|Year : 2014 | Volume
| Issue : 2 | Page : 83-89
Detection of bacteria in endodontic samples and its association with defined clinical signs and symptoms of endodontic infection
Shaikha Al-Samahi1, Mohammad A Al-Omari2
1 Ministry of Health, Fujairah Dental Center, Fujairah, United Arab Emirates
2 Department of Restorative Dentistry, Riyadh Colleges of Dentistry and Pharmacy, Riyadh, Kingdom of Saudi Arabia
|Date of Web Publication||12-Aug-2014|
Prof. Mohammad A Al-Omari
Riyadh Colleges of Dentistry and Pharmacy, P.O. Box 84891, Riyadh 11681
Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
Introduction : The role of bacteria in pulpal and periradicular lesions is well established. However, the association of bacterial species with signs and symptoms of endodontic infection is not well elucidated. The purpose of this study was to investigate the types of bacterial species in infected root canals (using MicroSeq 500 16S ribosomal ribonucleic acid, rRNA) and the association of bacterial species with clinical signs and symptoms of different pulpal and periapical infections.
Materials and Methods: Microbial samples were obtained from 92 adult patients (one tooth with single root canal for each patient). Clinical data including signs and symptoms were collected. Polymerase chain reaction (PCR) (MicroSeq 500 16S rRNA) was used to detect bacteria in the infected root canals.
Results: A total of 179 bacterial species were identified. Actinomyces viscosus and Streptococcus sanguis were associated with the presence of pain. While Staphylococcus haemolyticus and Veillonella spp. were associated with pain of palpation, the pain to percussion was associated with Actinomyces spp., Actinomyces naeslundii, and Actinomyces viscosus. Also, Streptococcus mitis, Bacteroides spp., and Veillonella spp. were associated with periapical swelling. The presence of sinus tract was found to be significantly associated with Neisseria spp. and Staphylococcus haemolyticus. The wet canal was significantly associated with the presence of Propionibacterium acnes and Bacteroides spp.
Conclusions: There are significant associations between the presence of some bacterial species with clinical signs and symptoms of infected root canals.
Keywords: Endodontic microbiology, polymerase chain reaction, root canals
|How to cite this article:|
Al-Samahi S, Al-Omari MA. Detection of bacteria in endodontic samples and its association with defined clinical signs and symptoms of endodontic infection. Saudi J Oral Sci 2014;1:83-9
|How to cite this URL:|
Al-Samahi S, Al-Omari MA. Detection of bacteria in endodontic samples and its association with defined clinical signs and symptoms of endodontic infection. Saudi J Oral Sci [serial online] 2014 [cited 2019 May 21];1:83-9. Available from: http://www.saudijos.org/text.asp?2014/1/2/83/138470
| Introduction|| |
It is now well known that presence of microorganisms in the root canal leads to the development of periapical periodontitis. , Also, micro-organisms are found to be the main cause of development or persistence of a periapical lesion in a well endodontically treated tooth. ,,
The microbiota of primary endodontic infections consists of a mixture of a large variety of bacterial species, with predominance of anaerobic bacteria, such as Porphyromonas, Prevotella, Fusobacterium, Peptostreptococcus, and Campylobacter species, as well as facultative organisms, such as Streptococcus species. , However, the root canal microbiota after failed treatment consists of a monoinfection with predominance of gram-positive facultative anaerobes (especially Enterococcus faecalis). ,,,,
Many studies have confirmed the association of signs and symptoms of root canal infection with some bacterial species. ,,,,,
Different methods have been used to detect and identify microorganisms in the infected root canals, such as; culture, ,,,, deoxyribonucleic acid (DNA)-DNA hybridization,  polymerase chain reaction (PCR), , or denaturing gradient gel electrophoresis fingerprint techniques.  The microbiological molecular diagnostic methods have improved the identification of microbiota related to endodontic infections. PCR amplification of conserved regions of the 16S rRNA gene (rDNA) has been widely applied to study bacterial species in the endodontic infections. One of molecular methods available in the market is MicroSeq 500 (Applied Biosystems, USA) 16S rRNA sequencing. In this system, the first 500 bp fragment of the 16S rRNA gene of the bacterial strains is amplified, sequenced and analyzed using the database of the system.  This system has not been used before in the investigation of microflora of the infected root canal.
The aim of this study was to investigate the bacterial species most commonly isolated from infected canals with different clinical signs and symptoms of primary (irreversible and necrotic pulp) and secondary endodontic infections (pulpless or failed root canal treatment) with different clinical periapical diagnosis using MicroSeq 500 16S rRNA in order to find new associations and/or confirm the other association reported in previous studies.
| Materials and Methods|| |
Subject selection and sample collection
Samples were taken from patients attending the Endodontic clinic in Fujairah Dental Center. Patients were informed about the study's nature and goals and they signed the consent form before participating in the study.
This work was approved by The Institutional Research Board (IRB) of Jordan University of Science and Technology (JUST). The total number of patients in the study was ninety two (92), including 55 (59.8%) females and 37 (40.2%) males with ages ranging between 15-50 years. One tooth with a single canal for each patient was included in the study.
Patients with periodontal pockets greater than 4 mm, who were pregnant or nursing or had difficulty in application of the rubber dam were excluded from the study.
Medical and dental history was reviewed and clinical and radiographic examination was performed. The following data were recognized for each patient: Age, gender, chief complaint, tooth type, presence of caries, restoration, nature of pain, previous pain, tenderness to percussion, pain on palpation, mobility, sinus and its origin, swelling of periodontal tissues, depth of periodontal pocket, previous antibiotic therapy, and internal condition of the root canal (presence of exudates or pus).
