, catalase and oxidase positivity, and the ability to grow in air supplemented with 10% CO2. The obligate and facultative anaerobes were further characterized using RapID ANA II identification kits (Innovative Diagnostic Systems, Norcross, GA), and RapID NH identification kits (Innovative Diagnostic Systems) were used for facultative anaerobic gram-negative rods and cocci. For the facultative anaerobic gram-positive, and catalase negative cocci, additional characterization was performed using API 20 Strep (Analytical Profile Index; bio Merieux, Marcy-lEtoile, France). Conventional biochemical tests were also used to supplement the commercial kits when necessary. The colony morphologies on MacConkey and Sabouraud plates were observed 1 day after aerobic incubation at 37C. Any growth was subcultured and identified with commercial kits, i.e. API 20E (Analytical Profile Index; Bio Merieux SA, France) for coliforms, and API 20C Aux (Analytical Profile Index) and CHROMagar Candida for yeasts. The germ tube test was also used to identify the yeasts isolated. All data were entered and calculated with computer software (Excel 7.0; Microsoft, Redmond, WA) and statistical software (SPSS 11.5 for Windows; SPSS, Chicago, IL). 2 tests were used to compare the prevalence of four genera and 25 bacterial species most commonly found in each group. Pearson 2 tests were used to determine whether there was an association between the prevalence of bacterial species and symptoms. Twenty-five tests were conducted for 25 bacterial species most commonly found and each symptom. Considering the number of tests, the significance level at 0.05 was used. majority of isolates (68.5%) and 31.5% were facultative anaerobes. Fifty-four bacterial species belonging to 28 genera were isolated from these canals. The average number of bacterial species in each canal was 4.2 (ranging from 0 7). The four most common genera were Prevotella (30/185), Actinomyces (21/185), Campylobacter (19/185), Peptostreptococcus (19/185). Table 3 shows the distribution of the bacterial isolates from both the exposed and the unexposed canals according to Gram staining, morphology, and oxygen requirements. 2 tests were used to compare the numbers of isolates in each group. The genus Prevotella was significantly more common in the exposed canals compared with the unexposed root canals (p 0.049). However, there were no differences in the prevalence of the other three most common genera namely, Peptostreptococcus (48/396), Actinomyces (41/396), and Campylobacter (33/396). At species level, the prevalence of F. nucleatum and P. acnes were significantly more common in unexposed canals (p 0.047 and p 0.0051, respectively). Pearson 2 tests were used to determine whether there was an association between the prevalence of bacterial species and symptoms. In the group of exposed canals, Fusobacterium varium (2 7.031, p 0.008), Gemella morbillorum (2 4.07, p 0.04), and P. gingivalis (2 4.02, p 0.05) were associated with swelling, while Lactobacillus spp. (2 4.27, p 0.04) and Actinomyces meyeri (2 16.32, p 0.0001) were associated with existing pain and tenderness to percussion, respectively. In the unexposed group, P. gingivalis (2 6.06, p 0.014) and Eggerthella lenta (formerly, Eubacterium lentum) (2 3.84, p 0.05) were associated with swelling, while Actinomyces spp. were associated with tenderness to percussion (2 5.98, p 0.014).

Discussion
The current microbiologic sampling procedure has been shown to be effective and widely employed (23, 24), but there are still a few shortcomings in this study. There was a potential risk of leakage of disinfectants into the pulp space. Care was taken to seal off the exposure sites during disinfection of the operation fields and access cavities, but its efficacy was not clear. Moreover, a brief exposure to air during the sampling procedure might be sufficient to kill some anaerobic microorganisms and the recovery of obligate anaerobes might be affected by the saline used during sampling. The paper points also may not capture all bacteria for subsequent isolation and identification (25). However, the short time span between sampling and microbiological processing in this study would guarantee the survival of the collected bacteria in the transport medium (1). The frequencies of obligate anaerobe isolates recovered from both exposed and unexposed canals in the present study were similar. These figures (68.9% and 68.5%) are comparable to the findings of recent studies employing anaerobic culture techniques (10, 18, 24 26). Sundqvist (26) recovered 353 bacterial isolates from 65 teeth with intact pulp chambers and found 90% of the isolates were obligate anaerobes. Wasfy et al. (10) also reported that 78 out of 85 traumatized, symptomatic anterior teeth with unexposed pulp space contained positive bacterial growth, and 73% (259 isolates) were obligate anaerobes. Gomes et al. (18) examined 70 canals associated with periapical radiolucencies or pain (47 primary infection, 21 root-filled, and two teeth with pulpitis), and reported that in 60 canals with positive growth, 155 (64%) of 242 cultivable isolates, belonging to 65 species, were obligate anaerobes. Peters et al. (23) investigated the microbiological status of 58 canals associated with asymptomatic apical lesions and they found that 87% of the 131 isolates were obligate anaerobes. More recently, Jacinto et al. (24) reported that 75% of the 218 isolates obtained from 48 teeth with apical periodontitis were obligate anaerobes.

