Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization

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1 Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization José F. Siqueira, Jr, DDS, MSc, PhD, a Isabela N. Rôças, DDS, b Renata Souto, BMic, b Milton de Uzeda, DDS, MSc, PhD, c and Ana Paula Colombo, DDS, PhD, a Rio de Janeiro, Brazil FEDERAL UNIVERSITY OF RIO DE JANEIRO AND STATE UNIVERSITY OF RIO DE JANEIRO Objective. The purpose of the present investigation was to examine the microbiota of acute periradicular abscesses of endodontic origin by using a molecular genetic method. Study design. Pus was collected by aspiration from 27 cases diagnosed as acute abscesses of endodontic origin, and DNA was extracted to evaluate the occurrence of 49 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The presence of bacterial DNA in clinical samples was confirmed by polymerase chain reaction with ubiquitous bacterial 16S rrna gene primers. Results. The results of the checkerboard DNA-DNA hybridization analysis revealed that 37 of the 49 DNA probes tested were reactive with one or more samples. The number of bacterial species in the pus samples ranged from 1 to 33 (mean, 5.9). Eighteen of the 27 pus samples were positive for at least one DNA probe. The most prevalent species found were: Bacteroides forsythus (29.6% of the cases); Porphyromonas gingivalis (29.6%); Streptococcus constellatus (25.9%), Prevotella intermedia (22.2%), Prevotella nigrescens (22.2%), Fusobacterium periodonticum (18.5%), Fusobacterium nucleatum subspecies nucleatum (18.5%), and Eikenella corrodens (18.5%). Conclusions. The microbiologic data of the present investigation indicated that molecular genetic methods could provide additional knowledge regarding the microbiota of acute periradicular abscesses by detecting bacterial species that are difficult or even impossible to grow. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:451-7) The egress of microorganisms and their products from the root canal system into the periradicular tissues can cause tissue damage and precipitate periradicular inflammation. 1,2 The severity of the tissue injury will depend on the number and the virulence of microorganisms that gain access to the periradicular tissues. The intensity of the host response is usually directly proportional to the intensity of the injury. An acute periradicular abscess can develop as a result of the excessive and sudden microbial invasion of periradicular tissues. Pus formation results from the release of oxygen-derived free radicals, such as superoxide and hydrogen peroxide, and lysosomal enzymes by polymorphonuclear neutrophils, such as elastase, collagenase, and gelatinase, which are the major mechanisms involved in the destruction of the connective tissue extracellular matrix. 1 Acute periradicular abscesses are clinically characterized by pain or swelling, or both, a Associate Researcher, Oral Microbiology Laboratory, Institute of Microbiology, Federal University of Rio de Janeiro, Brazil. b Postgraduate Student in Microbiology, Department of Microbiology, State University of Rio de Janeiro, Brazil. c Chairman, Institute of Microbiology, Federal University of Rio de Janeiro, Brazil. Received for publication Mar 7, 2001; returned for revision May 30, 2001; accepted for publication Jul 2, Copyright 2001 by Mosby, Inc /2001/$ /15/ doi: /moe and have the potential to spread to sinuses and other facial spaces of the head and neck. Several cultural studies have revealed that the microbiota associated with acute periradicular abscesses are usually polymicrobial, with a mean number of species ranging from <3 to 8.5 per specimen Obligate anaerobic bacteria are the most common isolates, including members of the genera Porphyromonas, Prevotella, Fusobacterium, Peptostreptococcus, and Eubacterium. In addition, some facultative bacteria, such as streptococci, have also been frequently isolated from acute abscesses of endodontic origin. A new era has been announced for diagnostic microbiology. Methodology that is based on nucleic acid detection has been introduced in both research and clinical laboratories and is revolutionizing the knowledge about several infectious diseases and allowing effective and rapid diagnosis of several diseases. 11,12 Recently, a method was introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single support membrane the checkerboard DNA-DNA hybridization. 13 It permits the simultaneous determination of the presence of a multitude of bacterial species in single or multiple clinical samples. The method does not require bacterial viability and is particularly applicable in epidemiologic research. Various studies using this molecular method have considerably enhanced the understanding of the microbiology of periodontal diseases

