Oral Microbiology Immunology 2003: 18: 285292 Printed in Denmark.

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Microbiological analysis of infected root canals from symptomatic and asymptomatic teeth with periapical periodontitis and the antimicrobial susceptibility of some isolated anaerobic bacteria
Jacinto RC, Gomes BPFA, Ferraz CCR, Zaia AA, Souza Filho FJ. Microbiological analysis of infected root canals from symptomatic and asymptomatic teeth with periapical periodontitis and the antimicrobial susceptibility of some isolated anaerobic bacteria. Oral Microbiol Immunol 2003: 18: 285292. Blackwell Munksgaard, 2003. The purpose of the present study was to investigate the correlation between the composition of the bacterial ora isolated from infected root canals of teeth with apical periodontitis with the presence of clinical signs and symptoms, and to test the antibiotic susceptibility of ve anaerobic bacteria mostly commonly found in the root canals of symptomatic teeth against various substances using the E-test. Microbial samples were taken from 48 root canals, 29 symptomatic and 19 asymptomatic, using adequate techniques. A total of 218 cultivable isolates were recovered from 48 different microbial species and 19 different genera. Root canals from symptomatic teeth harbored more obligate anaerobes and a bigger number of bacterial species than the asymptomatic teeth. More than 70% of the bacterial isolates were strict anaerobes. Statistical analysis used a Pearson Chi-squared test or a one-sided Fisher's Exact test as appropriate. Suggested relationships were found between specic microorganisms, especially gram-negative anaerobes, and the presence of spontaneous or previous pain, tenderness to percussion, pain on palpation and swelling amoxicillin, amoxicillin clavulanate and cephaclor were effective against all the strains tested. The lowest susceptibility rate was presented by Prevotella intermedia/nigrescens against Penicillin G. Our results suggested that specic bacteria are associated with endodontic symptoms of infected teeth with periapical periodontitis and the majority of the anaerobic bacterial species tested were susceptible to all antibiotics studied. Apical periodontitis is induced by bacteria of infected root canals (15). Clinically, apical periodontitis starts as an acute inammation of the apical periodontal ligament followed by symptoms such as pain, tenderness to percussion and swelling (24). If the presence of irritants extends for long, the lesion

R. C. Jacinto, B. P. F. A. Gomes, C. C. R. Ferraz, A. A. Zaia, F. J. Souza Filho

Endodontic Area, Dental School of Piracicaba, University of Campinas, Piracicaba, SP , Brazil

Key words: endodontics; microbiology; signs and symptoms; antimicrobial susceptibility Brenda P .F . A. Gomes, Endodontia, Faculdade de Odontologia de Piracicaba FOP-UNICAMP , Avenida Limeira, 901, Piracicaba, SP 13414-018, Brazil Tel.: 55 19 3412 5215; fax: 55 19 3412 5218; e-mail: bpgomes@fop.unicamp.br Accepted for publication February 10, 2003

may become chronic without causing dramatic clinical symptoms to the patient (20). However, the occurrence of spontaneous pain may be a result of an increased

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Jacinto et al.
Clinical features Microbial isolation

virulence of microorganisms in the root canal (36). Root canals of symptomatic teeth with necrotic pulps and periapical bone destruction tend to harbor a larger number of bacteria and a more complex anaerobic bacterial ora than the asymptomatic teeth with apical periodontitis (36). Positive correlations have been found between the number of bacteria and clinical symptoms (4, 37). Several species of bacteria have been found in symptomatic infected root canals, with a predominance of obligate anaerobes, especially Fusobacterium, Peptostreptococcus and ``black-pigmented bacteria'' (9, 10, 42). However, endodontic symptomatology itself is not an indication for systemic antimicrobial treatment (13), except in the presence of classic signs of infection, such as swelling, surface erythema, trismus, fever or lymphadenopathy (41). Systemic antibiotics act as a coadjuvant to the conventional surgical methods and should be used with restraint because of the possibilities of allergic reactions, toxicity, side effects and development of resistant strains of microbes (1). The increasing resistance of anaerobic bacteria to some widely used antibiotics and the introduction of new antibiotics for the treatment and prophylaxis of anaerobic infections ensure the need of monitoring susceptibility patterns periodically by using susceptibility tests. Therefore, the E-test (AB Biodisk, Solna, Sweden), an agar diffusion susceptibility test, holds the promise of being accurate and exible enough to be performed in the most clinical laboratories (29). The purpose of the present study was to investigate the correlation between the composition of the bacterial ora isolated from infected root canals of teeth with apical periodontitis with the presence of clinical signs and symptoms, and test the antibiotic susceptibility of ve anaerobic bacteria most commonly found in the root canals of symptomatic teeth to various substances using the E-test.
Material and methods
Patient selection

