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Ribotyping of Bacteria from Root Canal and Blood of Patients Receiving Endodontic Therapy
Anaerobe (1997) 3, 237–243
CLINICAL MICROBIOLOGY
Ribotyping of Bacteria from Root Canal and Blood of Patients Receiving Endodontic Therapy Gilberto J. Debelian1,2, Emenike R. Eribe2, Ingar Olsen2 and Leif Tronstad1 1
Division of Endodontics Department of Oral Biology, Dental Faculty, University of Oslo, Oslo, Norway 2
(Received 21 February 1997, accepted in revised form 25 May 1997) Key Words: anaerobic bacteria, bacteremia, endodontic infection, ribotyping
DNA restriction patterns and corresponding ribotypes of 26 bacterial isolates (Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Actinomyces israelii, Streptococcus intermedius, and Streptococcus sanguis) recovered from patients with infected root canals and their peripheral blood, collected during and after endodontic therapy, were examined. Eleven additional reference strains including type strains were also examined. Purified DNA was digested with BglI, EcoRI, and HindIII. Hybridization was carried out with a digoxigenin-labeled cDNA probe obtained by reverse transcription of Escherichia coli 16S + 23S rRNA. Ribotypes of the bacteria recovered from root canal and blood showed identical characteristics within the patients, and differed between the patients. The results were confirmed when the similarity coefficient (SAB) of the ribotypes from the isolates were assessed by the Dendron computer-assisted system. These results suggested that the bacteria isolated from the blood originated from the root canal. © 1997 Academic Press
Introduction Surgical intervention in oral tissues may lead to spreading of microorganisms from the oral cavity into the bloodstream [1–3]. Several studies have assessed the occurrence and frequency of bacteremia following dental treatment and bacteria isolated from the blood have been examined [4–7]. However, few efforts have been made to compare isolates in the blood and in the oral cavity. Address correspondence to: G. J. Debelian, Division of Endodontics, Faculty of Dentistry, University of Oslo, P.O. Box 1109 Blindern, N-0317 Oslo, Norway. E-mail: [email protected]
1075-9964/97/040237 + 07 $25.00/0/an970108
Recently, our group has studied bacteremia following endodontic therapy of teeth with asymptomatic apical periodontitis [3]. The results show that anaerobic bacteria are the most frequent organisms isolated from infected root canals and blood. Phenotypic characterisation of the isolates recovered from the same patient suggests that the microorganisms isolated from blood originate from the root canal [8,9]. Phenotypic characteristics may be inconsistently expressed usually due to variations in the bacterial growth conditions [10]. Therefore, the present study was performed to determine whether the identity of the root canal and blood isolates, as suggested by phenotypic criteria, could be verified by a genetic method. Restriction endonuclease analysis and corresponding ribotypes were used to determine the © 1997 Academic Press
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genetic similarities between the microorganisms isolated from the root canal and the blood within the same patients, and to further characterize the microorganisms recovered during endodontic bacteremias.
Material and Methods Bacterial strains The study comprised of 26 clinical strains of bacteria (Table 1). Eleven of these strains were isolated from the root canal of patients having teeth with asymptomatic apical periodontitis and the other 15 strains were recovered from the peripheral blood of these patients during and after endodontic treatment of the affected teeth [3]. An additional 11 reference strains were included for comparison.
Bacterial culture conditions All bacterial strains were cultivated as described previously [8]. Briefly, the isolates were transferred from frozen stock cultures in Todd Hewitt broth supplemented with dimethyl sulfoxide (Sigma, St. Louis, MO, U.S.A.) kept in liquid nitrogen to prereduced Trypticase Soy agar plates supplemented with 5% defibrinated whole human blood, 5 mg/L of hemin and 0.5 mg/L of menadione. The bacteria were cultured under anaerobic conditions (10% CO2, 10% H2 and 80% N2) at 37°C for 48 to 72 h. The Gramnegative strains were transferred to 10 mL of pre-
reduced anaerobically sterilized (PRAS) brain heart infusion broth (Biokar Diagnostics, Beauvais, France) supplemented with 0.1% calcium carbonate (Kebo, Stockholm, Sweden) (BHIC), and the Gram-positive strains were transferred to PRAS BHIC tubes supplemented with 0.025% of Tween-80 (Sigma). Seeding was performed while an anaerobic gas mixture (10% CO2, 10% H2, 80% N2) was flushed continuously into the culture tubes using a Virginia Polytechnic Institute (V.P.I.) Anaerobic Culture System (Bellco, Vineland, NJ, U.S.A.). All microorganisms were cultured in duplicate at 37°C for 48 h.
DNA extraction Chromosomal DNA was extracted using a modification of the method of Popovic et al. [11]. After culturing, the bacterial cells were harvested in 30 mL Corex tubes (Corning, New York, NY, U.S.A.) by centrifugation, 8000 3 g for 10 min at 4°C. A pellet of approximately 100 µL was transferred to an Eppendorf tube and suspended in 1.5 mL of TE buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8)]. The mixture was centrifuged at 13 000 3 g for 4 min at 4°C. The recovered pellet was resuspended in 100 µL of TE containing 5 µL/mL of lysozyme (Sigma) (stock solution 50 mg/mL) and 3 µL/mL of proteinase K (Sigma) (stock solution 20 mg/mL), and the mixture was incubated for 30 min at 37°C. Five-hundred µL of GES reagent (5 M guanidium thiocyanate, 100 mM EDTA, 0.5% Sarkosyl) were added to the mixture and the tubes were inverted several times until lysis was observed (1–5 min). Thereafter the tubes were placed
Table 1. Bacterial strains studied Root canal
Blood 1*
Blood 2*
Reference strains
Actinomyces israelii
Act. israelii
Act. israelii
Act. israelii CCUG 18307
Peptostreptococcus prevotii
Pept. prevotii
NR+
Pept. prevotii CCUG 4946
Fusobacterium nucleatum
Fus. nucleatum
NR
F. nucleatum subsp. nucleatum ATCC 23726 F. nucleatum subsp. vincentii ATCC 49256 F. nucleatum subsp. polymorphum ATCC 10953 F. nucleatum subsp. fusiforme CCUG 32880
Streptococcus intermedius
Strep. intermedius
NR
Strep. intermedius CCUG 17827
Streptococcus sanguis
Strep. sanguis
NR
Strep. sanguis CCUG 17826
Prop. acnes Prop. acnes Prop. acnes
Prop. acnes Prop. acnes Prop. acnes
Prop. acnes CCUG 1794
Prev. intermedia Prev. nigrescens Prev. intermedia
NR NR NR
Prev. intermedia ATCC 25611 Prev. nigrescens ATCC 33563
Propionibacterium acnes Propionibacterium acnes Propionibacterium acnes
(P1)1 (P2)2 (P3)3
Prevotella intermedia (P1) Prevotella nigrescens (P2) Prevotella intermedia (P3)
*Blood samples taken during endodontic treatment. **Blood samples taken 10 min after endodontic treatment. +Not recovered. 1Patient 1. 2Patient 2. 3Patient 3.