2 452 Siqueira et al ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY October 2001 Table I. Strains used for the preparation of DNA probes Acinetobacter baumannii ci Leptotrichia buccalis ATCC Actinobacillus actinomycetemcomitans ATCC Neisseria mucosa ATCC Actinomyces gerencseriae ATCC Peptostreptococcus anaerobius ATCC Actinomyces israelli ATCC Peptostreptococcus micros ATCC Actinomyces naeslundii genospecies 1 ATCC Porphyromonas endodontalis ATCC Actinomyces naeslundii genospecies 2 ATCC Porphyromonas gingivalis ATCC Actinomyces odontolyticus ATCC Prevotella intermedia ATCC Bacteroides forsythus ATCC Prevotella melaninogenica ATCC Bartonella species ci Prevotella nigrescens ATCC Bifidobacterium dentium ci Propionibacterium acnes ATCC Campylobacter rectus ATCC Pseudomonas aeruginosa ATCC Campylobacter showae ATCC Ralstonia species ci Capnocytophaga gingivalis ATCC Selenomonas noxia ATCC Capnocytophaga sputigena ATCC Staphylococcus aureus ci Corynebacterium matruchotii ATCC Streptococcus anginosus ATCC Eikenella corrodens ATCC Streptococcus constellatus ATCC Enterococcus faecalis ci Streptococcus gordonii ATCC Escherichia coli ATCC Streptococcus intermedius ATCC Eubacterium nodatum ATCC Streptococcus mitis ATCC Fusobacterium nucleatum subspecies nucleatum ATCC Streptococcus oralis ATCC Fusobacterium nucleatum subspecies polymorphum ATCC Streptococcus sanguis ATCC Fusobacterium periodonticum ATCC Treponema denticola B1 (FDC) Gemella morbillorum ATCC Treponema socranskii S1 (FDC) Haemophilus aphrophilus ci Veillonella parvula ATCC Lactobacillus oris ATCC ci, Clinical isolate; ATCC, American Type Culture Collection; FDC, Forsyth Dental Center. The purpose of this study was to investigate the microbiota associated with acute periradicular abscesses of endodontic origin using the checkerboard DNA-DNA hybridization assay. MATERIAL AND METHODS Specimen sampling The examined material was selected from adult patients who had been referred for emergency treatment to 3 hospitals in Rio de Janeiro, Brazil. Samples were collected from 27 teeth, all of them having carious lesions, necrotic pulps, and radiographic evidence of periradicular bone loss. Pain and swelling were present in all cases, which were diagnosed as acute periradicular abscesses according to criteria established by Torabinejad and Walton. 2 Three patients reported fever; 2 made use of antibiotics. Patients ages ranged from 18 to 60 years. Samples were collected by using strict asepsis. After disinfection of the oral mucosa with 2% chlorhexidine digluconate, pus from the abscessed teeth was collected by aspiration with a sterile syringe, transferred to cryotubes containing 1 ml of 5% dimethyl sulfoxide in trypticase soy broth (Difco, Detroit, Mich, USA) and immediately frozen at 20 C. DNA extraction and preparation of DNA probes Bacterial strains from lyophilized stocks (Table I) were grown anaerobically on the surface of blood agar plates for 3 to 7 days. The growth was harvested and placed in 1.5-mL microcentrifuge tubes containing 1 ml of TE buffer (10 mmol/l Tris-HCl, 0.1 mmol/l EDTA, ph 7.6). Cells were washed 2 times by centrifugation in TE buffer at 2500 g for 10 minutes. The cells were resuspended and lysed with either 10% SDS and Proteinase K (20 mg/ml; Sigma Chemical Co, St Louis, Mo, USA) for gram-negative strains or in 150 µl of an enzyme mixture containing 15 mg/ml of lysozyme (Sigma) and 5 mg/ml of achromopeptidase (Sigma) in TE buffer (ph 8.0) for gram-positive strains. The pelleted cells were resuspended by 15- second sonication and incubated at 37 C for 1 hour. DNA was isolated and purified by using the method described by Smith et al. 17 The concentration of the purified DNA was determined by spectrophotometric measurement of the absorbance at 260 nm. The purity of the preparations was assessed by the ratio of DNA to protein as measured by the ratio of the absorbances at 260 nm and 280 nm. Whole-genomic DNA probes were prepared from each of the test species by labeling 1 µg of DNA with digoxigenin (Boehringer Manheim, Indianapolis, Ind, USA) using a random primer technique. 18 Clinical samples were thawed to 37 C for 10 minutes and vortexed for 30 seconds. Microbial suspension was washed 3 times with 100 µl of bidistilled water by centrifugation for 2 minutes at 2500 g. Pellets were then resuspended in 100 µl of bidistilled water, boiled

