Basic Research—Biology

New Bacterial Composition in Primary and Persistent/ Secondary Endodontic Infections with Respect to Clinical and Radiographic Findings Christian Tennert, DDS,* Maximilian Fuhrmann,* Annette Wittmer,† Lamprini Karygianni, DDS,* Markus J. Altenburger, DDS, PD,* Klaus Pelz, DDS,† Elmar Hellwig, DDS, Prof,* and Ali Al-Ahmad, DDS, PD* Abstract Introduction: The aim of the present study was to analyze the microbiota of primary and secondary/ persistent endodontic infections of patients undergoing endodontic treatment with respect to clinical and radiographic findings. Methods: Samples from the root canals of 21 German patients were taken using 3 sequential sterile paper points. In the case of a root canal filling, gutta-percha was removed with sterile files, and samples were taken using sterile paper points. The samples were plated, and microorganisms were then isolated and identified morphologically by biochemical analysis and sequencing the 16S rRNA genes of isolated microorganisms. Results: In 12 of 21 root canals, 33 different species could be isolated. Six (50%) of the cases with isolated microorganisms were primary, and 6 (50%) cases were endodontic infections associated with root-filled teeth. Twelve of the isolated species were facultative anaerobic and 21 obligate anaerobic. Monomicrobial infections were found for Enterococcus faecalis and Actinomyces viscosus. E. faecalis was most frequently isolated in secondary endodontic infections (33%). Moraxella osloensis was isolated from a secondary endodontic infection that had an insufficient root canal filling accompanied by a mild sensation of pain. A new bacterial composition compromising Atopobium rimae, Anaerococcus prevotii, Pseudoramibacter alactolyticus, Dialister invisus, and Fusobacterium nucleatum was recovered from teeth with chronic apical abscesses. Conclusions: New bacterial combinations were found and correlated to clinical and radiographic findings, particularly to chronic apical abscesses. M. osloensis was detected in root canals for the second time and only in German patients. (J Endod 2014;40:670–677)

Key Words Apical periodontitis, dental trauma, endodontic infection, endodontic microorganisms, root canal treatment

T

he pulp is a very important tissue in teeth and serves several functions (eg, dentine formation and immune response). Microorganisms can enter the pulp cavity through a deep carious lesion, through cracks in fillings or cracks in the tooth, through blood vessels from the apical region, or from the periodontium. Invasion by bacteria will lead to necrosis of the pulp. In this case, a root canal treatment is indicated. The objectives of root canal treatment are adequate cleaning and shaping of the root canal system and elimination of all portals of entry between the root canal and the periodontium by filling the root canal system using a biologically inert and physically stable material (1). The goal of chemomechanical preparation of root canals is to eliminate the pulp tissue and all microorganisms in the pulp cavity. Because of the complex anatomy of the pulp with its ramifications, isthmi, apical deltas, and accessory canals, it is hard to assess the pulp cavity completely. In the literature, different estimates about the prognosis of root canal treatment range from about 62%–98% depending on pre-, intra-, and postoperative parameters (2). The main cause of endodontic failure is the persistence of microorganisms in the root canal system. Recontamination of the root canal system by insufficient coronal restorations or root canal fillings can also lead to endodontic failure (3, 4). It is known that microorganisms can gain resistance against disinfecting agents and endodontic medicaments, increasing the challenge to completely eliminate them during root canal treatment (5, 6). In endodontic infections, gram-positive and gram-negative species could be detected, whereas obligate anaerobes dominated (7, 8). During infection, these species can form biofilms, making the microorganisms up to 1,000 times harder to eliminate using disinfecting agents (9). Secondary endodontic infections are dominated by gram-positive species and facultative and obligate anaerobes (10). Enterococcus faecalis has gained attention by its ability to persist after root canal treatment and has been isolated from both primary and secondary infections although it has been recovered most frequently from secondary/persistent infections (11). The aim of the present study was to analyze the microbiota of primary and secondary/persistent endodontic infections of patients undergoing endodontic treatment with respect to both clinical and radiographic findings. So far, only 40 German patients with post-treatment apical periodontitis have been studied. However, there is

From the *Medical Center, Department of Operative Dentistry and Periodontology and †Department of Microbiology and Hygiene, Institute of Medial Microbiology and Hygiene, Freiburg, Germany. Address requests for reprints to Dr Christian Tennert, Department of Operative Dentistry and Periodontology, University School and Dental Hospital, Hugstetter Straße 55, D-79106 Freiburg, Germany. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.10.005

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Basic Research—Biology no previous study correlating isolated microorganisms with clinical and radiographic findings in a German population.