Ribotyping in Endodontic Bacteremia on ice for 5 min and 250 µL of cold 7.5 M ammonium acetate solution were added. The tubes were shaken gently until the two phases had been mixed and thereafter kept on ice for 10 min. An 0.5 mL aliquot of chloroform-2-pentanol (24:1) was added to the mixture and the tubes were vigorously shaken to blend the two phases, which were then centrifuged for 10 min at 13 000 3 g. The aqueous phase (top phase) was carefully collected with a sterile Pasteur pipette and transferred to a new 1.5 mL Eppendorf tube, and 459 µL of cold isopropanol (–20°C) were added to the supernatant. The tube was inverted gently until the DNA precipitate appeared at the top of the solution. The DNA pellet was recovered by centrifugation at 13 000 3 g for 4 min. The aqueous solution was aspirated with a Pasteur pipette and the DNA pellet was washed with 300 to 500 µL of 70% ethanol. The tubes were centrifuged again at 13 000 3 g for 4 min. This step was repeated two more times. The pellets were dried in a vacuum desiccator for 30 min and suspended in 100 µL of sterile water kept overnight at room temperature. DNA was stored at 4°C. The quantity and purity of the isolated DNA were determined by OD measurement at 260nm and 280nm (Lambda 11 UV/Vis spectrometer, Perkin Elmer, Norwalk, CT, U.S.A.). The quality of the DNA was checked by gel electrophoresis according to Popovic et al. [11].
Restriction enzymes Three restriction endonucleases were used to digest the DNA: BglI, EcoRI, and HindIII (Boehringer Mannheim, Mannheim, Germany). Three µL of DNA were transferred to a solution containing 1 µL of restriction enzyme, 2 µL of restriction buffer and 14 µL of sterile water. After incubation at 37°C for 2 h, the DNA digestion was stopped by incubating the mixture at 65°C when the mixture contained BglI or EcoRI, and at 85°C when the mixture contained HindIII. Two µL of 10 3 gel-loading buffer (0.25% 10 3 bromophenol blue, 0.25% xylene cyanol, and 30% glycerol in water) were added to each mixture.
Gel electrophoresis Restriction fragments were subjected to electrophoresis in 0.7% (w/v) agarose (Boehringer Mannheim) gels in 1X TBE buffer, and run at a constant voltage of 40 V for 16 h at room temperature. A digoxigeninlabeled DNA molecular-weight marker II (Boehringer Mannheim) was included in each run.
239
Southern blotting The DNA in the gel was depurinated in 0.25 M HCl for 7 min, denaturated in 0.5 M NaOH-1.5 M NaCl for 7 min, and neutralized in 0.5 M Tris-HCl-1.5 M NaCl for 7 min. It was then transferred to a nylon membrane (Genebind 45, Pharmacia, Uppsala, Sweden) with 20X SSC (0.5 mol/L NaCl, 0.05 mol/L sodium citrate, pH 7.0) by using a vacuum blotter system (VacuGene XL, Pharmacia) under a constant negative pressure of 50 mm Hg.
Ribotyping After transfer, the membranes were air-dried (for 30 min to 1 h) and then exposed to UV illumination (10 s) or baked for 30 min at 80°C. The membranes were prehybridized in screw-cap hybridization tubes (Hybritube 20, Gibco, Paisley, U.K.) at 68°C for 1 h in a hybridization solution containing 5X SSC (0.75 M NaCl plus 0.075 M sodium citrate), 1% blocking reagent, 0.1% Sarkosyl and 0.02% (w/v) SDS. Thereafter, the membranes were hybridized at 68°C overnight in the hybridization solution containing 50 µL of freshly denatured digoxigenin-labeled cDNA probe obtained from reverse transcription of 16S + 23S rRNA of Escherichia coli (Boehringer Mannheim). The probe was made as described by Popovic et al. [11]. Posthybridization washes were made with 2X SSC and 0.1% SDS for 3 3 5 min followed by 0.1% SSC and 0.1% SDS at 68°C for 2 3 15 min. The hybridizationpositive fragments were detected by a color reaction between nitroblue tetrazolium (NBT) and 5-bromo4-chloro-3-indolyl phosphatase (BCIP) with alkaline phosphatase-labeled anti-digoxigenin antibodies. This solution was made by adding 180 µL of NBT and 140 µL of BCIP to 40 mL of a buffer containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2. The reaction took place for 3 to 12 h and was stopped by washing the membranes in 100 mL of TE solution for 10 min. The membranes were then photographed. The ribotyping was repeated at least twice.
Assessment of ribotype patterns The ribotype patterns of each strain were compared and analysed by the Dendron computer-assisted system (Dendron 2.2, Solltech, Oakdale, IA, U.S.A.) [12]. The original membranes were scanned on a flatbed scanner (ScanMaker III, Microtek, Redondo Beach, CA, U.S.A.) and the digitized image was processed. The Dendron software recorded all bands in each lane. This information was categorized by the position of the bands. Once the ribotype of each lane had been logged into the database of the Dendron
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system, a similarity coefficient (SAB) was computed for every pair of strains, generating a matrix of values and a dendrogram. An SAB of 1.0 indicated that the strains compared were genetically identical, and of 0.0 indicated that the strains were non-identical.