3 ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Siqueira et al 453 Volume 92, Number 4 for 10 minutes, and chilled on ice. After centrifugation to remove cell debris for 10 seconds at 9000 g at 4 C, the supernatant was collected for testing. Microbiologic assessment The checkerboard DNA-DNA hybridization technique used was a modification 19 of that described by Socransky et al. 13 Briefly, denatured DNA from the clinical samples was fixed in individual lanes on a nylon membrane by using a Minislot (Immunetics, Cambridge, Mass, USA). A Miniblotter 45 apparatus was used to hybridize the digoxigenin-labeled whole chromosomal DNA probes (Table I) at 90 to the lanes of the clinical samples. After overnight hybridization at 42 C, membranes were washed at low and high stringency and bound probes were then detected with phosphatase-conjugated antibody to digoxigenin and with chemiluminescence. The sensitivity of this assay was adjusted to permit detection of 10 4 cells of a given species by adjusting the concentration of each DNA probe. This procedure was carried out to provide the same sensitivity of detection for each species. Amplification by the polymerase chain reaction To confirm the presence of bacterial DNA in pus samples, a polymerase chain reaction (PCR) was performed. A pair of ubiquitous primers (5 -GAT TAG ATA CCC TGG TAG TCC AC-3 and 5 -CCC GGG AAC GTA TTC ACC G-3 ; Oligos Etc Inc, Wilsonville, Ore, USA) that match almost all bacterial 16S rrna genes at the same position but not 18S rrna gene from eukaryotic cells was used to indicate the presence of bacteria in the clinical samples. 20 Aliquots of 5 µl of the supernatant from clinical samples were amplified. PCR was performed in 50 µl of reaction mixture containing 1 µl of each primer (40 pmol), 5 µl of 10X PCR buffer (Gibco BRL, Gaithersburg, Md, USA), 1.25 unit of Taq DNA polymerase (Gibco BRL), and 0.2 mmol/l of each deoxynucleoside triphosphate (datp, dctp, dgtp, and dttp; Gibco BRL). Earlier experiments found optimal MgCl 2 concentration in the mixture to be 2.0 mmol/l. Preparations were overlaid with 2 droplets of mineral oil and amplified in a DNA thermocycler (PTC-100; MJ Research, Inc, Watertown, Mass, USA). The PCR temperature profile included an initial denaturation step at 95 C for 2 minutes, followed by 36 cycles of a denaturation step at 95 C for 30 seconds, a primer annealing step at 60 C for 1 minute, an extension step at 72 C for 1 minute, and a final step of 72 C for 2 minutes. PCR products were analyzed by 1.5% agarose gel electrophoresis performed at 4 V/cm in Tris-borate EDTA buffer. The gel was stained with 0.5 µg/ml ethidium bromide and photographed under ultraviolet Table II. Prevalence of bacterial species in 27 abscess samples as detected by checkerboard DNA-DNA hybridization Number of Bacterial species samples % Bacteroides forsythus Porphyromonas gingivalis Streptococcus constellatus Prevotella intermedia Prevotella nigrescens Fusobacterium periodonticum Fusobacterium nucleatum subspecies nucleatum Eikenella corrodens Actinomyces gerencseriae Neisseria mucosa Porphyromonas endodontalis Gemella morbillorum Staphylococcus aureus Streptococcus gordonii Streptococcus intermedius Acinetobacter baumannii Actinomyces israelli Bifidobacterium dentium Corynebacterium matruchotii Campylobacter rectus Haemophilus aphrophilus Ralstonia species Streptococcus anginosus Streptococcus sanguis Veillonella parvula Actinomyces odontolyticus Actinobacillus actinomycetemcomitans Capnocytophaga gingivalis Enterococcus faecalis Escherichia coli Eubacterium nodatum Fusobacterium nucleatum subspecies polymorphum Peptostreptococcus micros Pseudomonas aeruginosa Selenomonas noxia Treponema denticola Treponema socranskii light. A 100 base-pair DNA ladder digest (Gibco BRL) served as the molecular weight marker. RESULTS All 27 aspirates of pus from periradicular abscesses contained bacteria as demonstrated by PCR using the ubiquitous bacterial primer pair. Only one band of the predicted size (602 base-pairs) was present for each root canal sample (data not shown). Such results indicated that bacteria were present in all cases sampled. The results of the checkerboard DNA-DNA hybridization analysis revealed that 37 of the 49 DNA probes tested were reactive with one or more pus samples. Eighteen of the 27 samples (66.7%) were positive for at least one DNA probe. One or more anaerobic bacterial species were found in 16 of the 18 positive cases. Of the