Materials and Methods Patient Selection Twenty-one patients (25–75 years of age) who had been referred to the University of Freiburg–Medical Center, Freiburg, Germany, for endodontic treatment took part in this study. Patients were excluded from the study if they showed 1 of the following criteria: severe systemic diseases, pregnancy or lactation, use of any antibiotics within the past 30 days, participation in another clinical study during the previous 3 months, teeth that could not be isolated with a rubber dam, teeth without a coronal seal or with restorations with leaky margins, or teeth with large intraradicular posts. Before the beginning of the study, the patients gave their written informed consent to the study protocol, which was reviewed and approved by the ethics committee of the University of Freiburg (140/09). For each patient, a preassembled data sheet was filled out. For primary endodontic infections, clinical and radiographic findings were assessed. Clinical parameters included the presence of a tissue swelling associated with the tooth, pulp vitality via cold testing using CO2, percussion sensitivity, and the quality of coronal restoration. The coronal restoration was examined for cracks/fracture lines, caries, or other defects. Radiographic findings included the presence of periradicular radiolucencies and the diameter of periradicular radiolucency; signs of periapical, external, or internal radicular resorption; caries; and cracks/fracture lines of the root. In case of secondary infections, the radiographic quality of the root canal filling (length and homogeneity) was evaluated. Additionally, if the tooth was symptomatic, the following parameters were recorded: onset of pain (time and duration) and quality of pain (throbbing, stabbing, dull, and so on). With all these diagnostic findings a diagnosis of the tooth was made. Table 1 constitutes an overview of primary and secondary/persistent endodontic infections examined. Sampling Procedure All samples were taken by the same endodontic specialist under strictly aseptic conditions. Each tooth was isolated with a rubber dam. The tooth and the surrounding area were then disinfected with 30% hydrogen peroxide (H2O2) and decontaminated with a 3% sodium hypochlorite (NaOCl) solution. Endodontic access was achieved with a sterile high-speed carbide bur until the pulp cavity or the root filling was exposed. After access was achieved, the tooth and the adjacent rubber dam were disinfected with 30% H2O2 and 3% NaOCl again. NaOCl was then inactivated by swabbing the cavity with 5% sodium thiosulfate solution. To assess the efficacy of the disinfection procedure, 2 sequential sterile foam pellets were moistened in sterile saline solution (0.9% NaCl) and used to swab the access cavity and the tooth surface. They were then transferred into a vial containing 0.75 mL reduced transport

fluid (RTF) (12) and sampled for bacterial growth (quality control). If growth occurred, the patient sample was disqualified from the study. The working length was established radiographically using an electronic apex locator (Raypex 5; VDW, Munich, Germany). The canal was enlarged up to 1–2 mm from the radiographic apex with a minimum size of ISO 35 nickel-titanium K-type file. Teeth that could not be instrumented to this length were excluded from the study. No solvent was used at any time. Approximately 40 mL sterile saline solution was introduced to the canal with a sterile syringe. A sterile Hedstrom file corresponding to the last instrument used was placed in the canal at the working length and pumped vigorously with minimal reaming motion to disrupt the canal contents. Three sequential sterile paper points were placed at the working length and used to soak up the fluid in the canal. Each paper point was retained in position for 1 minute and transferred into a vial containing 0.75 mL RTF. In cases in which the root canal treatment had been previously initiated, the root canals contained an intracanal dressing. The intracanal dressing was either a calcium hydroxide slurry or Ledermix (RIEMSER Pharma GmbH, Greifswald, Germany). The intracanal dressing was removed using 5 mL sterile Ringer solution and sampled as described previously. In root-filled teeth, coronal gutta-percha was removed by using Gates-Glidden drills; the apical material was retrieved with Hedstrom files and, if possible, was then transferred to a sterile tube containing 0.75 mL RTF. After removing gutta-percha, the working length was determined radiographically using an electronic apex locator (Raypex 5; VDW, Munich, Germany). Root canals were enlarged apically and sampled as described earlier. Finally, conventional root canal treatment was finished after root canal disinfection, and the root canal system was filled using the vertical compaction technique.