The following features were noted for each patient: age, gender, tooth and pulp status, nature of pain, previous pain, tenderness to percussion, pain on palpation, mobility, presence of a sinus and its origin (`endodontic' or `periodontic'), presence of swelling of the periodontal tissues, depth of the periodontal pocket, history of previous and present antibiotic therapy or other medication, radiographic ndings and status of root canal such as dry canal or the presence of clear, haemorrhagic or purulent exudates.
Patient sampling

A total of 48 patients, who attended the State University of Campinas UNICAMP, with the need for endodontic treatment due to the necrosis of the dental pulp tissues, were involved in this study. The patients signed a document according to the Ethical Committee in Research (State University of Campinas UNICAMP). The age of the patients ranged from 13 to 63 years.

In each case a single root canal was sampled, in order to conne the microbial evaluation to a single ecologic environment. Samples were collected using strict asepsis. A rubber dam was used to isolate the tooth. The tooth and the surrounding eld were then cleansed with 30% hydrogen peroxide and decontaminated with a 2.5% sodium hypochlorite solution for 30 s each. The solution was inactivated with sterile 5% sodium thiosulfate. Access to the root canal was made using sterile burs without water spray. Aseptic techniques were used for instrumentation, during access to and removal of the contents from the pulp space, and sample collection. After initial entry to the pulp space, the enlargement of the root canal was established with minimal instrumentation, where possible, and without the use of any irrigant. A sterile paper point was then introduced in the full length of the canal and retained in position for 60 s for microbial sampling. If the root canal was dry, a small amount of sterile saline solution was introduced into the canal to ensure viable sample acquisition. Chemical active irrigants were never used before sampling. To maintain the necessary anaerobiosis to ensure the survival of anaerobic bacteria, a set clinical routine was established. The paper point was inserted in the canal to the approximate apex under 99.9% N2 gas ow. A sterilized prereduced transport uid (RTF) (38) was used to transport the sample to the laboratory, where the samples were placed in an anaerobic chamber (Don Whitley Scientic, Bradford, UK) and processed in a maximum of 4 h. The anaerobic atmosphere, consisting of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide, was maintained at 378C. All culturing procedures were accomplished under these conditions.

Inside the anaerobic chamber the transport media was shaken thoroughly in a mixer o Paulo, SP, for 60 s (Vortex, Marconi, Sa Brazil). The transport media contained glass beads with a diameter of 3 mm to facilitate mixing and homogenization of the sample prior to cultivation. Serial 10fold dilutions were made up to 1 : 104 in tubes containing Fastidious Anaerobe Broth (FAB, Lab M, Bury, UK). Fifty ml of the serial dilutions 1 : 102, 1 : 103 and 1 : 104 were plated, using sterile plastic spreaders, into 5% debrinated sheep blood Fastidious Anaerobe Agar (FAA, Lab M), in which 1 ml/l of hemin and 1 ml/l of vitamin K1 were added, so as to culture non-selectively obligate anaerobes and facultative anaerobes. Selective culture media were also used as follows: 5% sheep blood FAA NAL (0.001% w/v nalidixic acid) VAN (0.5 mg/l vancomycin) to select gram-negative anaerobic bacteria; 5% sheep blood FAA KAN (kanamycin) VAN to select ``black-pigmented bacteria''; 5% sheep blood FAA NEO (0.0075% w/v neomycin) for clostridia and other anaerobes; 5% sheep blood FAA NAL (0.001% w/v nalidixic acid) for gram-positive anaerobes and Actinomyces involved. The plates were incubated at 378 C in an anaerobic atmospherefor up to 14 days to permit detection of very slowly growing strains. The same dilutions were plated in 5% sheep blood Brain Heart Infusion (BHI) agar (Oxoid, Basingstoke, UK), to allow aerobic or facultative microorganism growth, and in Agar Dextrose Sabouraud (Oxoid, Basingstoke, UK) supplemented with 100 mg/ml of chloramphenicol (Medley, Campinas, SP, Brazil) for yeasts, both incubated aerobically at 378C for 2 days.
Microbial identication

Preliminary characterization of microbial species was based on the features of the colonies (i.e. size, color, shape, high, lip, surface, texture, consistency, brightness and hemolysis), visualized under an stereoscopic lens (Lambda Let 2, Atto instruments Co., Hong Kong). Isolates were then puried by subculture, gramstained, tested for catalase production, and their gaseous requirements established by incubation for 2 days aerobically and anaerobically. Based on this information it was possible to select appropriate procedures for identication of the species as follows:

Microbiology of symptomatic and asymptomatic teeth  Rapid ID 32A (BioMe rieux SA, Marcyl'Etoile, France) for strict anaerobic, gram-negative or gram-positive rods.  RapID ANA II System (Innovative Diagnostic Systems Inc., Atlanta, GA) for gram-positive cocci and gram-positive or gram-negative rods, strictly anaerobic.  API Staph (BioMe rieux SA) for staphylococci and micrococci (gram-positive cocci, catalase-positive).  Rapid ID 32 Strep (BioMe rieux SA) for streptococci (gram-positive cocci, catalase-negative).  RapID NH System (Innovative Diagnostic Systems Inc.) for Eikenella, Haemophilus, Neisseria and Actinobacillus.  API C Aux (BioMe rieux SA) for yeast.  Three complementary tests were also used to ensure the identication of bacteria from the genus Prevotella and Porphyromonas: 1. uorescence under long-wave (366 nm) UV light Black-Ray lamp, model UVL-56 (UVP Inc, San Gabriel CA); 2. lactose fermentation by the application of the uorogenic substrate 4methylumbelliferyl-b-galactoside (Sigma Chemical Co. St Louis, MO, M1633), according to Alcoforado et al. (2); 3. trypsin-like activity by the application of the synthetic uorogenic peptide Larginin-7-amino-4-methylocoumarin amido-HCl (C9396) (23, 33). The reference strains included were: Porphyromonas gingivalis ATCC 33277; Porphyromonas endodontalis ATCC 35406; Porphyromonas asaccharolytica ATCC 25260; Prevotella intermedia ATCC 25611; Prevotella nigrescens ATCC 33536; Prevotella melaninogenica ATCC 25845; Prevotella loescheii ATCC 15930; Prevotella corporis ATCC 33457 and Prevotella denticola ATCC 33185.
Antimicrobial susceptibility testing

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The E-test was evaluated by using prereduced plates containing 5% debrinated sheep blood Brucella broth agar enriched with 1 ml/l of hemin and 1 ml/l of vitamin K1. The inoculum was approximately 3 108 cfu/ml (McFarland 1) and it was spread over the agar plate with a sterile cotton swab, using a fresh swab for each plate. One strip of each antibiotic was then applied to each plate, after allowing the agar surface to dry for 10 min. The plates were then immediately incubated in an anaerobic chamber (Don Whitley Scientic). The elliptic zone of inhibition was examined after 24 h and 48 h of incubation if sufcient growth had been obtained and the ellipse was clearly visible. For slow growers, reading of the elliptic zone was performed after 48 h and 72 h of incubation. The reading at the intersection of the bacterial zone of inhibition and the E-strip represented the minimal inhibitory concentration (MIC) of the organism. The breakpoints used for interpretation were generally those recommended by the NCCLS (25), as follows: for amoxicillin <4 mg/ml, for amoxicillin clavulanate <2 mg/ml, for clindamycin <2 mg/ml, for metronidazole <8 mg/ml, for benzylpenicillin <0.5 mg/ml, for erythromycin <4 mg/ ml and for cephaclor <16 mg/ml. The breakpoints of azythromycin and erythromycin against strict anaerobes have not as yet been determined by the NCCLS (25). The breakpoint for erythromycin, in this study, was determined to be <4 mg/ml according to Kuriyama et al. (16).
Statistical analysis

Fig. 1. Prevalence of obligate anaerobes, facultative anaerobes, gram-positives and gramnegative bacteria in symptomatic and asymptomatic teeth from 48 root canals.

The antimicrobial susceptibility test was performed using the E-test method (AB Biodisk). The antibiotics used were amoxicillin, amoxicillin clavulanate, clindamycin, metronidazole, benzylpenicillin, erythromycin, cephaclor and azythromycin. These antibiotics were tested against the strains of Fusobacterium necrophorum, Fusobacterium nucleatum, Peptostreptococcus micros, Peptostreptococcus prevotii, and all ``black-pigmented bacteria'' as a group, especially P. intermedia/nigrescens, isolated from the 29 root canals investigated in this study. These anaerobic microorganisms were those most commonly found in the root canals of symptomatic teeth.