4 454 Siqueira et al ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY October 2001 Table III. Microbiologic findings and clinical information for the 18 patient cases with positive DNA probes Sample Clinical features Microbiologic findings 1 Pain, swelling C matruchotii, H aphrophilus, F nucleatum nucleatum, E corrodens, G morbillorum, E nodatum, S constellatus 2 Pain, swelling B forsythus, P gingivalis, C matruchotii, E faecalis, H aphrophilus, P intermedia, F nucleatum nucleatum, F nucleatum polymorphum, F periodonticum, C gingivalis, C rectus, Ralstonia species, E corrodens, G morbillorum, N mucosa, S anginosus, S intermedius, P aeruginosa, A actinomycetemcomitans, E coli, A baumanii, S constellatus, T denticola, T socranskii, B dentium, V parvula, S sanguis, A gerencseriae, A israelii, A odontolyticus, S gordonii, S noxia, S aureus 3 Pain, swelling P nigrescens 4 Pain, swelling B forsythus, P gingivalis, P endodontalis, P nigrescens, F periodonticum, E corrodens, S gordonii 5 Pain, swelling B forsythus, P gingivalis, P endodontalis, P intermedia, P nigrescens, F periodonticum, Fever E corrodens, A gerencseriae, S gordonii, N mucosa, S aureus Tetracycline 6 Pain, swelling A baumanii, P gingivalis, P intermedia, F periodonticum, A gerencseriae, S aureus Fever B forsythus, P gingivalis, P endodontalis, P intermedia, P nigrescens, F nucleatum nucleatum, Ampicillin C rectus, S constellatus, S intermedius 7 Pain, swelling 8 Pain, swelling N mucosa 9 Pain, swelling P gingivalis 10 Pain, swelling P nigrescens 11 Pain, swelling P gingivalis 12 Pain, swelling B forsythus, P gingivalis, P endodontalis, F nucleatum nucleatum, F periodonticum, P intermedia, P nigrescens, E corrodens, G morbillorum, Ralstonia species 13 Pain, swelling S constellatus, A gerencseriae 14 Pain, swelling B forsythus, S constellatus, B dentium, P micros, A israelii, S anginosus, S sanguis, S intermedius 15 Pain, swelling P intermedia, V parvula, N mucosa 16 Pain, swelling B forsythus, S constellatus, F nucleatum nucleatum 17 Pain, swelling B forsythus 18 Pain, swelling S constellatus target bacterial species, the most prevalent were as follow: Bacteroides forsythus (8 of 27 cases, 29.6%), Porphyromonas gingivalis (29.6%), Streptococcus constellatus (25.9%), Prevotella intermedia (22.2%), Prevotella nigrescens (22.2%), Fusobacterium periodonticum (18.5%), Fusobacterium nucleatum subspecies nucleatum (18.5%), and Eikenella corrodens (18.5%). Results are shown in Tables II and III. The number of bacterial species per pus sample ranged from 1 to 33 (mean, 5.9). Excluding a very discrepant case containing 33 bacterial species, the mean number of species per sample was 4.3 (range, 1-11). Mixed bacterial populations were observed in 11 of the 18 positive cases. DISCUSSION Culture procedures have traditionally been used as the reference standard in the assessment of the microbiota associated with various infectious diseases, including infections of endodontic origin. It has been revealed that the serial dilution anaerobic culture procedure of plaque samples recovered a relatively small percentage of the microscopic count obtained on the same sample. 16,21,22 Species identification is undertaken on agar plates from the highest sample dilution, which may not be adequately representative of the obtained sample. In addition to the shortcomings of dilution, nonviable, uncultivable, or fastidious bacteria contribute to the failure of a culture method. Loesche et al 22 compared the ability of several detection methods (ie, a serial dilution anaerobic culture, a microscopic procedure, a DNA probe procedure, and immunologic reagents using both ELISA and indirect immunofluorescence assay) to detect Treponema denticola, P gingivalis, B forsythus, and Actinobacillus actinomycetemcomitans in subgingival plaque samples. They defined as a true positive result the consensus of at least 3 or 4 of the detection methods. The culture procedure was the poorest detection method. They concluded that there are no gold standards for the detection of those 4 species in plaque samples. Papapanou et al 16 compared checkerboard DNA- DNA hybridization methodology with culture techniques for identification of subgingival microbiota. Their study demonstrated a reasonable degree of agreement between the techniques. They found that the checkerboard technology resulted in higher prevalence figures for half of the species tested when compared