Isolation of Microorganisms The microorganisms originating from the samples were isolated and identified morphologically by biochemical analysis and sequencing the 16S rRNA genes of the pure isolates as described in earlier studies (12, 13). The undiluted samples were plated. This corresponds to a dilution of 101 of the original root canal bacteria referring to sampling using 3 paper points and stored in 0.75 mL RTF. Additionally, serial dilutions (101–103) were prepared in peptone yeast medium containing cysteine hydrochloride. Each dilution was plated on yeast-cysteine blood agar plates, Columbia blood agar plates, and bile esculin plates. The yeast-cysteine blood agar plates were used to cultivate anaerobic bacteria at 37 C for 10 days (anaerobic jar, Anaerocult A; Merck, Darmstadt, Germany). The Columbia blood agar plates were incubated at 37 C and 5%–10% CO2 atmosphere for 3 days to cultivate facultative anaerobic bacteria. The bile esculin agar plates were incubated at 37 C and 5%–10% CO2 atmosphere for 3–5 days to cultivate E. faecalis. The colonies were differentiated by morphology, color, size, and hemolytic reaction, and colonies were counted to

TABLE 1. Analyzed Cases and Diagnoses of Endodontic Infections Type of infection

Diagnosis

Cases

Primary infection Primary infection Primary infection

Asymptomatic apical periodontitis Crown fracture with exposed pulp After intracanal dressing

Primary infection Primary infection Secondary/persistent infection Secondary/persistent infection

Chronic apical abscess Symptomatic apical periodontitis Insufficient root canal filling Asymptomatic apical periodontitis with insufficient root canal filling

1 2 2 4 1 1 1 9

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Ledermix Calcium hydroxide

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Basic Research—Biology calculate the number of colony-forming units (CFUs) per milliliter. Subsequently, subcultures from all colony types were made to obtain pure cultures.

Differentiation by Morphologic and Biochemical Analysis The bacterial cell morphology was determined using Gram stains and visualized using light microscopy (Axioscope; Zeiss, Jena, Germany; 1,000 magnification). Isolated anaerobic species were differentiated biochemically using routine anaerobic methods including commercially available tests (rapid ID 32 A; Bio Merieux, Marcy-1’Etoile, France and rapid ANA II; Innovativ Diagnostics Systems, Innogenetics, Heiden, Germany). The rapid ID 32 A and the rapid ANA II system use miniaturized enzymatic tests and databases. They are qualitative micromethods using conventional and chromogenic substrates for the identification of medically important anaerobic bacteria isolated from human clinical specimens. The commercially available tests were performed according to the manufacturer’s instructions. Biochemical analysis of facultative anaerobic/aerobic bacteria was performed using enzymatic activities and fermentation of sugars. Commercially available tablets for microbial identification (Rosco Diagnostics, Taastrup, Denmark) and API 20 Strep (Bio Merieux, Marcy-l’Etoile, France) were used for these tests. All tests were performed according to the manufacturer’s instructions. Diagnostic tablets were used for the detection of alkaline phosphatase (differentiation of staphylococci, nonfermenters, viridans streptococci, and anaerobes), indoxyl acetate (differentiation of Campylobacter spp.), and glycosidases (differentiation of Enterobacteriaceae, nonfermenters, staphylococci, streptococci, anaerobes, Neisseria, and Haemophilus). API 20 Strep contains 20 biochemical tests to show enzymatic activities and fermentation of sugars and enables genus or species differentiation of most streptococci and enterococci as well as the most common related microorganisms. Identification by 16S rRNA Gene Sequencing All bacterial isolates were identified using 16S rRNA gene sequencing in order to confirm or to advance the identification. Pure gram-negative bacterial isolates, which were previously identified using biochemical methods, were picked using a sterile inoculating loop and placed into 200 mL lysis buffer (10 mmol/L Tris-HCl buffer, 1 mmol/L EDTA, and 1% Triton X-100; pH = 8.0) followed by boiling for 10 minutes. After centrifugation at 12,000g for 10 minutes, 1 mL supernatant was used for amplification of the 16S rRNA gene. DNA from grampositive bacterial isolates was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 16S rRNA gene amplification was performed in a total volume of 50 mL containing 5 mL 10 polymerase chain reaction (PCR) buffer (Qiagen), MgCl2 (2.5 mmol/L), 200 mmol/L each deoxyribonucleoside triphosphate, 2 U Taq Polymerase (Qiagen), and 300 nmol/L reverse and forward primer. For amplification of the 16S rRNA gene, a set of primers (forward primer TP16U1: 50 -AGAGTTT GATC[C/A]TGGCTCAG-30 and reverse-primer RT16U6: 50 -ATTGTAG CACGTGTGT[A/C]GCCC-30 ) was used as published in a previous study (12). The 1,018–base pair–long PCR products were extracted and purified using the GFX PCR DNA and gel band purification kit (Amersham Biosciences Europe GmbH, Freiburg, Germany). Purified PCR products were sequenced using the BigDye terminator kit v1.1 cycle sequencing kit (Applied Biosystem, Darmstadt, Germany) and the ABI 310 Genetic Analyzer (GMI, Inc, Ramsey, MN). TP16U1 was used as a sequencing primer. Sequencing was repeated up to 3 times. Sequences were 672