Results
All swab samples over the wall of access cavities were negative. Bacteria were present in all but one of the canals that had not been exposed, whereas yeasts were only found in one of the exposed canals. The identification and frequency of bacterial strains isolated from the canals are shown in Table 2. The number of canals with different total colony forming units (i.e. No growth, 3, 3 to 4, 4 to 5, 5 to 6 and 6 to 7, and 7 Log (10) CFU/ml) of the bacteria on CBABS medium were 0, 4, 7, 7, 8, 13, 8 for the exposed, and 1, 2, 2, 8, 14, 14, 2 for the unexposed groups, respectively. There were 211 isolates of bacteria recovered from the 45 exposed root canals, of which 68.9% were obligate anaerobes. Fifty-five bacterial species belonging to 28 genera were identified. The four most frequently recovered bacterial genera were Prevotella (51/211), Peptostreptococcus (29/211), Actinomyces (20/211), and Campylobacter (14/ 211). The mean number of bacterial species in each canal was 4.6 (ranging from 110). In the 43 unexposed canals, 185 isolates of bacteria were recovered. As in the exposed canals, obligate anaerobes accounted for the 426

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TABLE 2. Number of bacterial strains isolated from canals with exposed and unexposed pulp space* (Percentage of canals) No. of canals Exposed n 45 (%)
GRAM NEGATIVE RODS Obligate anaerobes Bacteroides capillosus B. fragilis B. uniformis Campylobacter gracilus# (formerly Bacteroides) Campylobacter spp. Fusobacterium necrophorum F. nucleatum F. varium Fusobacterium spp. Prevotella buccae P. corporis P. denticola P. loescheii P. intermedia P. melaninogenica P. oralis gp P. oris Porphyromonas endodontalis P. gingivalis Facultative anaerobes Capnocytophaga spp. Eikenella corrodens Haemophilus parainfluenzae Kingella kingae GRAM POSITIVE RODS Obligate anaerobes Actinomyces israelii A. meyeri A. odontolyticus Atopobium minutum (formerly Lactobacillus minutus) Bifidobacterium spp. Clostridium subterminale Collinsella aerofaciens (formerly Eubacterium) Eggerthella lenta (formerly Eubacterium lentum) Eubacterium limosum Facultative anaerobes Actinobacillus actinomycetemcomitans Actinomyces naeslundii A. pyogenes A. viscosus A. spp. Corynebacterium spp. Enterococcus faecalis Lactobacillus acidophilus L. jensenii Lactobacillus spp. Propionibacterium acnes P. granulosum P. propionicus GRAM NEGATIVE COCCI Obligate anaerobes Veillonella dispar Veillonella spp.

TABLE 2. Continued No. of canals Exposed n 45 (%)

Facultative anaerobes Neisseria lactamica N. meningitidis N. mucosa N. sicca/subflava Neisseria spp. GRAM POSITIVE COCCI Obligate anaerobes Peptostreptococcus anaerobius P. asaccharolyticus P. magnus P. micros P. prevotii Facultative anaerobes Aerococcus viridans Gemella haemolysans G. morbillorum Staphylococcus saccharolyticus Staphylococcus spp. Streptococcus constellatus S. equines S. mitis S. milleri I S. sanguius Streptococcus spp. 1 (2) 1 (2) 2 (4) 3 (7) 4 (9) 2 (4) 2 (4) 14 (31) 7 (16) 1 (2) 2 (4) 11 (24) 1 (2) 5 (11) 1 (2) 3 (7) 1 (2) 1 (2)

Unexposed n 43(%)

Unexposed n 43(%)
3 (7) 1 (2) 3 (7) 4 (9) 1 (2) 2 (5) 9 (21) 7 (16) 1 (2) 5 (12) 2 (5) 1 (2) 1 (2) 1 (2) -