Results The three restriction enzymes provided clear and reproducible restriction endonuclease patterns. However, the great number of fragments made it difficult to analyse the similarities between the strains. The transfer of the fragments to a membrane followed by hybridization with the probe containing rRNA genes gave fewer bands. The number of bands from the different strains varied from 1 to 15. The ribotypes of the bacteria recovered from the root canal and blood showed identical genetic characteristics within the patients (Figures 1–5). The dendrogram derived from the similarity coefficients (SAB) of the clinical isolates, showed identity between the bacteria from the root canal and the blood with all enzymes tested (SAB = 1.0) (Figures 3 and 5). The four reference strains of Fusobacterium nucleatum showed both similar and dissimilar ribotypes Bgl I kbp
mw 1 2 3 4
EcoR I 5
6
7
(lanes 3, 4, 5 and 6 for BglI, lanes 9, 10, 11 and 12 for EcoRI and lanes 15, 16, 17 and 18 for HindIII) (Figure 2). Identical ribotypes were observed for the same restriction enzyme between the clinical strains of F. nucleatum recovered from the root canal and blood, and the type strain of F. nucleatum subsp. vincentii (lane 4 for BglI, lane 10 for EcoRI, and lane 16 for HindIII). The clinical isolates from the root canal and blood and the type strain F. nucleatum subsp. vincentii showed a similarity coefficient (SAB) of 1.0 (Figure 3). When BglI was used, the type strain of Prevotella intermedia (lane 1) presented both similar and dissimilar ribotype patterns to those of the clinical strains of P. intermedia (lanes 3 and 4, 7 and 8) (Figure 4). However, the type strain of P. nigrescens (lane 2) presented identical ribotype to those of the clinical strains of P. nigrescens (lanes 5 and 6). When EcoRI was used, the type strain of P. intermedia (lane 9) gave both similar and dissimilar ribotype patterns to those of the clinical strains in lane 11 and 12 and identical ribotype to the clinical strains in lanes 15 and 16. The type strain of P. nigrescens (lane 10) presented both similar and dissimilar ribotypes patterns to the clinical strains of P. nigrescens in lanes 13 and 14. When HindIII was used, the type strain of P. intermedia (lane 17) gave both similar and dissimilar ribotype patterns to those of the clinical strains of P. intermedia shown in lanes 19 and 20, 23 and 24. The type strain of P. nigrescens (lane 18) showed identical ribotype when compared to the
Hind III 8 9 10 11 12
Bgl I kbp
mw 1 2 3 4 5 6
EcoR I
Hind III
7 8 9 10 11 12 13 14 15 16 17 18
23.1
23.1 9.4
9.4 6.5
6.5
4.3
4.3
2.3 2.0
Figure 1. Ribotypes of Actinomyces israelii after digesting with BglI (lanes 1–4, EcoRI (lanes 5–8) and HindIII (lanes 9–12); mw = molecular weight of λ DNA; lanes 1,5 and 9: type strain of A. israelii = CCUG 18307; lanes 2,6 and 10: isolates from root canal; lanes 3,4 and 11: isolates from blood during treatment; lanes 4,8 and 12: isolates from blood 10 min after treatment. Note that the clinical isolates of A. israelii present identical ribotypes when the same restriction enzyme is used.
2.3 2.0
Figure 2. Ribotypes of Fusobacterium nucleatum after cutting with BglI (lanes 1–6), EcoRI (lanes 7–12) and HindIII (lanes 13–18); mw = molecular weight of λ DNA; lanes 1,7 and 13: isolates from root canal; lanes 2,8 and 14: isolates from blood during treatment; lanes 3,9 and 15: reference strains of F. nucleatum subsp. nucleatum = ATCC 23726; lanes 4,10 and 16: type strain of F. nucleatum subsp. vincentii = ATCC 49256; lanes 5,11 and 17: type strain of F. nucleatum subsp. polymorphum = ATCC 10953; lanes 6,12 and 18: type strain of F. nucleatum subsp. fusiforme = CCUG 32880. Note identity of ribotypes of clinical strains of F. nucleatum recovered from root canal and blood, and reference strain of F. nucleatum subsp. vincentii ATCC 49256 when BglI and HindIII were used. When EcoRI was used, an extra band at 2.3 kbp is present in lane 10 and absent in the ribotypes of the clinical isolates (lanes 8 and 9).
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L3 L6 L2 L1 L4 L5
0
0.1
0.2
0.3
0.4 0.5 0.6 Fn–Bgl/I
0.7
0.8
0.9
1 SAB
Figure 3. Dendrogram based on similarity coefficients (sAB) of Fusobacterium nucleatum strains when BglI was used. L1 and L2: isolates of F. nucleatum, respectively from root canal and blood, L3: reference strain of F. nucleatum subsp. nucleatum = ATCC 23726, L4: type strain of F. nucleatum subsp. vincentii = ATCC 49256, L5: type strain of F. nucleatum subsp. polymorphum = ATCC 10953, L6: type strain of F. nucleatum subsp. fusiforme = CCUG 32880. Note identity (SAB = 1.0) of ribotypes between clinical isolates (L1 and L2) and type strain of F. nucleatum subsp. vincentii (L4). Bgl I P1 kbp
mw
1
2
3
4
EcoR I P2
5
6
P3 7
8
P1
P2
Hind III P3
9 10 11 12 13 14 15 16
P1
P2
P3
17 18 19 20 21 22 23 24
23.1 9.4 6.5
4.3
2.3 2.0
Figure 4. Ribotype patterns of Prevotella intermedia (P1 and P3) and Prevotella nigrescens (P2) from three patients after cutting with BglI (lanes 1–8), EcoRI (lanes 9–16) and HindIII (lanes 17–24); mw = molecular weight of λ DNA; lanes 1,9 and 17: type strain of P. intermedia = ATCC 25611; lanes 2,10 and 18: type strain of P. nigrescens = ATCC 33563; P. intermedia from root canal P1-lanes 3, 11, 19 and P3-lanes 7, 15, 23); P. intermedia from blood (P1-lanes 4, 12, 20 and P3-lanes 8, 16, 24); P. nigrescens from root canal (P2-lanes 5, 13, 21); P. nigrescens from blood (P2-lanes 6, 14, 22). Note identity of ribotypes of isolates of P. intermedia and P. nigrescens from root canal and blood of same patient for all enzymes used.