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analyzed using the BLAST program from the NCBI (http://www.ncbi. nih.gov/BLAST). Sequence comparison to GenBank data entries served as a confirmation or revision of the detected and identified bacterial strains.

Results In 12 of 21 root canals, 33 different bacterial species could be isolated (Table 2). Six (50%) of the cases with isolated microorganisms were associated with primary endodontic infections, and 6 (50%) with secondary/persistent endodontic infections. The CFUs ranged from 1  103 to 1.49  107 CFU/mL. The largest number of microorganisms was isolated from a tooth with a chronic apical abscess. In 2 of 6 cases with an intracanal dressing, either Ledermix or calcium hydroxide, microorganisms were detected. Fifty percent of the examined teeth presented monomicrobial primary infections, and 50% presented a polymicrobial primary infection. Looking at monomicrobial infections, 2 of 3 cases had an E. faecalis monoinfection, and 1 was a monoinfection with Actinomyces viscosus. Looking at secondary infections, 2 of 6 cases (33%) were monomicrobial infections, and 4 of 6 (66%) cases were polymicrobial infections. E. faecalis could be isolated from 2 polymicrobial infections. Table 3 shows the isolated species with respect to the diagnosis of each tooth. Nineteen different species could be isolated from root canals with asymptomatic apical periodontitis and insufficient root canal filling. A monoinfection of E. faecalis was found after intracanal dressing with calcium hydroxide, and a monoinfection of A. viscosus was found after intracanal dressing with Ledermix. Atopobium rimae, Anaerococcus prevotii, Pseudoramibacter alactolyticus, Dialister invisus, and Fusobacterium nucleatum were recovered from teeth with a chronic apical abscess. Moraxella osloensis was isolated from a symptomatic secondary endodontic infection with an insufficient root canal filling and mild pain sensation. Aerobic and anaerobic species, most frequently gram-positive bacteria, could be isolated from teeth with asymptomatic apical periodontitis with or without insufficient root canal filling, with or without fistula, and with crown fracture and exposed pulp. The greatest variety of microbiota was found among teeth with insufficiently filled root canals and asymptomatic apical periodontitis. Twelve of the isolated species were facultative anaerobic, and 21 were obligate anaerobic. Most root canals showed a polymicrobial infection containing gram-negative bacilli and gram-positive facultative-anaerobic cocci. E. faecalis and Streptococcus mutans were isolated most frequently (19%). Lactobacillus rhamnosus, Parvimonas micra, and A. rimae were isolated in 9.5% of the cases. Streptococcus mitis, Streptococcus sanguis, Streptococcus constellatus, Lactobacillus gasseri, Actinomyces naeslundii, M. osloensis, Eikenella corrodens, A. viscosus, A. prevotii, Bifidobacterium sp., D. invisus, Dialister pneumosintes, Veillonella dispar, Mogibacterium pumilum, Prevotella intermedia, Mogibacterium timidum, Filifactor alocis, Tannerella forsythia, Porphyromonas gingivalis, Campylobacter rectus, and Propionibacterium acidifaciens were isolated in 4.7% of the cases.