The data collected were typed onto a spreadsheet (QUATTRO PRO, Borland International Inc., Scotts Valley, CA) and statistically analyzed using SPSS for Windows (SPSS Inc., Chicago, IL). Either a Pearson Chi-squared test or a one-sided Fisher's exact test, as appropriate, was chosen to test the null hypothesis that there is no relationship between endodontic symptoms and signs, and the presence of specic bacterial species.
Results

In all, 48 root canals were examined, 29 from patients with spontaneous pain and 19 that were painless at the time the sample was taken. The total number of cultivable isolates recovered was 218, 150 in symptomatic teeth and 68 in painless teeth, belonging to 48 different microbial species and to 19 different genera. The total number of microbes in the samples ranged from

3.8 104 to 2.5 105 cfu (colony former unit) the mean cfu was 2 105. Individual root canals yielded a maximum of nine bacterial species. Only one root canal had no cultivable bacteria; this canal was painless, without tenderness to percussion, pain on palpation or history of previous pain and was caries-free. Obligate anaerobes accounted for 74.77% of total species isolation (Fig. 1) and were present in 45 root canals, 72.4% of them in painful teeth and 27.6% in the spontaneous pain-free teeth. There was a statistically signicant difference between obligate anaerobes and spontaneous pain and tenderness to percussion (both P < 0.01). Only in one root canal related to pain was no obligate anaerobe found. The most commonly anaerobes isolated were: F. necrophorum (6.9%), P. prevotii (i.e. Anaerococcus prevotii) (6.4%), P. micros (6.0%), F. nucleatum (5.0%) and P. intermedia/nigrescens (4.6%) (Table 1). Gram-negative bacteria accounted for 39% of total species isolations (Fig. 1) and they were associated with spontaneous pain, previous episodes of pain, pain on palpation and swelling (all P < 0.05). ``Black-pigmented bacteria'' were found in 41.7% of the root canals, 75% of these isolated from teeth with spontaneous pain and 20% from teeth painless at the moment of the sample teeth but with previous pain. ``Black-pigmented bacteria'' comprised the species P. intermedia/nigrescens, P. corporis, P. gingivalis, P, loescheii, P. denticola and P. melaninogenica (Table 1). Associations were found between spontaneous pain (n 29) and the following bacteria: Prevotella spp. and Bacteroides spp. (both P < 0.05). Previous episodes of pain were reported by 36 patients and were associated with P. loescheii and Peptostreptococcus spp., (especially P. prevotii, i.e. Anaerococcus prevotii) (both P < 0.05).

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Jacinto et al. P. intermedia/nigrescens group, n 20), P. micros (n 10) and P. prevotii (i.e. Anaerococcus prevotii, n 9) are shown in Tables 27, respectively. The E-test results after 24-h incubation were considered readable or interpretable for 80% of the F. necrophorum, for 67% of the F. nucleatum, for 90% of the P. micros, for 88% of the P. prevotii and for 20% of the ``black-pigmented bacteria''. Nevertheless, after 48 h, they were readable for 90% of the total isolates tested. The values of MIC50 and MIC90 refer to the minimal inhibitory concentration that was effective against 50% and 90% of the tested strains. Tables 27 also show the range of MIC for each antibiotic against the strains tested, as well as the susceptibility rate of the strains against each antibiotic according to the susceptibility breakpoints previously determined by the NCCLS criteria (25). As the breakpoint of azythromycin against strict anaerobes has not been determined by the NCCLS, it was impossible to analyze the susceptibility rate of the strains against this antibiotic. Nevertheless, we can report that the MIC of azythromycin for 60% of F. necrophorum was <0.75 mg/ml, for 30% it was <4 mg/ml and for 10% it was 8 mg/ml. The MIC of azythromycin for 90% of the Fusobacterium nucleatum was <8 mg/ml while for one strain of F. nucleatum it was 16 mg/ml. The MIC of azythromycin for 85% of ``blackpigmented bacteria'' was <0.5 mg/ml, for 10% it was <2 mg/ml and for 5% it was 12 mg/ml. The MIC of azythromycin for 87% of P. intermedia/nigrescens was <0.5 mg/ml and for 13% it was 12 mg/ml. The MIC of azythromycin for 90% of the P. prevotii and for 100% of the P. micros was <1.0 mg/ml.
Discussion