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clinical strains of P. nigrescens in lanes 21 and 22. The dendrogram derived from the SAB of the reference strains and clinical isolates of P. intermedia and P. nigrescens, when BglI was used, showed that the clinical isolates from the root canal and blood within the patients were identical (SAB = 1.0) (P1 for lanes 3 and 4, P2 for lanes 5 and 6, P3 for lanes 7 and 8) (Figure 5).
Discussion The present study investigated the suitability of using ribotyping to characterize and trace bacteria involved in endodontic bacteremia. Ribotyping is a general and widely used DNA hybridization method based on restriction fragment length polymorphism in the chromosomal DNA containing rRNA genes [10,13–20]. In this study, we successfully used ribotyping for 26 strains of eight different bacterial species and four subspecies. It proved to be a simple, reproducible and practical method to trace microorganisms from the blood to their origin in the root canal and the limited number of bands revealed by ribotyping facilitated the visual inspections of the membranes. The use of different restriction enzymes gave different ribotypes of the same strain. This enabled us to determine with certainty that the isolates from the blood and the root canal were identical and also to identify different strains belonging to the same species. This was the case with F. nucleatum isolates, where a comparison of the ribotypes of the clinical isolates with the reference strains enabled us to identify the isolates as F. nucleatum subsp. vincentii (Figures 2 and 3). The isolates of P. intermedia and P.
nigrescens were also differentiated by their ribotypes, supporting and confirming previous findings [10,20]. The selection of the enzymes BglI, EcoRI and HindIII were based on previous ribotyping studies of oral bacteria [10,20], and in this study the enzymes digested all DNAs from the strains tested. rRNA is ubiquitous in bacteria [21]. The molecule’s ribonucleotide sequences are conserved and the bacterial rRNA is present in multiple copies [21]. Therefore, the nonradioactive oligonucleotide cDNA probe prepared from commercially available E. coli rRNA can be applied for a diversity of bacteria. An additional advantage of the cDNA probe was that it could be reused in several runs. We found that it hybridized well to the DNA fragments of the bacterial strains tested up to five times. The use of the Dendron system proved to be of great value and enabled us to compare the strains in an objective way. Thus, in many instances the system indicated differences between ribotypes that were not evident by visual inspection. The Dendron system confirmed that the root canal and blood isolates were identical. Moreover, the system proved that the clinical isolates of F. nucleatum and the type strain of F. nucleatum subsp. vincentii were identical [8]. The system also made it possible to differentiate between the clinical isolates of P. intermedia and P. nigrescens (Figure 5), again supporting previous results [8,9]. In conclusion, ribotyping used in conjunction with the Dendron computer-assisted system proved to be a valuable and rapid method for characterization and tracing of microorganisms, even closely related ones. The results of previous phenotypic studies were confirmed [3,89,22], leaving little doubt that the bacteria isolated from the blood during and after endodontic therapy had their origin in the root canal.
L2 L5 L6 L7
P1 P3
L8 L1 L4
P2
L3
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 PixPn–Bgl/I
1 SAB
Figure 5. Dendrogram based on similarity coefficient (sAB) of Prevotella intermedia and Prevotella nigrescens isolates when BglI was used. L1: type strain of P. intermedia = ATCC 25611; L2: type strain of P. nigrescens = ATCC 33563; L3, L4 and L7, L8: clinical isolates of P. intermedia respectively from patients 1 (P1) and 3 (P3); L5, L6: clinical isolates of P. nigrescens from patient 2 (P2). Note identity of ribotypes (SAB = 1.0) of clinical isolates derived from root canal and blood from same patient. The strains of P1 (L3 and L4) show identity (SAB = 1.0) to P. intermedia ATCC 25611 (L1).
Ribotyping in Endodontic Bacteremia This battery of phenotypic and genetic tests would also be appropriate for tracing other blood isolates to their origin in the body. By doing this systematically the discussion whether focal infection is real or not might be founded more on sound scientific evidence rather than on isolated case reports.
11.
12.
13.
References 1. Heimdahl A., Josefsson K., Konow L.V. and Nord C.E. (1985) Detection of anaerobic bacteria in blood cultures by lysis filtration. J Clin Microbiol 4: 404–407 2. Flemmig T. and Nachnami S. (1991) Bacteremia following subgingival irrigation and scaling and root planing. J Periodontol 62: 602–607 3. Debelian G.J., Olsen I. and Tronstad L. (1995) Bacteremia in conjunction with endodontic therapy. Endod Dent Traumatol 11: 142–146 4. Asikainen S. and Alaluusua S. (1993) Bacteriology of dental infections. Eur Heart J 14: 43–50 5. Debelian G.J., Olsen I. and Tronstad L. (1994) Systemic diseases caused by oral microorganisms. Endod Dent Traumatol 10: 57–65 6. Navazesh M. and Mulligan R. (1995) Systemic dissemination as a result of oral infection in individuals 50 years of age and older. Spec Care Dentist 15: 11–19 7. Okabe K., Nakagawa K. and Yamamoto E. (1995) Factors affecting the occurrence of bacteremia associated with tooth extraction. Int J Oral Maxillofac Surg 24: 239–242 8. Debelian G.J., Olsen I. and Tronstad L. (1996) Electrophoresis of whole-cell soluble proteins of microorganisms isolated from bacteremias in endodontic therapy. Eur J Oral Sci 104: 540–546 9. Debelian G.J., Olsen I. and Tronstad L. (1997) Distinction of Prevotella intermedia and Prevotella nigrescens from endodontic bacteremia through their fatty acid contents. Anaerobe 3: 61–68 10. Pearce M.A., Dixon R.A., Gharbia S.E., Shah H.N. and Devine D.A. (1996) Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonu-