Discussion Although there are many reports addressing the microbiota in endodontic infections up to now, only few studies described the bacterial prevalence in German patients (12–15). Moreover, only few studies on the endodontic infections correlated with chronic apical abscesses have been published (15–18). In the present clinical in vivo study, microorganisms were isolated from teeth with a primary or secondary/persistent endodontic infections, and the isolated JOE — Volume 40, Number 5, May 2014

Basic Research—Biology TABLE 2. Detected Species in Primary and Secondary/Persistent Endodontic Infections Frequency

CFU/mL (range)

Diagnosis of tooth

Anaerococcus prevotii Actinomyces naeslundii Actinomyces viscosus Atopobium rimae

Species

1/21 1/21 1/21 2/21

3.0 E + 06 2.0 E + 03 2.0 E + 03 1.2 E + 07

Bifidobacterium sp. Black-pigmented Bacteroides

1/21 2/21

1.0 E + 07 5.0 E + 04

Campylobacter rectus Dialister invisus Dialister pneumosintes Eikenella corrodens Enterococcus faecalis

1/21 1/21 1/21 1/21 4/21

2.0 E + 05 8.0 E + 05 1.0 E + 05 3.0 E + 05 2.8E + 06

Filifactor alocis Fusobacterium nucleatum

1/21 4/21

8.0 E + 05 9.3 E + 06

Lactobacillus casei Lactobacillus gasseri Lactobacillus rhamnosus

1/21 1/21 1/21

2.0 E + 03 3.0 E + 03 2.2 E + 04

Mogibacterium pumilum Mogibacterium timidum Moraxella osloensis Parvimonas micra Porphyromonas gingivalis Prevotella buccae Prevotella intermedia Propionibacterium acidifaciens Propionibacterium propionicus Pseudoramibacter alactolyticus

1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 4/21

5.0 E + 05 4.0 E + 05 5.0 E + 03 1.1 E + 06 3.0 E + 06 5.0 E + 04 7.0 E + 05 5.0 E + 06 6.0 E + 05 9.1 E + 06

Solobacterium moorei Streptococcus anginosus Streptococcus constellatus Streptococcus mitis Streptococcus mutans

1/21 1/21 1/21 1/21 1/21

3.0 E + 06 7.0 E + 05 2.0 E + 05 1.0 E + 03 3.2 E + 06

Tannerella forsythia Veillonella dispar

1/21 1/21

1.0 E + 06 1.0 E + 04

AAP with fistula CF with exposed pulp Intracanal dressing (Ledermix) CAA CF with exposed pulp CF with exposed pulp CAA CF with exposed pulp AAP CAA AAP AAP with insuff RCF AAP with insuff RCF intracanal dressing (calcium hydroxide) CF with exposed pulp AAP with insuff RCF AAP with insuff RCF CAA AAP CF with exposed pulp AAP with insuff RCF CF with exposed pulp AAP with insuff RCF CF with exposed pulp AAP with insuff RCF AAP with insuff RCF Insuff RCF AAP CF with exposed pulp AAP AAP with insuff RCF AAP with insuff RCF AAP with insuff RCF AAP with insuff RCF AAP with insuff RCF CAA AAP AF with exposed pulp AAP with insuff RCF AAP with insuff RCF AAP with insuff RCF AAP AAP with insuff RCF CF with exposed pulp AAP with insuff RCF AAP with insuff RCF

AAP, asymptomatic apical periodontitis; CAA, chronic apical abscess; Insuff RCF, insufficient root canal filling; CF, crown fracture.

microorganisms were correlated with clinical and radiographic signs of the infected teeth. To date, there is no other study correlating isolated microorganisms with clinical and radiographic findings in a German population. In the present study, samples were taken from the main root canal with paper points as described previously (12, 14, 19). Bacteria present in apical ramifications or in dentinal tubules might not have been soaked up by the paper points. Furthermore, microorganisms may be covered by the remaining obturation material even after the removal of the root canal filling because it has been shown that the complete retrieval of the old obturation materials from root canal walls is impossible (12). Thus, because of technical restrictions during sampling, microorganisms causing apical periodontitis may not be detected by culture or molecular techniques and therefore lead to an underestimation of the detected microorganisms in root canals. In Table 4, all isolated species are listed and compared with isolates found in previous studies concerning primary and secondary endodontic infections of patients with different geographic origin. This study showed that polymicrobial infections were detected twice as often in monomicrobial infections compared with secondary/persistent infections. This might be explained by the fact that a secondary JOE — Volume 40, Number 5, May 2014