Table 1. Prevalence of bacteria found in 48 root canals (``black-pigmented bacteria'' are in boldface) Species Fusobacterium necrophorum Anaerococcus prevotii (P. prevotii) Peptostreptococcus micros Streptococcus sanguis Fusobacterium nucleatum Prevotella intermedia/nigrescens Gemella morbillorum Veillonella spp. Streptococcus constellatus Actinomyces odontolyticus Streptococcus mitis Prevotella corporis Prevotella oralis Eggerthella lenta (E. lentum) Clostridium spp. Peptostreptococcus anaerobius Campylobacter gracilis (B. gracilis) Bifidobacterium adolescentis Enterococcus faecalis Actinomyces meyeri Bacteroides fragilis Tissierela praecuta Streptococcus anginosus Prevotella loescheii Prevotella melaninogenica Propionibacterium acnes Clostridium acetobutylicum Eubacterium limosum Lactobacillus acidophilus Staphylococcus saccharolyticus Porphyromonas gingivalis Streptococcus oralis Staphylococcus epidermides Prevotella denticola Bifidobacterium spp. Prevotella buccae Actinomyces viscosus Symptomatic 10 9 10 4 9 8 8 7 5 5 3 5 5 5 4 4 4 3 2 1 3 3 1 2 2 1 1 1 1 1 2 2 2 2 2 2 1 Asymptomatic 5 5 3 7 2 2 2 3 4 2 3 1 1 1 1 1 0 1 1 2 0 0 2 1 0 1 1 1 1 1 2 2 2 1 1 0 1 Total 15 14 13 11 11 10 10 10 9 7 6 6 6 6 5 5 4 4 3 3 3 3 3 3 2 2 2 2 2 2 4 4 4 3 3 2 2

The following species were isolated in only one instance: Staphylococcus aureus, Haemophylus parainfluenzae, Streptococcus mutans, Bifidobacterium breve, Peptostreptococcus saccharolyticus, Neisseria spp., Staphylococcus lentus, Gemella haemolysans, Actinomyces israelii, Clostridium butirycum, Fusobacterium varium, Prevotella bivia, Clostridium hastiforme, Enterococcus faecium.

Tenderness to percussion (n 34) was related to Bidobacterium spp., Actinomyces spp. and S. constellatus (all P < 0.05). Relationship was found between pain on palpation (n 22) and the following bacteria: Porphyromonas gingivalis and Peptostreptococcus spp. (both P < 0.05). Swelling (n 20) was statistically related to Fusobacterium spp. (especially

F. nucleatum) (both P < 0.05). Wet canal (n 29) was not associated with any specic bacteria; however anaerobic bacteria were found in all of them. The results of the antimicrobial susceptibility testing against F. necrophorum (n 10), F. nucleatum (n 9), P. intermedia/nigrescens n 8), all ``black-pigmented bacteria'' strains isolated (including the

Table 2. Antimicrobial susceptibility of Fusobacterium necrophorum strains isolated from 30 root canals of symptomatic teeth to eight antibiotics Fusobacterium necrophorum (n 10) MIC (mg/ml) Antibiotics Benzylpenicillin Amoxicillin clavunate Amoxicillin Clindamycin Metronidazole Erythromycin Cephaclor Azythromycin

50% 0.016 0.016 0.023 0.023 0.032 0.38 0.094 0.32

90% 0.38 0.75 1 4 0.047 3 0.75 4

Range of MIC (mg/ml) 0.0163 0.0161.5 0.0161.5 0.01616 0.01664 0.0328 0.0162 0.0168

Susceptibility rate (%) 90% 100% 100% 90% 90% 80% 100%

50% and 90% at which 50% and 90% of the isolates are inhibited, respectively.

The development of endodontically related pain and swelling has some etiologic factors such as alteration of the local adaptation syndrome, which can also be caused by endodontic therapy, changes in the periapical tissue pressure, microbial factors, effect of chemical mediators, changes in cyclic nucleotides, immunologic phenomena and various psychological factors (30). Microbial infection is one of the most signicant (9). During this microbiological analysis, cultivable microorganisms were found in all cases, except in one, which corresponded to an asymptomatic caries-free tooth. The prevalence of four to six microorganisms in necrotic root canals was found, supporting the polymicrobial fea-

Microbiology of symptomatic and asymptomatic teeth

Table 3. Antimicrobial susceptibility of Fusobacterium nucleatum strains isolated from 29 root canals of symptomatic teeth to eight antibiotics Fusobacterium nucleatum (n 9) MIC (mg/ml) Antibiotics Benzylpenicillin Amoxicillin clavunate Amoxicillin Clindamycin Metronidazole Erythromycin Cephaclor Azythromycin

289

50% 0.023 0.023 0.047 0.047 0.016 0.19 0.38 1.5

90% 0.064 0.094 0.19 0.094 0.016 32.0 0.5 8.0

Range of MIC 0.0160.19 0.0160.19 0.0160.25 0.0160.25 0.0160.023 0.01632.0 0.0160.75 0.01616.0

Susceptibility rate 100% 100% 100% 100% 100% 55.6% 100%

50% and 90% at which 50% and 90% of the isolates are inhibited, respectively.