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clease and ribosomal RNA gene restriction analyses. Oral Microbiol Immunol 11: 135–141 Popovic T., Bopp C.A., Olsvik Ø. and Kiehlbauch J.A. (1992) Ribotyping in molecular epidemiology. In Persing D.H., Smith T.F., Tenover F.C. and White T.F. (eds) Diagnostic molecular microbiology. Principles and applications, pp. 573–583. Am Soc Microbiol, Washington, DC Schmid J., Odds F.C., Wiselka M.J., Nicholson K.G. and Soll D.R. (1992) Genetic similarities and maintenance of Candida albicans strains from a group of AIDS patients, demonstrated by DNA fingerprinting. J Clin Microbiol 30: 935–941 Grimont F. and Grimont P.A.D. (1986) Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann Inst Pasteur Microbiol 137: 165–175 Lawson P.A., Gharbia S.E., Shah H.N. and Clark D.R. (1989) Recognition of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 by ribosomal RNA gene restriction patterns. FEMS Microbiol Lett 65: 41–46 Bowden G., Johnson J. and Schachtele C. (1993) Characterization of Actinomyces with genomic DNA fingerprints and rRNA gene probes. J Dent Res 72: 1171–1179 Hillman J.D., Maiden M.F., Pfaller S.P., Martin L., Ducan M.J. and Socransky S.S. (1993) Characterization of hemolytic bacteria in subgingival plaque. J Periodontal Res 28: 173–179 van Steenbergen T.J., Bosch-Tijhof C.J., van Winkelhoff A.J., Gmur ¨ R. and de Graaff J. (1994) Comparison of six typing methods for Actinobacillus actinomycetemcomitans. J Clin Microbiol 32: 2769–2774 Fiehn N-E., Bangsborg J.M. and Colding H. (1995) Ribotyping on small-sized spirochetes isolated from subgingival plaque. Oral Microbiol Immunol 10: 13–18 Okwumabua O., Tan Z., Staats J., Oberst R.D., Chengappa M.M. and Nagaraja T.G. (1996) Ribotyping to differentiate Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme from bovine ruminal contents and liver abscesses. Appl Environ Microbiol 62: 469–472 M¨atto¨ J., Saarela M., von Troil-Lind´en B., Kon ¨ onen ¨ E., JousimiesSomer H., Torkko H., Alaluusua S. and Asikainen S. (1996) Distribution and genetic analysis of oral Prevotella intermedia and Prevotella nigrescens. Oral Microbiol Immunol 11: 96–102 Woese C.R. (1987) Bacterial evolution. Microbiol Rev 51: 221–271 Debelian G.J., Olsen I. and Tronstad L. (1992) Profiling of Propionibacterium acnes recovered from root canal and blood during and after endodontic treatment. Endod Dent Traumatol 8: 248–254
CLINICAL MICROBIOLOGY
Ribotyping of Bacteria from Root Canal and Blood of Patients Receiving Endodontic Therapy Gilberto J. Debelian1,2, Emenike R. Eribe2, Ingar Olsen2 and Leif Tronstad1 1
Division of Endodontics Department of Oral Biology, Dental Faculty, University of Oslo, Oslo, Norway 2
(Received 21 February 1997, accepted in revised form 25 May 1997) Key Words: anaerobic bacteria, bacteremia, endodontic infection, ribotyping
DNA restriction patterns and corresponding ribotypes of 26 bacterial isolates (Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Actinomyces israelii, Streptococcus intermedius, and Streptococcus sanguis) recovered from patients with infected root canals and their peripheral blood, collected during and after endodontic therapy, were examined. Eleven additional reference strains including type strains were also examined. Purified DNA was digested with BglI, EcoRI, and HindIII. Hybridization was carried out with a digoxigenin-labeled cDNA probe obtained by reverse transcription of Escherichia coli 16S + 23S rRNA. Ribotypes of the bacteria recovered from root canal and blood showed identical characteristics within the patients, and differed between the patients. The results were confirmed when the similarity coefficient (SAB) of the ribotypes from the isolates were assessed by the Dendron computer-assisted system. These results suggested that the bacteria isolated from the blood originated from the root canal. © 1997 Academic Press
Introduction Surgical intervention in oral tissues may lead to spreading of microorganisms from the oral cavity into the bloodstream [1–3]. Several studies have assessed the occurrence and frequency of bacteremia following dental treatment and bacteria isolated from the blood have been examined [4–7]. However, few efforts have been made to compare isolates in the blood and in the oral cavity. Address correspondence to: G. J. Debelian, Division of Endodontics, Faculty of Dentistry, University of Oslo, P.O. Box 1109 Blindern, N-0317 Oslo, Norway. E-mail: [email protected]
1075-9964/97/040237 + 07 $25.00/0/an970108
Recently, our group has studied bacteremia following endodontic therapy of teeth with asymptomatic apical periodontitis [3]. The results show that anaerobic bacteria are the most frequent organisms isolated from infected root canals and blood. Phenotypic characterisation of the isolates recovered from the same patient suggests that the microorganisms isolated from blood originate from the root canal [8,9]. Phenotypic characteristics may be inconsistently expressed usually due to variations in the bacterial growth conditions [10]. Therefore, the present study was performed to determine whether the identity of the root canal and blood isolates, as suggested by phenotypic criteria, could be verified by a genetic method. Restriction endonuclease analysis and corresponding ribotypes were used to determine the © 1997 Academic Press
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genetic similarities between the microorganisms isolated from the root canal and the blood within the same patients, and to further characterize the microorganisms recovered during endodontic bacteremias.
Material and Methods Bacterial strains The study comprised of 26 clinical strains of bacteria (Table 1). Eleven of these strains were isolated from the root canal of patients having teeth with asymptomatic apical periodontitis and the other 15 strains were recovered from the peripheral blood of these patients during and after endodontic treatment of the affected teeth [3]. An additional 11 reference strains were included for comparison.
Bacterial culture conditions All bacterial strains were cultivated as described previously [8]. Briefly, the isolates were transferred from frozen stock cultures in Todd Hewitt broth supplemented with dimethyl sulfoxide (Sigma, St. Louis, MO, U.S.A.) kept in liquid nitrogen to prereduced Trypticase Soy agar plates supplemented with 5% defibrinated whole human blood, 5 mg/L of hemin and 0.5 mg/L of menadione. The bacteria were cultured under anaerobic conditions (10% CO2, 10% H2 and 80% N2) at 37°C for 48 to 72 h. The Gramnegative strains were transferred to 10 mL of pre-
reduced anaerobically sterilized (PRAS) brain heart infusion broth (Biokar Diagnostics, Beauvais, France) supplemented with 0.1% calcium carbonate (Kebo, Stockholm, Sweden) (BHIC), and the Gram-positive strains were transferred to PRAS BHIC tubes supplemented with 0.025% of Tween-80 (Sigma). Seeding was performed while an anaerobic gas mixture (10% CO2, 10% H2, 80% N2) was flushed continuously into the culture tubes using a Virginia Polytechnic Institute (V.P.I.) Anaerobic Culture System (Bellco, Vineland, NJ, U.S.A.). All microorganisms were cultured in duplicate at 37°C for 48 h.