endodontic infection might take more time to accumulate microorganisms to show radiographic or clinical signs. Microorganisms might enter the filled root canal space and slowly penetrate fins, voids, or tubules. Additionally, microorganisms may enter the pulp cavity during root canal treatment by any contamination of instruments and material used after final irrigation and during root canal filling (20, 21). In the present study, M. osloensis has been isolated from root canals of a German patient for the second time (13). Moraxellae are aerobic, oxidase-positive, nonmotile, and gram-negative coccobacilli and part of the normal flora of human skin and mucosal surfaces. M. osloensis is a rare causative organism of infections in humans. There are reports of M. osloensis causing bacteremia, central venous catheter-related infection, pneumonia, meningitis, and endophthalmitis (22). In this study, M. osloensis was isolated from a symptomatic secondary endodontic infection with mild pain sensation. It was identified by 16S rRNA gene sequencing, which has been reported as a useful method for precise identification (23, 24). In previous studies, E. faecalis has been isolated from primary and secondary/persistent endodontic infections (11) and was also found in secondary infections in monoculture (12). The prevalence of E. faecalis varied among different studies. E. faecalis was found in 9.5% in patients New Bacterial Composition in Endodontic Infections

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Basic Research—Biology TABLE 3. Diagnoses of Teeth and Isolated Species No. of cases

Species

Primary infection

Type of infection

Asymptomatic apical periodontitis

Diagnosis

1

Primary infection

Crown fracture with exposed pulp

2

Primary infection

After intracanal dressing

Primary infection

Chronic apical abscess

Secondary infection

Asymptomatic apical periodontitis with insufficient root canal filling

5

Secondary infection

Insufficient root canal filling

1

Campylobacter rectus Dialister pneumosintes Fusobacterium nucleatum Parvimonas micra Porphyromonas gingivalis Streptococcus mitis Actinomyces naeslundii Atopobium rimae Bifidobacterium sp. Enterococcus faecalis Fusobacterium nucleatum Lactobacillus gasseri Parvimonas micra Pseudoramibacter alactolyticus Streptococcus mutans Actinomyces viscosus Enterococcus faecalis Anaerococcus prevotii Atopobium rimae Dialister invisus Fusobacterium nucleatum Anaerococcus prevotii Pseudoramibacter alactolyticus Eikenella corrodens Enterococcus faecalis Eubacterium tidium Filifactor alocis Fusobacterium nucleatum Lactobacillus casei Lactobacillus rhamnosus Mogibacterium pumilum Prevotella buccae Prevotella intermedia Propionibacterium acidifaciens Propionibacterium propionicus Pseudoramibacter alactolyticus Solobacterium moorei Streptococcus mutans Streptococcus constellatus Streptococcus anginosus Tannerella forsythia Veillonella dispar Moraxella osloensis

Ledermix Calcium hydroxide

with marginal periodontitis (11) and with a range from 29%–77% in patients with apical periodontitis (11, 25). In the present study, E. faecalis was found in 33% of the cases. It was recovered from rootfilled teeth with secondary/persistent apical periodontitis and from root canals with a calcium hydroxide dressing. These findings are consistent with previous results in which E. faecalis was most frequently isolated from root canals with secondary/persistent infections (26). Its resistance against calcium hydroxide, tetracycline, chlorhexidine digluconate, and disinfectants has been described previously (21, 27). E. faecalis is able to synthesize toxins against gram-positive and gramnegative bacteria and is able to form a monomicrobial infection by eliminating other microorganisms (28). Actinomyces viscosus has been found in previous studies in 42% of the cases (29), whereas in the present study, A. viscosus was found in only 5% of the cases. Previously, P. gingivalis, Leptotrichia buccalis, and Porphyromonas endodontalis have been isolated from cases of a chronic apical abscess (16). In the present study, a new bacterial combination consisting of A. rimae, A. prevotii, P. alactolyticus, D. invisus, and F. nucleatum was isolated from root canals with a chronic apical abscess. This underlines the polymicrobial character of endodontic infections and adds new etiologic aspects for the development of fistula accompanying root canal infection. D. invisus was previously found 674

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1 1 1

in primary and secondary endodontic infections with 16S rRNA genebased nested and seminested PCR assays (30, 31). Moreover, in the aforementioned studies, D. invisus was revealed to be the most prevalent species (81% of the teeth). In another study with PCR, D. invisus was found in 48% of the samples of failing root-filled teeth and was one of the most prevalent species (12). As far as the application of PCR is concerned, the incapability of this method to differentiate between living and dead microorganisms has to be considered. Especially in cases in which endodontic microorganisms form biofilm, extracellular DNA could be detected by PCR because DNA has been detected as part of the extracellular polymeric substances of biofilms (32). In previous studies, F. nucleatum has been isolated from root canals and the periodontium, showing a prevalence of 4%–62% (33). In combination with P. micra and Capnocytophaga sputigena, F. nucleatum is assumed to correlate with periodontal-endodontic infections (34). F. nucleatum has previously been isolated from root canals with apical periodontitis in 53% of the cases (35). Recently, F. nucleatum showed a high prevalence of 70% in primary and secondary root canal infections (36). This is consistent with the findings of the current study in which F. nucleatum could be isolated from primary endodontic infections and root canals with a periapical lesion. P. JOE — Volume 40, Number 5, May 2014