2 (3) 1 (2) 6 (13) 8 (18) 1 (2) 2 (4) 5 (11) 2 (4) 10 (22) 5 (11) 1 (2) 13 (29) 9 (20) 5 (11) 6 (13) 2 (4) 1 (2) 3 (7) 11 (24) 1 (2) 1 (2) 3 (7) 6 (13) 5 (11) 1 (2) 2 (4) 1 (2) 4 (9) 5 (11) 1 (2) 5 (11) 1 (2) 6 (13) 1 (2) 5 (11) 3 (7) 5 (9)

1 (2) 2 (5) 1 (2) 6 (14) 13 (30) 1 (2) 8 (19) 1 (2) 3 (7) 6 (14) 3 (7) 1 (2) 7 (16) 8 (19) 2 (5) 2 (5) 1 (2) 6 (14) 7 (16) 2 (5) 1 (2) 6 (14) 8 (19) 8 (19) 1 (2) 4 (9) 1 (2) 1 (2) 1 (2) 2 (5) 2 (5) 1 (2) 2 (5) 5 (12) 2 (5) 7 (16) 1 (2) 2 (5) 1 (2) 7 (16)

* Bacteria were cultured on Columbia blood agar base for total cultivable microflora. Other selective media were also used to culture specific microorganisms, e.g. Mitis salivarius Bacitracin agar for Streptococcus mutans, and Sabouraud dextrose agar for yeasts.

* Bacteria were cultured on Columbia blood agar base for total cultivable microflora. Other selective media were also used to culture specific microorganisms, e.g. Mitis salivarius Bacitracin agar for Streptococcus mutans, and Sabouraud dextrose agar for yeasts.

The current study has confirmed the polymicrobial nature and the predominance of Gram-negative rods in primary endodontic infections. The most commonly identified bacterial species in this study were similar to other studies of primary endodontic infections, except Streptococcus, which was isolated from only nine canals in this study (10, 18, 27). In addition, two other bacterial species associated with caries, A. meyeri and A. odontolyticus, were found in 20% of unexposed, and more than 10% of exposed canals. This high prevalence of Actinomyces spp. is in agreement with a previous study where molecular techniques were employed to investigate the presence of this organism in infected root canals in ethnic Chinese patients (28). P. gingivalis has been reported to be closely associated with acute symptoms in endodontic infections (27), and this was confirmed. In addition, E. lenta (formerly, Eubacterium lentum), F. varium, and G. morbillorum are thought to be associated with swollen lesions in this study. These microorganisms were often found in primary endodontic infections, and efficiently removed by conventional chemo-mechanical treatment. There were significant differences between the two groups in the prevalence of Prevotella, F. nucleatum, and P. acnes. One explanation for this may be that exposed canals acquire a flora that is more representative of the oral commensals while the flora found in unexposed canals could be considered to be truly pathogenic. It is also noteworthy in this context that F. nucleatum has been reported to be associated with the more severe form of inter-appointment flare-ups (29) and it was isolated from approximately 20% of the unexposed canals in the current study. Similarly, P. acnes was also found to be more prevalent in the unexposed canals, and this may have systemic clinical relevance. A previous study reported that the isolates of P. acne recovered from infected root canals and systemic circulation during endodontic treat427

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TABLE 3. Distribution of bacterial isolates from canals with exposed and unexposed pulp space according to Gram-staining, morphology and oxygen requirements. Exposed No. of isolates (n 211) (%)
Gram negative rods Obligate anaerobes Facultative anaerobes Gram negative cocci Obligate anaerobes Facultative anaerobes Gram positive rods Obligate anaerobes Facultative anaerobes Gram positive cocci Obligate anaerobes Facultative anaerobes 82 (38.9%) 13 (6.2%) 8 (3.8%) 7 (3.3%) 27 (12.8%) 19 (9.0%) 29 (13.7%) 26 (12.3%)

Unexposed No. of isolates (n 185) (%)