5 ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Siqueira et al 455 Volume 92, Number 4 with culture data. In addition, the checkerboard method resulted in statistically significantly higher bacterial counts for the majority of the species. Conversely to the conventional culture procedures, molecular techniques do not depend on sampling under carefully controlled anaerobic conditions, do not require special transport media, and do not require cultivation of the isolates. These advantages ensure the detection of uncultivable or difficult-to-grow bacteria. DNA-DNA hybridization technology has the additional advantage that DNA is not amplified. 23 Thus, microbial contaminants are not cultured, nor is their DNA amplified. Because the numbers of contaminating microorganisms are not increased, one may assume that, if present, they would be in numbers below detection limits of the checkerboard DNA-DNA hybridization method, which was established at 10 4 bacterial cells. On the basis of these reports, one should bear in mind that, because of the absence of an indisputable reference standard, no definitive conclusions may be drawn with respect to the ability of a technique to better reflect reality. However, as stated by Papapanou et al, 16 because DNA-DNA hybridization usually detects target microbial species, the major advantage of the culture procedure is its capability to detect the unexpected. The sensitivity of the DNA-DNA hybridization assay used in this study was set to 10 4 cells of a bacterial species by adjusting the concentration of each DNA probe in the hybridization buffer. With regard to the specificity of the method, Socransky et al 14 reported that more than 92% of all test probes/heterologous species reactions did not exhibit cross-reactions under the conditions of the assay. Some probes as those to B forsythus, P gingivalis, and T denticola did not exhibit cross-reactions with any heterologous species tested. When cross-reactions were observed, they were always within genera and were quite limited. For example, cross-reactions within the genus Streptococcus were minimal, always exceeding 100:1 for homologous/heterologous species, although Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis showed no cross-reactions with Streptococcus intermedius and S constellatus. Mixed bacterial populations were observed in 11 of the 18 positive cases. Nonetheless, it does not necessarily mean that monoinfections occurred in the other 7 cases. Because the DNA-DNA hybridization method used in this study provides information regarding only the target bacterial species, it is possible that other non target species could have been present. In addition, the possibility that even some target species were present but not detected cannot be discarded, because negative results indicate that the target bacteria either was absent or was present in numbers below the threshold of detection. The same explanation is valid for the 9 cases that were negative for the 49 probes used but were positive in PCR for ubiquitous bacterial DNA. The number of bacterial species per purulent sample ranged from 1 to 33 (mean, 5.9). Excluding a very discrepant case containing 33 bacterial species, the mean number of species per sample was 4.3 (range, 1-11). This figure is comparable with those of other studies, which have observed a mean number of species ranging from 2.1 to 4.86, 3-8,10 except for the study of Sundqvist et al, 9 which reported a mean number of 8.5 species per sample. Although there is as yet no definitive documentation that a single microbial species is involved in the etiology of periradicular diseases, evidence suggests that acute periradicular abscesses may be associated with particular microbial species. 