Basic Research—Biology TABLE 4. Comparison of Microbial Profiles of Primary and Secondary Endodontic Infections Found in Previous Studies Comparing Their Geographic Origin Current study isolation Species

Primary infections

Secondary infection

Isolation from primary infections

Anaerococcus prevotii Actinomyces naeslundii

X X

Brazil China US

Actinomyces viscosus

X

China US

Atopobium rimae

X

Brazil

Bifidobacterium sp.

X

Black-pigmented Bacteroides

X

Campylobacter rectus Dialister invisus

X X

Brazil Netherlands Sweden Sweden US Brazil Brazil United Kingdom

Dialister pneumosintes Eikenella corrodens

X

Enterococcus faecalis

X

X

Filifactor alocis Fusobacterium nucleatum

X

X X

Brazil Brazil Japan Netherlands US

Lactobacillus casei Lactobacillus gasseri

X X

Lactobacillus rhamnosus

X

Brazil Brazil Sweden Japan US Japan Japan

X

Mogibacterium pumilum Mogibacterium timidum

X X X

Moraxella osloensis Parvimonas micra

X

X

Porphyromonas gingivalis

X

Brazil Japan Brazil Romania Brazil Italy United Kingdom US

— Brazil Brazil US

Prevotella buccae

X

Prevotella intermedia

X

Propionibacterium acidifaciens Propionibacterium propionicus

X X

Brazil Germany Brazil China Brazil Sweden

X

Brazil

Pseudoramibacter alactolyticus

X

Isolation from secondary/ persistent infection — Brazil Italy Japan United Kingdom US Brazil China Lithuania US Germany Japan US Brazil — Brazil Brazil Germany Japan Brazil Germany USA Brazil Germany Japan Lithuania Sweden Switzerland UK US Brazil Brazil Germany Japan Sweden UK Latvia Germany Latvia Latvia Japan Japan UK Germany Germany US Brazil China Germany Italy Japan Brazil Brazil US — Brazil China Italy Sweden Brazil Japan Sweden US (Continued )

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Basic Research—Biology TABLE 4. (Continued ) Current study isolation Species

Primary infections

Secondary infection

Isolation from primary infections

Solobacterium moorei

X

Brazil

Streptococcus anginosus

X

Brazil USA

Streptococcus constellatus

X

Brazil

Streptococcus mitis

X

Streptococcus mutans

X

Brazil

X

Brazil Sweden

Tannerella forsythia

X

Bosnia and Herzegovina Brazil

Veillonella dispar

X

Japan

alactolyticus has been associated with acute periradicular periodontitis (32%) and acute apical abscesses (62%) (17). In another previous study, it has been isolated in 33.3% of 489 root canals (37). In the present study, P. intermedia and Prevotella buccae were found in 1 asymptomatic tooth with secondary/persistent endodontic infection with periapical radiolucency without pain sensation. In previous studies, they have been associated with persistent infections, pain (positive percussion sensitivity), swelling, and chronic apical abscess (15, 18). In the root canal, fastidious microorganisms (eg, spirochetes, members of Deferribacteriaceae or Atopobium spp.) may also be present. These microorganisms cannot be detected by culture methods. Therefore, specific DNA probes (PCR) have been used to detect unculturable microorganisms (31). However, gene detection using PCR is a very sensitive method of bacterial identification and detecting endodontic microorganisms using this method can lead to an overestimation of the recovered species (38). Moreover, DNA of dead microorganisms in the root canal system or the diffusion of DNA from the oral cavity into the root canal may lead to false-positive results. This underlines the importance of culture methods to cultivate viable microorganisms. In a recent comprehensive study, culture method was combined with independent cloning technique (13). Taking into account that culture media select for certain bacteria, some fastidious species, especially anaerobic bacteria, might have escaped detection using the culture technique. Hence, monoinfections should be considered critically. Only a combination of the culture as well as culture-independent techniques would achieve a comprehensive microbial analysis of endodontic infections (13). In this study, only the combination of morphologic and biochemical analysis with identification by 16S rRNA gene sequencing revealed a realistic composition of infectious microorganisms in root canals. Therefore, a combination of methods for studying the etiologic agents involved in root canal infections is recommended. Alternate identifications by commercially available microbial identification kits may lead to false determination of the microbiota of endodontic infections. This was extensively reported by our group for Vagococcus fluvialis, which 676