DNA extraction Chromosomal DNA was extracted using a modification of the method of Popovic et al. [11]. After culturing, the bacterial cells were harvested in 30 mL Corex tubes (Corning, New York, NY, U.S.A.) by centrifugation, 8000 3 g for 10 min at 4°C. A pellet of approximately 100 µL was transferred to an Eppendorf tube and suspended in 1.5 mL of TE buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8)]. The mixture was centrifuged at 13 000 3 g for 4 min at 4°C. The recovered pellet was resuspended in 100 µL of TE containing 5 µL/mL of lysozyme (Sigma) (stock solution 50 mg/mL) and 3 µL/mL of proteinase K (Sigma) (stock solution 20 mg/mL), and the mixture was incubated for 30 min at 37°C. Five-hundred µL of GES reagent (5 M guanidium thiocyanate, 100 mM EDTA, 0.5% Sarkosyl) were added to the mixture and the tubes were inverted several times until lysis was observed (1–5 min). Thereafter the tubes were placed
Table 1. Bacterial strains studied Root canal
Blood 1*
Blood 2*
Reference strains
Actinomyces israelii
Act. israelii
Act. israelii
Act. israelii CCUG 18307
Peptostreptococcus prevotii
Pept. prevotii
NR+
Pept. prevotii CCUG 4946
Fusobacterium nucleatum
Fus. nucleatum
NR
F. nucleatum subsp. nucleatum ATCC 23726 F. nucleatum subsp. vincentii ATCC 49256 F. nucleatum subsp. polymorphum ATCC 10953 F. nucleatum subsp. fusiforme CCUG 32880
Streptococcus intermedius
Strep. intermedius
NR
Strep. intermedius CCUG 17827
Streptococcus sanguis
Strep. sanguis
NR
Strep. sanguis CCUG 17826
Prop. acnes Prop. acnes Prop. acnes
Prop. acnes Prop. acnes Prop. acnes
Prop. acnes CCUG 1794
Prev. intermedia Prev. nigrescens Prev. intermedia
NR NR NR
Prev. intermedia ATCC 25611 Prev. nigrescens ATCC 33563
Propionibacterium acnes Propionibacterium acnes Propionibacterium acnes
(P1)1 (P2)2 (P3)3
Prevotella intermedia (P1) Prevotella nigrescens (P2) Prevotella intermedia (P3)
*Blood samples taken during endodontic treatment. **Blood samples taken 10 min after endodontic treatment. +Not recovered. 1Patient 1. 2Patient 2. 3Patient 3.
Ribotyping in Endodontic Bacteremia on ice for 5 min and 250 µL of cold 7.5 M ammonium acetate solution were added. The tubes were shaken gently until the two phases had been mixed and thereafter kept on ice for 10 min. An 0.5 mL aliquot of chloroform-2-pentanol (24:1) was added to the mixture and the tubes were vigorously shaken to blend the two phases, which were then centrifuged for 10 min at 13 000 3 g. The aqueous phase (top phase) was carefully collected with a sterile Pasteur pipette and transferred to a new 1.5 mL Eppendorf tube, and 459 µL of cold isopropanol (–20°C) were added to the supernatant. The tube was inverted gently until the DNA precipitate appeared at the top of the solution. The DNA pellet was recovered by centrifugation at 13 000 3 g for 4 min. The aqueous solution was aspirated with a Pasteur pipette and the DNA pellet was washed with 300 to 500 µL of 70% ethanol. The tubes were centrifuged again at 13 000 3 g for 4 min. This step was repeated two more times. The pellets were dried in a vacuum desiccator for 30 min and suspended in 100 µL of sterile water kept overnight at room temperature. DNA was stored at 4°C. The quantity and purity of the isolated DNA were determined by OD measurement at 260nm and 280nm (Lambda 11 UV/Vis spectrometer, Perkin Elmer, Norwalk, CT, U.S.A.). The quality of the DNA was checked by gel electrophoresis according to Popovic et al. [11].
Restriction enzymes Three restriction endonucleases were used to digest the DNA: BglI, EcoRI, and HindIII (Boehringer Mannheim, Mannheim, Germany). Three µL of DNA were transferred to a solution containing 1 µL of restriction enzyme, 2 µL of restriction buffer and 14 µL of sterile water. After incubation at 37°C for 2 h, the DNA digestion was stopped by incubating the mixture at 65°C when the mixture contained BglI or EcoRI, and at 85°C when the mixture contained HindIII. Two µL of 10 3 gel-loading buffer (0.25% 10 3 bromophenol blue, 0.25% xylene cyanol, and 30% glycerol in water) were added to each mixture.
Gel electrophoresis Restriction fragments were subjected to electrophoresis in 0.7% (w/v) agarose (Boehringer Mannheim) gels in 1X TBE buffer, and run at a constant voltage of 40 V for 16 h at room temperature. A digoxigeninlabeled DNA molecular-weight marker II (Boehringer Mannheim) was included in each run.
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Southern blotting The DNA in the gel was depurinated in 0.25 M HCl for 7 min, denaturated in 0.5 M NaOH-1.5 M NaCl for 7 min, and neutralized in 0.5 M Tris-HCl-1.5 M NaCl for 7 min. It was then transferred to a nylon membrane (Genebind 45, Pharmacia, Uppsala, Sweden) with 20X SSC (0.5 mol/L NaCl, 0.05 mol/L sodium citrate, pH 7.0) by using a vacuum blotter system (VacuGene XL, Pharmacia) under a constant negative pressure of 50 mm Hg.