3-10,24-28 Blackpigmented anaerobic rods have been closely associated with acute symptoms of endodontic infections. 3,7,9,10,24-28 van Winkelhoff et al 28 found that all abscesses of endodontic origin examined harbored one or more species of the genera Prevotella and Porphyromonas. P intermedia were found in 63%, Porphyromonas endodontalis in 53%, and P gingivalis in 12% of the examined abscesses. Sundqvist et al 9 reported that one or more species of black-pigmented anaerobes were present in 94.1% of the cases diagnosed as acute periradicular abscesses. P intermedia were recovered from 58.8% of the cases, P endodontalis from 23.5%, and P gingivalis from 5.9%. Oliveira et al 29 detected P endodontalis in 66.7% of the abscessed cases by using a highly sensitive PCR assay. Siqueira et al 30 examined pus from acute periradicular abscesses through a 16S rrna based PCR and reported the occurrence of P endodontalis in 70% and P gingivalis in 40% of the cases. In the present study, one or more species of black-pigmented anaerobic rods were found in 11 cases (40.7%). P gingivalis was one of the most prevalent species present in 29.6% of the pus samples. In decreasing order of prevalence, the most-oftendetected black-pigmented bacteria were P intermedia and P nigrescens (both occurring in 22.2% of the cases), as well as P endodontalis (14.8%). Prevotella melaninogenica was not detected in any abscess sample. Although we found a relatively smaller prevalence when compared with most of the previous studies, our results confirmed the suggestion that black-pigmented bacteria may be involved in the pathogenesis of the acute periradicular abscess. Members of the Streptococcus anginosus group and Fusobacterium species were also among the mostoften-detected bacteria in the abscessed cases examined herein. These findings are in agreement with those

6 456 Siqueira et al ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY October 2001 of previous studies. 3-5,8,10,31,32 Fisher and Russell 33 investigated 45 samples from periradicular abscesses. Members of the S anginosus group were isolated from 16 patients (37%), with 15 being identified as S anginosus and 1 as S intermedius. Schuman and Turner 24 cultured the aspirates from 50 patients with oral abscesses and observed that streptococci of the anginosus group were recovered from 22% of the abscesses. According to them, patients with oral abscesses infected with these streptococci are likely to have more rapid onset of infection. Heimdahl et al 32 reported that the occurrence of F nucleatum (56% of the cases) appeared to be associated with the severity of acute orofacial infections of odontogenic origin. Whereas F nucleatum has frequently been found in infections of endodontic origin, F periodonticum had rarely or never been detected in such infections. We detected each of these species in 18.5% of the abscessed cases examined. Because studies have shown that strains of streptococci of the anginosus group and F nucleatum can induce abscesses in experimental animals when in pure and/or in mixed cultures, they might be involved in the pathogenesis of acute periradicular infections The role of F periodonticum in the pathogenesis of acute periradicular lesions awaits further investigation. With P gingivalis, B forsythus was the most prevalent bacterial species in the abscesses evaluated in this study. B forsythus is also considered an important periodontopathogen. 14 Because B forsythus is a slow-growing, fastidious microorganism that is difficult to culture, it had not been carefully investigated in endodontic infections. It is apparent that the prevalence of B forsythus in infected root canals had been underestimated because of its poor growth on culture media and its occurrence at levels below the levels of detection by the culture method. Recent molecular studies have detected it in root canal infections. 19,38,39 Up until now, the current study was probably the first study to report a relatively high prevalence of this species in acute periradicular abscesses. B forsythus produces a trypsin-like enzyme, which can be an important virulence factor in endodontic abscesses. Other possible virulence factors include lipopolysaccharide, alkaline phosphatase, acid phosphatase, propionate, isovalerate, phenylacetate, and butyrate. It has been reported that B forsythus strains did not cause abscess formation by monoinoculation. 