72 (38.9%) 10 (5.4%) 8 (4.3%) 11 (5.9%) 28 (15.1%) 26 (14.1%) 19 (10.3%) 11 (6.0%)

ment had identical biochemical profiles (30). Some authors have therefore surmised that dental infection might provoke a follicular inflammatory response in the skin, leading to recalcitrant acne (31). The age of necrotic process is a factor affecting the microbiota of infected canals (32). However, there is no reliable means to measure the age of necrotic process of a pulp, and it is impossible to specify the age of necrotic process in human study because of ethical reasons. It is unclear if the age of necrotic process is equally distributed between the two groups of infected canals in this study. Although all the sampled canals had apical periodontitis indicating a later stage of disease process, the reported differences in the microbiota between the two groups might be partially because of the differences in the age of necrotic process. Bacterial and yeast species associated with refractory periapical lesions, such as E. facaelis, P. aeruginosa, and C. albicans were recovered from a very small number of canals in the present study. The sparse findings of these three microorganisms in primary endodontic infections were also noted in other investigations (18, 23, 26). Because of the ecological factors such as oxygen tension, availability of nutrients and bacterial interactions, only selected groups of microflora are found in infected canals. For example, the growth of one bacterial species may depend on the combination of other species, which may supply nutrients as metabolic byproducts. On the other hand, the difference in salivary carriage of microorganisms was found to be another confounding factor. Egan et al. (33) found that the presence of yeasts in canals was significantly associated with their presence in saliva. This may explain why yeasts were present in some teeth and not in others. It is possible that some microorganisms were favored by ecological changes because of treatment measures, and became the causative factor of endodontic failure despite their low prevalence in primary endodontic infections. Theoretically, the exposure of pulp space to the oral environment may allow the direct entry of bacteria, nutrients and oxygen into the canals, creating an environment that is radically different from teeth with no exposure since they will have a low redox potential and lack of nutrients. Therefore, it was postulated that significant qualitative and quantitative differences might exist between exposed and unexposed canals. The present study however suggests that the predominant cultivable microorganisms in exposed and unexposed canals are similar. In a histological study, no inflammation can be seen when caries/bacteria is 1.1 mm from the pulp. Once the remaining dentin approached 0.5 mm, mild to severe inflammation and microorganisms can be seen in the pulp (34). In fact, this concept has been indirectly confirmed in this study as microbial flora isolated from clinically exposed and unexposed canals appeared to be similar. Furthermore, recent in vivo studies have 428

shown that in the presence of bacteria, the severity of pulpal inflammation increased with reduced dentin thickness following tooth preparation. This may be explained by the increased dentin permeability with reduced dentin thickness, which allows differential penetration of bacteria and their byproducts along dentinal tubules into the unexposed pulp space (35). Similarly, micro-cracks in enamel and dentin may also lead to the penetration of bacteria and oral fluids into the dentin-pulp complex. In conclusion, similarity in bacterial species in these two groups suggests that exposure of the pulp space may not be a significant factor in determining the selection of bacteria present in infected canals.

References
1. Weiger R, Manncke B, Werner H, Lost C. Microbial flora of sinus tracts and root canals of non-vital teeth. Endod Dent Traumatol 1995;11:159. 2. Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathol 1994;78:52230. 3. Siqueira JF Jr, Ro c a IN, Andrade AF, de Uzeda M. Peptostreptococcus micros in primary endodontic infections as detected by 16S rDNA-based polymerase chain reaction. J Endod 2003;29:1113. 4. Siqueira JF Jr, Ro c a IN. Bacteroides forsythus in primary endodontic infections as detected by nested PCR. J Endod 2003;29:390 3. 5. Siqueira JF Jr, Ro c a IN. Positive and negative bacterial associations involving Dialister pneumosintes in primary endodontic infections. J Endod 2003;29:438 41. 6. Siqueira JF Jr. Taxonomic changes of bacteria associated with endodontic infections. J Endod 2003;29:619 23. 7. Moraes SR, Siqueira JF Jr, Colombo AP, Ro c a IN, de S, Domingues RM. Comparison of the effectiveness of bacterial culture, 16S rDNA directed polymerase chain reaction, and checkerboard DNA-DNA hybridization for detection of Fusobacterium nucleatum in endodontic infections. J Endod 2002;28:86 9. 8. Wittgow WC Jr, Sabiston CB Jr. Microorganisms from pulpal chambers of intact teeth with necrotic pulps. J Endod 1975;1:168 71. 9. Hoshino E, Ando N, Sato M, Kota K. Bacterial invasion of non-exposed dental pulp. Int Endod J 1992;25:25. 10. Wasfy MO, McMahon KT, Minah GE, Falkler WA Jr. Microbiological evaluation of periapical infections in Egypt. Oral Microbiol Immunol 1992;7:100 5. 11. Lana MA, Ribeiro-Sobrinho AP, Stehling R, Garcia GD, Silva BK, Hamdan JS, Nicoli JR, Carvalho MA, Farias L de M. Microorganisms isolated from root canals presenting necrotic pulp and their drug susceptibility in vitro. Oral Microbiol Immunol 2001;16:100 5. 12. Goncalves RB, Robitaille M, Mouton C. Identical clonal types of Porphyromonas gingivalis or Prevotella nigrescens recovered from infected root canals and subgingival plaque. Oral Microbiol Immunol 1999;14:197200. 13. Rupf S, Kannengiesser S, Merte K, Pfister W, Sigusch B, Eschrich K. Comparison of profiles of key periodontal pathogens in periodontium and endodontium. Endod Dent Traumatol 2000;16:269 75. 14. Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:17. 15. Sundqvist G, Figdor D, Persson S, Sjo gren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative retreatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86 93.