Ribotyping After transfer, the membranes were air-dried (for 30 min to 1 h) and then exposed to UV illumination (10 s) or baked for 30 min at 80°C. The membranes were prehybridized in screw-cap hybridization tubes (Hybritube 20, Gibco, Paisley, U.K.) at 68°C for 1 h in a hybridization solution containing 5X SSC (0.75 M NaCl plus 0.075 M sodium citrate), 1% blocking reagent, 0.1% Sarkosyl and 0.02% (w/v) SDS. Thereafter, the membranes were hybridized at 68°C overnight in the hybridization solution containing 50 µL of freshly denatured digoxigenin-labeled cDNA probe obtained from reverse transcription of 16S + 23S rRNA of Escherichia coli (Boehringer Mannheim). The probe was made as described by Popovic et al. [11]. Posthybridization washes were made with 2X SSC and 0.1% SDS for 3 3 5 min followed by 0.1% SSC and 0.1% SDS at 68°C for 2 3 15 min. The hybridizationpositive fragments were detected by a color reaction between nitroblue tetrazolium (NBT) and 5-bromo4-chloro-3-indolyl phosphatase (BCIP) with alkaline phosphatase-labeled anti-digoxigenin antibodies. This solution was made by adding 180 µL of NBT and 140 µL of BCIP to 40 mL of a buffer containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2. The reaction took place for 3 to 12 h and was stopped by washing the membranes in 100 mL of TE solution for 10 min. The membranes were then photographed. The ribotyping was repeated at least twice.
Assessment of ribotype patterns The ribotype patterns of each strain were compared and analysed by the Dendron computer-assisted system (Dendron 2.2, Solltech, Oakdale, IA, U.S.A.) [12]. The original membranes were scanned on a flatbed scanner (ScanMaker III, Microtek, Redondo Beach, CA, U.S.A.) and the digitized image was processed. The Dendron software recorded all bands in each lane. This information was categorized by the position of the bands. Once the ribotype of each lane had been logged into the database of the Dendron
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system, a similarity coefficient (SAB) was computed for every pair of strains, generating a matrix of values and a dendrogram. An SAB of 1.0 indicated that the strains compared were genetically identical, and of 0.0 indicated that the strains were non-identical.
Results The three restriction enzymes provided clear and reproducible restriction endonuclease patterns. However, the great number of fragments made it difficult to analyse the similarities between the strains. The transfer of the fragments to a membrane followed by hybridization with the probe containing rRNA genes gave fewer bands. The number of bands from the different strains varied from 1 to 15. The ribotypes of the bacteria recovered from the root canal and blood showed identical genetic characteristics within the patients (Figures 1–5). The dendrogram derived from the similarity coefficients (SAB) of the clinical isolates, showed identity between the bacteria from the root canal and the blood with all enzymes tested (SAB = 1.0) (Figures 3 and 5). The four reference strains of Fusobacterium nucleatum showed both similar and dissimilar ribotypes Bgl I kbp
mw 1 2 3 4
EcoR I 5
6
7
(lanes 3, 4, 5 and 6 for BglI, lanes 9, 10, 11 and 12 for EcoRI and lanes 15, 16, 17 and 18 for HindIII) (Figure 2). Identical ribotypes were observed for the same restriction enzyme between the clinical strains of F. nucleatum recovered from the root canal and blood, and the type strain of F. nucleatum subsp. vincentii (lane 4 for BglI, lane 10 for EcoRI, and lane 16 for HindIII). The clinical isolates from the root canal and blood and the type strain F. nucleatum subsp. vincentii showed a similarity coefficient (SAB) of 1.0 (Figure 3). When BglI was used, the type strain of Prevotella intermedia (lane 1) presented both similar and dissimilar ribotype patterns to those of the clinical strains of P. intermedia (lanes 3 and 4, 7 and 8) (Figure 4). However, the type strain of P. nigrescens (lane 2) presented identical ribotype to those of the clinical strains of P. nigrescens (lanes 5 and 6). When EcoRI was used, the type strain of P. intermedia (lane 9) gave both similar and dissimilar ribotype patterns to those of the clinical strains in lane 11 and 12 and identical ribotype to the clinical strains in lanes 15 and 16. The type strain of P. nigrescens (lane 10) presented both similar and dissimilar ribotypes patterns to the clinical strains of P. nigrescens in lanes 13 and 14. When HindIII was used, the type strain of P. intermedia (lane 17) gave both similar and dissimilar ribotype patterns to those of the clinical strains of P. intermedia shown in lanes 19 and 20, 23 and 24. The type strain of P. nigrescens (lane 18) showed identical ribotype when compared to the
Hind III 8 9 10 11 12
Bgl I kbp
mw 1 2 3 4 5 6
EcoR I
Hind III
7 8 9 10 11 12 13 14 15 16 17 18
23.1
23.1 9.4
9.4 6.5
6.5
4.3
4.3
2.3 2.0
Figure 1. Ribotypes of Actinomyces israelii after digesting with BglI (lanes 1–4, EcoRI (lanes 5–8) and HindIII (lanes 9–12); mw = molecular weight of λ DNA; lanes 1,5 and 9: type strain of A. israelii = CCUG 18307; lanes 2,6 and 10: isolates from root canal; lanes 3,4 and 11: isolates from blood during treatment; lanes 4,8 and 12: isolates from blood 10 min after treatment. Note that the clinical isolates of A. israelii present identical ribotypes when the same restriction enzyme is used.
2.3 2.0
Figure 2. Ribotypes of Fusobacterium nucleatum after cutting with BglI (lanes 1–6), EcoRI (lanes 7–12) and HindIII (lanes 13–18); mw = molecular weight of λ DNA; lanes 1,7 and 13: isolates from root canal; lanes 2,8 and 14: isolates from blood during treatment; lanes 3,9 and 15: reference strains of F. nucleatum subsp. nucleatum = ATCC 23726; lanes 4,10 and 16: type strain of F. nucleatum subsp. vincentii = ATCC 49256; lanes 5,11 and 17: type strain of F. nucleatum subsp. polymorphum = ATCC 10953; lanes 6,12 and 18: type strain of F. nucleatum subsp. fusiforme = CCUG 32880. Note identity of ribotypes of clinical strains of F. nucleatum recovered from root canal and blood, and reference strain of F. nucleatum subsp. vincentii ATCC 49256 when BglI and HindIII were used. When EcoRI was used, an extra band at 2.3 kbp is present in lane 10 and absent in the ribotypes of the clinical isolates (lanes 8 and 9).