Table 4. Antimicrobial susceptibility of Prevotella intermedia/nigrecens strains isolated from 29 root canals of symptomatic teeth to eight antibiotics Prevotella intermedia/nigrescens (n 8) MIC (mg/ml) Antibiotics Benzylpenicillin Amoxicillin clavunate Amoxicillin Clindamycin Metronidazole Erythromycin Cephaclor Azythromycin

50% 1.5 0.016 1.5 0.19 0.016 0.047 0.25 0.125

90% 3 0.75 3 0.38 16 1.5 2 12

Range of MIC 0.0163 0.0160.75 0.0163 0.0164 0.01616 0.0161.5 0.0322 0.01612

Susceptibility rate (%) 62.5% 100% 100% 87.5% 87.5% 100% 100%

50% and 90% at which 50% and 90% of the isolates are inhibited, respectively.

Table 5. Antimicrobial susceptibility of ``black- pigmented bacteria'' strains isolated from 29 root canals of symptomatic teeth to eight antibiotics ``Black-pigmented bacteria'' (n 20) MIC (mg/ml) Antibiotics Benzylpenicillin Amoxicillin clavunate Amoxicillin Clindamycin Metronidazole Erythromycin Cephaclor Azythromycin

50% 0.023 0.016 0.047 0.016 0.016 0.047 0.094 0.25

90% 1.5 0.032 1.5 0.19 0.25 0.75 0.25 1

Range of MIC 0.0163 0.0160.75 0.0163 0.0164 0.016-16 0.0161.5 0.0162 0.01612

Susceptibility rate (%) 85% 100% 100% 95% 95% 100% 100%

50% and 90% at which 50% and 90% of the isolates are inhibited, respectively.

Table 6. Antimicrobial susceptibility of Peptostreptococcus micros strains isolated from 29 root canals of symptomatic teeth to eight antibiotics Peptostreptococcus micros (n 10) MIC (mg/ml) Antibiotics Benzylpenicillin Amoxicillin clavunate Amoxicillin Clindamycin Metronidazole Erythromycin Cephaclor Azythromycin

50% 0.016 0.064 0.047 0.125 0.125 0.047 0.5 0.75

90% 0.023 0.19 0.064 0.75 0.19 1.5 2.0 1.0

Range of MIC 0.0160.38 0.0160.19 0.0160.064 0.0161.5 0.0163.0 0.0161.5 0.0322 0.0161.5

Susceptibility rate 100% 100% 100% 100% 100% 100% 100%

50% and 90% at which 50% and 90% of the isolates are inhibited, respectively.

ture of root canal with primary infection (4, 9, 10, 26, 42). Among the bacterial species isolated in this experiment, obligate anaerobes predominated, accounting for over 74% of the total species isolated. The number of facultative anaerobes was slightly higher than obligate anaerobes in asymptomatic teeth, while in symptomatic teeth the obligate anaerobes outnumbered facultative anaerobes. Obligate anaerobes were statistically related to pain, tenderness to percussion and abscess, especially the gram-negative ones, corroborating the ndings of Gomes et al. (9, 10), Griffee et al. (11), Sundqvist et al. (37) and Yoshida et al. (42). Spontaneous or previous pain was associated with Prevotella spp. (especially P. intermedia), Fusobacterium spp. and Peptostreptococcus spp. Hashioka et al. (14) and Gomes et al. (9, 10) also reported the role of certain bacteria in the development of endodontic symptomatology. Percussion may determine the presence of pathology in the periapical tissues, a positive result indicating inammation in the periodontal ligament (9). In this study, Bidobacterium spp., Actinomyces spp. and S. constellatus were isolated more frequently in cases with pain to percussion. Despite the high incidence of Peptostreptococcus, Porphyromonas and Bacteroides, Hashioka et al. (14) did not associate specic bacteria with tenderness to percussion. Swelling or any soft tissue enlargement related to the tooth is frequently a clinical sign of an acute process in the periapical region, generally associated with the presence of microorganisms. Swelling was statistically related to P. gingivalis, Peptostreptococcus spp., (especially P. micros), F. nucleatum and F. necrophorum,. However, Makkar et al. (21) did not nd a relationship between swelling and Porphyromonas spp. in 90 non-vital root canals analyzed. On the other hand, Gomes et al. found a positive association between swelling and Peptostreptococcus spp. (9), P. micros, Eubacterium spp. and Prevotella spp. (10). Griffee et al. (11) related a possible association between pain on palpation, an important examination for dening the diagnosis of periapical abscess, and the presence of ``black-pigmented bacteria''. In this study, a relationship was found between pain on palpation and P. gingivalis, which is also a ``black-pigmented bacteria'', and Peptostreptococcus spp. Wet canals refers to the presence of a clear, hemorrhagic or purulent exudate, detected as a distinct dampening or stain on the sampling paper points. Our ndings