40 However, B forsythus coinoculated with F nucleatum caused abscess formation in rabbits. Combinations of B forsythus strains with P gingivalis were highly virulent and invasive, as revealed by the wound-chamber method in rabbits. In the present study, B forsythus was always found combined with other bacterial species, except in the instance of one case. Even in such a case, because of the inherent limitations of the evaluation method used as discussed earlier, one cannot state that it was present in monoinfection. The results of the present study support the current concept that the etiology of acute periradicular abscesses is polymicrobial. Molecular technology has at least extended the number of detectable species associated with infections of endodontic origin. REFERENCES 1. Siqueira JF Jr. Tratamento das infecções endodônticas. Rio de Janeiro: MEDSI; Torabinejad M, Walton RE. Periradicular lesions. In: Ingle JI, Bakland LK, editors. Endodontics. 4th ed. Baltimore: Williams & Wilkins; p Lewis MA, MacFarlane TW, McGowan DA. Quantitative bacteriology of acute dento-alveolar abscesses. J Med Microbiol 1986;21: Sakamoto H, Kato H, Sato T, Sasaki J. Semiquantitative bacteriology of closed odontogenic abscesses. Bull Tokyo Dent Coll 1998;39: Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol 1991;6: Sklavounos A, Legakis NJ, Ioannidou H, Patrikiou A. Anaerobic bacteria in dentoalveolar abscesses. Int J Oral Maxillofac Surg 1986;15: Wasfy MO, McMahon KT, Minah GE, Falkler WA Jr. Microbiological evaluation of periapical infections in Egypt. Oral Microbiol Immunol 1992;7: Williams BL, McCann GF, Schoenknecht FD. Bacteriology of dental abscesses of endodontic origin. J Clin Microbiol 1983;18: Sundqvist G, Johansson E, Sjögren U. Prevalence of blackpigmented Bacteroides species in root canal infections. J Endod 1989;15: Kuriyama T, Karasawa T, Nakagawa K, Saiki Y, Yamamoto E, Nakamura S. Bacteriologic features and antimicrobial susceptibility in isolates from orofacial odontogenic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90: Pitt TL, Saunders NA. Molecular bacteriology: a diagnostic tool for the millennium. J Clin Pathol 2000;53: Whelen AC, Persing DH. The role of nucleic acid amplification and detection in the clinical microbiology laboratory. Annu Rev Microbiol 1996;50: Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE. Checkerboard DNA-DNA hybridization. Biotechniques 1994;17: Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25: Ximénez-Fyvie LA, Haffajee AD, Socransky SS. Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis. J Clin Periodontol 2000;27: Papapanou PN, Madianos PN, Dahlén G, Sandros J. Checkerboard versus culture: a comparison between two methods for identification of subgingival microbiota. Eur J Oral Sci 1997;105: Smith GL, Socransky SS, Smith CM. Rapid method for the purification of DNA from subgingival microorganisms. Oral Microbiol Immunol 1989;4: Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132: Siqueira JF Jr, Rôças IN, Souto R, Uzeda M, Colombo AP. Checkerboard DNA-DNA hybridization analysis of endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;89:744-8.

7 ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Siqueira et al 457 Volume 92, Number Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 1996;11: Mombelli A, Minder CE, Gusberti FA, Lang NP. Reproducibility of microscopic and cultural data in repeated subgingival plaque samples. J Clin Periodontol 1989;16: Loesche WJ, Lopatin DE, Stoll J, van Poperin N, Hujoel PP. Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard? J Clin Microbiol 19920;30: Gatti JJ, Dobeck JM, Smith C, White RR, Socransky SS, Skobe Z. Bacteria of asymptomatic periradicular endodontic lesions identified by DNA-DNA hybridization. Endod Dent Traumatol 2000;16: Schuman NJ, Turner JE. The clinical significance of beta hemolytic streptococci of the milleri group in oral abscesses. J Clin Pediatr Dent 1999;23: Kulekci G, Inanc D, Kocak H, Kasapoglu C, Gumru OZ. Bacteriology of dentoalveolar abscesses in patients who have received empirical antibiotic therapy. Clin Infect Dis 1996; 23(suppl 1):S Wade WG, Lewis MA, Cheeseman SL, Absi EG, Bishop PA. An unclassified Eubacterium taxon in acute dento-alveolar abscess. J Med Microbiol 1994;40: Brook I, Frazier EH, Gher ME Jr. Microbiology of periapical abscesses and associated maxillary sinusitis. J Periodontol 1996; 67: van Winkelhoff AJ, Carlee AW, de Graaff J. Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses. Infect Immun 1985;49: Oliveira JCM, Siqueira JF Jr, Alves GB, Hirata R Jr, Andrade AFB. Detection of Porphyromonas endodontalis in infected root canals by 16s rrna gene-directed polymerase chain reaction. J Endod 2000;26: Siqueira JF Jr, Rôças IN, Oliveira JCM, Santos KRN. pathogens in acute periradicular abscesses by 16S rdna directed PCR. J Endod 2001;27: Goumas PD, Naxakis SS, Papavasiliou DA, Moschovakis ED, Tsintsos SJ, Skoutelis A. Periapical abscesses: causal bacteria and antibiotic sensitivity. J Chemother 1997;9: Heimdahl A, von Konow L, Satoh T, Nord CE. Clinical appearance of orofacial infections of odontogenic origin in relation to microbiological findings. J Clin Microbiol 1985;22: Fisher LE, Russell RR. The isolation and characterization of milleri group streptococci from dental periapical abscesses. J Dent Res 1993;72: Brook I, Walker RI. Infectivity of organisms recovered from polymicrobial abscesses. Infect Immun 1983;42: Sundqvist GK, Eckerbom MI, Larsson AP, Sjögren UT. Capacity of anaerobic bacteria from necrotic dental pulps to induce purulent infections. Infect Immun 1979;25: Baumgartner JC, Falkler Jr WA, Beckerman T. Experimentally induced infection by oral anaerobic microorganisms in a mouse model. Oral Microbiol Immunol 1992;7: Siqueira JF Jr, Magalhães FAC, Lima KC, Uzeda M. Pathogenicity of facultative and obligate anaerobic bacteria in monoculture and combined with either Prevotella intermedia or Prevotella nigrescens. Oral Microbiol Immunol 1998;11: Rôças IN, Siqueira JF Jr, Santos KR, Coelho AM. Red complex (Bacteroides forsythus, Porphyromonas gingivalis, and Treponema denticola) in endodontic infections: a molecular approach. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91: Conrads G, Gharbia SE, Gulabivala K, Lampert F, Shah HN. The use of a 16s rdna directed PCR for the detection of endodontopathogenic bacteria. J Endod 1997;23: Takemoto T, Kurihara H, Dahlen G. Characterization of Bacteroides forsythus isolates. J Clin Microbiol 1997;35: Reprint requests: José F. Siqueira, Jr R. Herotides de Oliveira 61/601 Icaraí, Niterói, RJ Brazil siqueira@estacio.br Abstract Cumulative effects of successive restorative procedures on anterior crown flexure: Intact versus veneered incisors P. Magne and W. H. Douglas Quintessence International 2000;31:5-18. Objective. This study sought to evaluate variations in crown flexure and the tooth-restorative marginal interface when successive restorative procedures were carried out on intact and veneered incisors. Study design. This in vitro study made a comparison between 7 mandibular incisors with dentin-bonded porcelain veneers and 7 intact mandibular incisors. Coronal flexure and morphology of tooth-restoration interface were assessed at 5 sequential stages: intact tooth, Class III preparations, Class III resin-composite restorations, endodontic access before restoration, and endodontic restoration (without posts). Interface morphology was assessed after functional and thermal cycling loads. Results. No significant differences in crown flexure were found between natural and veneered teeth. Among all specimens, the endodontic treatment step resulted in the highest crown flexure rate. Next highest were the unrestored Class III preparations and the endodontic restoration stage. This was followed by the Class III restoration. No measurable microleakage or gaps were detected at natural tooth restorative interfaces. Conclusions. Subsequent reductions in tooth structure resulted in an increase in crown flexibility, with partial recovery after restoration. Endodontic procedures demonstrated the greatest loss of crown stiffness. Clinical significance. The authors reported that unrestored endodontic access preparations can have a negative influence by increasing crown flexure of anterior teeth. The study is limited to mandibular incisors and does not address root canal anatomy nor does it indicate placement of any root-filling material. It does not compare effects on placement of a restoration at varying levels into the root canal system. The authors imply that loss of crown rigidity equates with loss of strength, although this was not demonstrated in the study. James L. Jostes, DDS, MS University of Iowa