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16. Cheung GS, Ho MW. Microbial flora of root canal-treated teeth associated with asymptomatic periapical radiolucent lesions. Oral Microbiol Immunol 2001;16:3327. 17. Waltimo TM, Siren EK, Torkko HL, Olsen I, Haapasalo MP. Fungi in therapy-resistant apical periodontitis. Int Endod J 1997;30:96 101. 18. Gomes BP, Drucker DB, Lilley JD. Positive and negative associations between bacterial species in dental root canal. Microbios 1994;80:231 43. 19. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbiol 1972;24:638 44. 20. Mo ller AJR. Microbiological examination of root canals and periapical tissues of human teeth. Methodological studies. Odontol Tidskr 1966;74:1380. 21. Langgartner J, Linde H-J, Lehn N, Reng M, Scho merich J, Glu ck T. Combined skin disinfection with chlorhexidine/propanolol and aqueous povidone-iodine reduces bacterial colonization of central venous catheters. Intensive Care Med 2004;30:1081 8. 22. Baumgartner JC, Siqueira JF Jr, Xia T, Ro c a IN. Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction. J Endod 2004; 30:141 4. 23. Peters LB, Wesselink PR, van Winkelhoff AJ. Combinations of bacterial species in endodontic infections. Int Endod J 2002;35:698 702. 24. Jacinto RC, Gomes BP, Ferraz CC, Zaia AA, Filho FJ. Microbiological analysis of infected root canals from symptomatic and asymptomatic teeth with apical periodontitis and the antimicrobial susceptibility of some isolated anaerobic bacteria. Oral Microbiol Immunol 2003;18:28592. 25. Matuso T, Shirakami T, Ozaki K, Nakanishi T, Yumoto J, Ebisu S. An immunohistological study of the localization of bacteria invading root pulpal walls of teeth with periapical lesions. J Endod 2003;194 200. 26. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257 62. 27. Haapasalo M. Black-pigmented gram-negative anaerobes in endodontic infections. FEMS Immunol Med Microbiol 1993;6:2137. 28. Tang G, Samaranayake LP, Yip HK, Chu FC, Tsang PC, Cheung BP. Direct detection of Actinomyces spp. from infected root canals in a Chinese population: a study using PCR-based, oligonucleotide-DNA hybridization technique. J Dent 2003;31:559 68. 29. Chavez de Paz Villanueva LE. Fusobacterium nucleatum in endodontic flare-ups. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:179 83. 30. Debelian GJ, Olsen I, Tronstad L. Profiling of Propionibacterium acnes recovered from root canal and blood during and after endodontic treatment. Endod Dent Traumatol 1992;8:248 54. 31. Boyd AS, King LE Jr. Recalcitrant acne vulgaris secondary to a dental abscess. Cutis 1999;64:116 8. 32. Fabricius L, Dahlen G, Ohman AE, Moller AJ. Predominant indigenous oral bacteria isolated from infected root canals after varied times of closure. Scand J Dent Res 1982;90:134 44. 33. Egan MW, Spratt DA, Ng YL, Lam JM, Moles DR, Gulabivala K. Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis. Int Endod J 2002;35:3219. 34. Reeves R, Stanley HR. The relationship of bacterial penetration and pulpal pathosis in carious teeth. Oral Surg Oral Med Oral Pathol 1965;22:59 65. 35. Camps J, Dejou J, Remusat M, About I. Factors influencing pulpal response to cavity restorations. Dent Mater 2000;16:432 40.

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