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Jacinto et al. penicillin as a result of b-lactamase production. On the other hand, in our study most strains presented a negative b-lactamase production. Kuriyama et al. (16) found that, despite the resistance rate, more than 70% of ``black-pigmented bacteria'' were susceptible to benzylpenicillin, agreeing with our ndings. F. necrophorum showed a susceptibility rate of 90% against benzylpenicillin, agreeing with Kuriyama et al. (16) despite a MIC90 of 1 mg/ml benzylpenicillin in that study. Erythromycin and clindamycin have been prescribed to patients who are allergic to penicillin. However, it has been noted that erythromycin is not effective against Fusobacterium spp. (16, 17). In this study, 55.6% of the F. nucleatum and 80% of F. necrophorum were susceptible to erythromycin. For strains of P. micros, P. prevotii and ``black-pigmented bacteria'', the susceptibility rate to erythromycin was 100%, 90% and 100%, respectively. Kuriyama et al. found that 100% of ``blackpigmented bacteria'' (16) and 96% of the Peptostreptococcus spp. (18) were susceptible to erythromycin. Clindamycin has been noted to be totally effective against obligate anaerobes, particularly against ``black-pigmented bacteria'' (16, 22, 34). In the present study, 90% of F. necrophorum, 100% of the F. nucleatum, 95% of ``black-pigmented bacteria'', 87.5% of P. intermedia/nigrescens 100% of the P. micros and 100% of the P. prevotii were susceptible to clindamycin. Metronidazole, as well as clindamycin, has demonstrated a strong antimicrobial power against anaerobic gram-negative rods (17, 34). In the present study, metronidazale showed results similar to clindamycin for all the strains tested, although the MIC90 for P. intermedia/nigrescens against metronidazole was extremely high (16 mg/ml), disagreeing with other studies (17, 27, 34), where 100% of P. intermedia/nigrescens were susceptible to this agent. All obligate anaerobes tested were susceptible to amoxicillin, amoxicillin clavulanate and cephaclor, which was also achieved by other authors (16, 17, 19). In spite of having few side effects, a broad antimicrobial spectrum and strong bactericidal activity, cephalosporin should not be prescribed for patients who have immediate hypersensitivity reactions to penicillin, as some of these patients may also be allergic to several other b-lactam antibiotics (16). Clindamycin and metronidazole may be recommended for patients whose antimi-

Table 7. Antimicrobial susceptibility of Peptostreptococcus prevotii strains isolated from 29 root canals of symptomatic teeth to eight antibiotics Peptostreptococcus prevotii (n 9) MIC (mg/ml) Antibiotics Benzylpenicillin Amoxicillin clavunate Amoxicillin Clindamycin Metronidazole Erythromycin Cephaclor Azythromycin

50% 0.016 0.064 0.032 0.016 0.016 0.016 0.047 0.023

90% 0.016 0.19 0.25 0.125 1.0 1.0 1.0 1.5

Range of MIC 0.0160.38 0.0161.0 0.0160.5 0.0160.125 0.0161.0 0.01664.0 0.0161.0 0.0168

Susceptibility rate 100% 100% 100% 100% 100% 90% 100%

50% and 90% at which 50% and 90% of the isolates are inhibited, respectively.

in these sorts of canals are in agreement with other studies (9, 10). ``Black-pigmented'' anaerobic rods (Porphyromonas spp. and some species of the genus Prevotella) have been strongly implicated in the etiology of infected dental root canals and apical periodontitis (4, 8, 26, 37, 40). It is probable that the proteolytic activity of ``black-pigmented bacteria'' is a highly signicant virulence factor because proteinases from these microorganisms have effects on plasma proteins involved in the defense processes (37). ``Black-pigmented bacteria'' were found in 34% of the root canals. Sundqvist (37) and Baumgartner et al. (4) found 30% of ``black-pigmented bacteria'' in necrotic root canals, Dougherty et al. (8) and Siqueira Jr et al. (31) found 67% and 59.3% of ``black-pigmented bacteria'', respectively. Nevertheless, we found ``black-pigmented bacteria'' in 50% of the painful teeth and in 16.7% of the pain-free teeth, and they were related to pain, pain on palpation and tenderness to percussion. ``Black-pigmented bacteria'' were alwaysassociatedwithotherbacteria,conrming the synergetic relationships between the bacteria found in polymicrobial infections, especially with the gram-positive microorganisms. ``Black-pigmented bacteria'' need very specic nutritional requirements that are made available by some specic bacteria such as P. micros, Eubacterium spp. and Campylobacter rectus (10, 35). The anaerobic strains, F. necrophorum, F. nucleatum, P. prevotii (i.e. Anaerococcus prevotii), P. micros, F. nucleatum, P. intermedia/nigrescens and the ``blackpigmented bacteria'' (which also included P. intermedia/nigrescens) that were isolated from symptomatic teeth in this study had their antimicrobial susceptibility tested to some antibiotics commonly used in endodontic treatment. F. necrophorum and F. nucleatum are species commonly