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Isolation from secondary/ persistent infection Brazil Germany UK Brazil Sweden UK US Brazil China Japan Norway Brazil China UK Sweden Brazil China Germany Japan Brazil Germany Israel Japan Norway UK

could be misidentified as the taxonomically related E. faecalis (39). Hence, we combined the biochemical identification with sequencing of the 16S rRNA genes for all isolates. This was particularly necessary for the following identified bacteria: A. prevotii, A. rimae, Bifidobacterium spp., Dialister spp., Filifactor alocis, Lactobacillus spp., Mogibacterium spp., P. intermedia, Propionibacterium spp., P. alactolyticus, Solobacterium moorei, and V. dispar. Previous studies associated certain microbial species with persistent infections as well as sensitivity to percussion pain, swelling, and chronic apical abscess (15, 18). In the present study, P. gingivalis was found in 1 root canal of a persistent endodontic infection with periapical radiolucency without pain sensation. P. gingivalis has been associated with pain in previous studies (16, 40). Nevertheless, in the present study P. gingivalis was isolated from a root canal with asymptomatic apical periodontitis. In the present study, there were 2 cases of positive percussion sensitivity in which microorganisms could be isolated (M. osloensis and A. viscosus), but no correlation between pain and the isolated microorganisms could be found. More than 400 different microbial taxa have been identified in endodontic samples from teeth with different forms of apical periodontitis. These taxa are usually found in combinations involving many species in primary infections and a few ones in secondary/persistent infections (41). A community concept has been published by Siqueira and Rocas (42). Some gram-negative anaerobic bacteria have been suggested to be involved with symptomatic lesions, but the same species may also be present in asymptomatic cases (43). Additionally, the high interindividual variability observed for samples from the same clinical disease indicates that different compositions of bacterial communities can result in similar disease outcome (42). The diversity of bacterial communities has been found to differ significantly when the microbiota of asymptomatic apical periodontitis and acute apical abscesses are compared (44). Differences are essentially represented by different dominant species in the communities and the large number of species in abscesses (43). Endodontic abscesses are polymicrobial infections whereby bacterial species that individually may have low virulence and are unable to cause disease JOE — Volume 40, Number 5, May 2014

Basic Research—Biology can do so when they occur in association with others as part of a mixed consortium (pathogenic synergism) (45, 46). There are indications in the literature of synergistic infections of anaerobic bacteria inducing encapsulation of other microorganisms to enhance their virulence (45). In another previous study, a synergism of a specific combination of bacteria and the presence of Bacteroides melaninogenicus and Bacteroides asaccharolyticus was described to cause the shift from an asymptomatic apical periodontitis to an apical abscess (46). Because most of the new microbial compositions include species that were previously found outside the root canal system, further studies should focus on virulence factors, pathogenicity, and the pathogenic role of certain strains, especially F. nucleatum, A. rimae, D. invisus, and M. osloensis. Additionally, many potent virulence factors of E. faecalis such as gelatinase, hyaluronidase, bacteriocins, aggregation substance, and lipoteichoic acid were suggested to play an important role in the pathogenicity, biofilm formation, and the expression of apical periodontitis (28). Hence, studies investigating the role of virulence factors and new isolates need to clarify the role of pathogenic microorganisms for the development of apical disease. Furthermore, the pathogenicity of combinations of different microorganisms should be investigated to reveal synergisms or antagonisms in the development of endodontic infections and apical periodontitis.

Acknowledgments The authors thank Dr Annette Anderson for her assistance in reviewing the manuscript. Supported by the German Research Foundation (DFG, AL 1179/1-1). The authors deny any conflicts of interest related to this study.

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New Bacterial Composition in Endodontic Infections

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secondary endodontic infections with respect to clinical and radiographic findings.

The aim of the present study was to analyze the microbiota of primary and secondary/persistent endodontic infections of patients undergoing endodontic...
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