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L3 L6 L2 L1 L4 L5
0
0.1
0.2
0.3
0.4 0.5 0.6 Fn–Bgl/I
0.7
0.8
0.9
1 SAB
Figure 3. Dendrogram based on similarity coefficients (sAB) of Fusobacterium nucleatum strains when BglI was used. L1 and L2: isolates of F. nucleatum, respectively from root canal and blood, L3: reference strain of F. nucleatum subsp. nucleatum = ATCC 23726, L4: type strain of F. nucleatum subsp. vincentii = ATCC 49256, L5: type strain of F. nucleatum subsp. polymorphum = ATCC 10953, L6: type strain of F. nucleatum subsp. fusiforme = CCUG 32880. Note identity (SAB = 1.0) of ribotypes between clinical isolates (L1 and L2) and type strain of F. nucleatum subsp. vincentii (L4). Bgl I P1 kbp
mw
1
2
3
4
EcoR I P2
5
6
P3 7
8
P1
P2
Hind III P3
9 10 11 12 13 14 15 16
P1
P2
P3
17 18 19 20 21 22 23 24
23.1 9.4 6.5
4.3
2.3 2.0
Figure 4. Ribotype patterns of Prevotella intermedia (P1 and P3) and Prevotella nigrescens (P2) from three patients after cutting with BglI (lanes 1–8), EcoRI (lanes 9–16) and HindIII (lanes 17–24); mw = molecular weight of λ DNA; lanes 1,9 and 17: type strain of P. intermedia = ATCC 25611; lanes 2,10 and 18: type strain of P. nigrescens = ATCC 33563; P. intermedia from root canal P1-lanes 3, 11, 19 and P3-lanes 7, 15, 23); P. intermedia from blood (P1-lanes 4, 12, 20 and P3-lanes 8, 16, 24); P. nigrescens from root canal (P2-lanes 5, 13, 21); P. nigrescens from blood (P2-lanes 6, 14, 22). Note identity of ribotypes of isolates of P. intermedia and P. nigrescens from root canal and blood of same patient for all enzymes used.
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clinical strains of P. nigrescens in lanes 21 and 22. The dendrogram derived from the SAB of the reference strains and clinical isolates of P. intermedia and P. nigrescens, when BglI was used, showed that the clinical isolates from the root canal and blood within the patients were identical (SAB = 1.0) (P1 for lanes 3 and 4, P2 for lanes 5 and 6, P3 for lanes 7 and 8) (Figure 5).
Discussion The present study investigated the suitability of using ribotyping to characterize and trace bacteria involved in endodontic bacteremia. Ribotyping is a general and widely used DNA hybridization method based on restriction fragment length polymorphism in the chromosomal DNA containing rRNA genes [10,13–20]. In this study, we successfully used ribotyping for 26 strains of eight different bacterial species and four subspecies. It proved to be a simple, reproducible and practical method to trace microorganisms from the blood to their origin in the root canal and the limited number of bands revealed by ribotyping facilitated the visual inspections of the membranes. The use of different restriction enzymes gave different ribotypes of the same strain. This enabled us to determine with certainty that the isolates from the blood and the root canal were identical and also to identify different strains belonging to the same species. This was the case with F. nucleatum isolates, where a comparison of the ribotypes of the clinical isolates with the reference strains enabled us to identify the isolates as F. nucleatum subsp. vincentii (Figures 2 and 3). The isolates of P. intermedia and P.
nigrescens were also differentiated by their ribotypes, supporting and confirming previous findings [10,20]. The selection of the enzymes BglI, EcoRI and HindIII were based on previous ribotyping studies of oral bacteria [10,20], and in this study the enzymes digested all DNAs from the strains tested. rRNA is ubiquitous in bacteria [21]. The molecule’s ribonucleotide sequences are conserved and the bacterial rRNA is present in multiple copies [21]. Therefore, the nonradioactive oligonucleotide cDNA probe prepared from commercially available E. coli rRNA can be applied for a diversity of bacteria. An additional advantage of the cDNA probe was that it could be reused in several runs. We found that it hybridized well to the DNA fragments of the bacterial strains tested up to five times. The use of the Dendron system proved to be of great value and enabled us to compare the strains in an objective way. Thus, in many instances the system indicated differences between ribotypes that were not evident by visual inspection. The Dendron system confirmed that the root canal and blood isolates were identical. Moreover, the system proved that the clinical isolates of F. nucleatum and the type strain of F. nucleatum subsp. vincentii were identical [8]. The system also made it possible to differentiate between the clinical isolates of P. intermedia and P. nigrescens (Figure 5), again supporting previous results [8,9]. In conclusion, ribotyping used in conjunction with the Dendron computer-assisted system proved to be a valuable and rapid method for characterization and tracing of microorganisms, even closely related ones. The results of previous phenotypic studies were confirmed [3,89,22], leaving little doubt that the bacteria isolated from the blood during and after endodontic therapy had their origin in the root canal.
L2 L5 L6 L7
P1 P3
L8 L1 L4
P2
L3
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 PixPn–Bgl/I
1 SAB
Figure 5. Dendrogram based on similarity coefficient (sAB) of Prevotella intermedia and Prevotella nigrescens isolates when BglI was used. L1: type strain of P. intermedia = ATCC 25611; L2: type strain of P. nigrescens = ATCC 33563; L3, L4 and L7, L8: clinical isolates of P. intermedia respectively from patients 1 (P1) and 3 (P3); L5, L6: clinical isolates of P. nigrescens from patient 2 (P2). Note identity of ribotypes (SAB = 1.0) of clinical isolates derived from root canal and blood from same patient. The strains of P1 (L3 and L4) show identity (SAB = 1.0) to P. intermedia ATCC 25611 (L1).
Ribotyping in Endodontic Bacteremia This battery of phenotypic and genetic tests would also be appropriate for tracing other blood isolates to their origin in the body. By doing this systematically the discussion whether focal infection is real or not might be founded more on sound scientific evidence rather than on isolated case reports.
11.
12.
13.
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