isolated from root canal abscesses (9, 10, 39), P. micros and ``black-pigmented bacteria'' have been associated with pain of endodontic origin (10, 14). The susceptibility of ``black-pigmented bacteria'' as a group was investigated because, apart from P. intermedia/nigrescens, the number of strains of other species was not signicantly enough to be analyzed separately. As a general rule, 24-h incubation is insufcient to obtain readable and/or accurate E-test results for the majority of obligate anaerobes. Previous studies related readable E-test results for 64% of anaerobes after 24-h incubation, and for 95% of the same anaerobic strains after 48h incubation (28) as conrmed in this study. The susceptibility breakpoints determined by NCCLS criteria (25) have been used in several bacterial studies (5, 1618, 22, 27, 28, 34) and were also used here. Nevertheless, NCCLS breakpoints may be too strict for some antibiotics because the breakpoints are below the typical serum or tissue concentrations of the antibiotics. The bacterial strains resistant to certain antibiotics according to the present criteria may be clinically susceptible to those antibiotics because they may be affected by other factors, such as infection site or dosage (16). Penicillin has traditionally been recommended as a rst-line antibiotic because it works well against most causative bacteria and has a low incidence of side effects. On the other hand, it has been suggested that the antimicrobial activity of penicillin has decreased against anaerobic bacteria related to endodontic infections (16). Dahle n et al. (6) have found most strains of P. intermedia/nigrescens to be sensitive to benzylpenicillin. However, in this study just 62.5% of P. intermedia/nigrescens were susceptible to benzylpenicillin. Appelbaum et al. (3) found that 52% of P. intermedia/nigrescens were resistant to

Microbiology of symptomatic and asymptomatic teeth crobial therapy with penicillin or amoxicillin, rst choice antibiotics, has failed (17, 19). Amoxicillin clavulanate can also be a good choice as antimicrobial therapy (5). On the other hand, the high cost of amoxicillin clavulanate is a major consideration, unless rst line treatment is not effective against an acute infection (19). The present study provided evidence that specic bacteria, mainly gram-negative anaerobic bacteria, are associated with endodontic signs and symptoms such as pain, previous episodes of pain, pain on palpation, tenderness to percussion, abscess and swelling of infected teeth with periapical pathology. However, the role of the gram-positive bacteria in inducing acute periapical inammatory reaction cannot be forgotten, as they can increase the pathogenicity of the gram-negative microorganisms (7). It also appears that certain bacteria isolated from symptomatic teeth are still susceptible to the most clinically used antibiotics. Furthermore, concerns about antibiotic resistance, at least for the species isolated in this study, are somewhat premature, although the resistance of P. intermedia/nigrescens tested against benzylpenicillin should be taken into account. Studies in which a greater number of species are tested are therefore required. Moreover, such studies should also consider the development of resistant bacteria according to the geographic areas. Usually, different methodologies bring different results. Nowadays, molecular assays have identied species from the infected root canals that are hard to culture, such as B. forsythus and T. denticola (32). Such works did not nd any correlation between clinical features and specic bacteria. However, it cannot be forgotten that the methodologies used by the above-mentioned authors determine the presence of the microorganism, but not the total number of bacteria collected in a sample. Griffen et al. (12) found higher levels of P. gingivalis in samples from subjects with periodontitis than in sites without the evidence of disease using the Real-Time PCR, a method that offers the ability to determine the absolute and relative amounts of a specic microorganism in a mixed sample without the need to culture it. This points to the importance of considering, when using molecular assays, not only the presence of an specic microorganism but also the number of strains in the sample, in order to associate it with the signs and symptoms. In culture methods, a microorganism, when present, cannot be detected if it is not present in a high enough concentration to grow on the culture medium. This may account for the association of specic microorganisms with endodontic signs and symptoms, when such assay methods are used.
Acknowledgments

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This research was supported by Brazilian grants from FAPESP (no. 2000/13689-7), CNPq (520277/99-6) and CAPES.
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