Vitality and periapical tests were performed and periapical radiographs were taken with parallel technique. Then pulpal and periapical diagnoses were carried out according to the clinical signs and symptoms  as follows:
- Pulpal Diagnosis; Irreversible pulpitis, Necrotic pulp, Treated root canal.
- Periapical Diagnosis; Acute periapical periodontitis, Chronic periapical periodontitis, Apical periodontitis with abscess, Apical periodontitis with sinus tract.
The crown of the involved tooth was cleaned with pumice and patients were asked to rinse with 0.2% chlorohexidine solution for 30 seconds. After that, tooth isolation was performed with rubber dam. Then, the crown of the tooth and the surrounding rubber dam were disinfected with 30% hydrogen peroxide (H2O2) for 30 seconds followed by 2.5% sodium hypochlorite (NaOCl) for an additional 30 seconds.  Subsequently, 5% sodium thiosulfate (Na2S2O3.5H2O) was applied to the tooth to neutralize the effect of chloride on sampling.
After that, access cavity was prepared under irrigation with sterile saline (in some cases local anesthesia was administrated). Access was performed with sterile Gates Glidden and burs. If the root canal was dry, a small amount of sterile saline solution was introduced into the canal, and then the canal was negotiated to the estimated length. In previously root canal treated teeth, gutta-percha was removed without using solvent.
Microbial samples were collected with two sterile paper points (Hygenic® Ster-ICell Paper Points), which were placed consequently into the canal to full length for 1 minute. The paper points of each root canal were pooled in a sterile tube containing 5 ml anaerobic dental transport medium (Dental Transport, Anaerobic System, USA).
Surface decontamination test
Swab cultures obtained from the walls of the access cavities before entering the pulp spaces were transported into the microbiology laboratory and processed within 10 minutes. The presence of bacterial colonies on the agar plates indicated contamination of the sampled site and the tooth was excluded from the study.
All samples were transported immediately to the microbiology laboratories for isolation. One of the paper point samples was plated onto Schaedler Anaerobe Agar (HOSPITECH, Dubai, UAE), which had been supplemented with Vitamin K1 and 5% sheep blood. The plates were incubated at 37°C in an anaerobic environment (5% H2, 5% CO2, 90% N2) (GasPak system, Microbiology Systems, UAE) for 5-7 days. The other paper point was plated into Columbia blood agar (HOSPITECH, Dubai, UAE) for incubation aerobically in 5 % CO2 at 37°C for 2 days and then stored at 4°C until the PCR procedure was performed.
Bacterial identification was performed by using 16S rRNA PCR (MicoSeq 50016S ribosomal ribonucleic acid, rRNA). The MicoSeq 500 (Applied Biosystems, USA) 16S rRNA bacterial identification kit was composed of PCR and cycle sequencing kits, analysis software, and a 500-bp 16S ribosomal deoxyribonucleic acid (rDNA) library of bacterial nucleic acid sequences. PCR and sequencing kits are designed with universal primers to cover all bacteria. The procedures were done according to the manufacturer's instructions.
All data were analyzed with the Statistical Package for Social Science (SPSS) computer software (version 11.0, SPSS, Inc., Chicago, IL, USA). A Pearson chi-square (χ2 ) test or a one sided Fisher's exact test, as appropriate, was used to test null hypothesis that there was no association between the prevalence of specific bacterial species and clinical signs and symptoms of endodontic infection. A probability value (P-value) of equal or less than 0.05 was considered statistically significant.
| Results|| |
The total number of the study sample was 92 patients (one tooth per patient) that included 55 (59.8%) females and 37 (40.2%) males. The teeth with single root were only included in this study. These teeth consisted of central incisors (18.5%); lateral incisors (22.8%); first premolar for the lower arch (3.3%); and second premolar (41.3%). Out of these teeth, 80.4 % were located in the upper arch and 19.6 % were located in the lower arch.
The signs, symptoms, and clinical test showed that 10 teeth presented with the history of pain, 41 teeth were presented with pain, 22 teeth were sensitive to cold, 21 teeth sensitive to heat, 22 teeth were presented with positive response to electrical pulp test, 38 teeth presented with pain to palpation, 58 teeth presented with pain to percussion, 9 teeth presented with swelling, 8 teeth presented with sinus tract and 13 teeth were presented with wet canal. The periapical radiographs revealed that; 36 teeth were present with normal periapical radiograph, 12 teeth were showed widening or thickening of periodontal lamina in the radiograph, 32 teeth showed 1-3 mm radiolucent change, 11 teeth were showed radiolucent change more than 3 mm, and 1 tooth showed radio-opaque changes in the periapical area. A total of 92 samples were subjected to PCR with 16S rRNA (MicroSeq 500 16S rRNA). Bacteria were present in the 92 teeth and the total bacterial species was 179 [Figure 1]. The species which were identified in this study were classified according to their phyla. The number of phyla found in this study was four; Actinobacteria (57.0%), Firmicute (27.4%), Proteobacteria (12.3%) and Bacteroides (3.4%).
In the irreversible pulpitis cases, the bacterial species number ranged from 1 to 4 species per canal while in necrotic pulp, the range was from 1 to 5 species per canal. On the other hand, the pulpless cases the range was from 1 to 2 species per canal. This association between the species number and pulpal diagnosis was considered statistically significant (P = 0.00). At the same time, there was a significant association (P = 0.00) between the species number per canal and the periapical diagnosis of endodontic infection.
The results showed significant association between the presence of some species with signs and symptoms of infected root canals [Table 1]. Also, there was a significant association between the presences of some bacterial species and pulpal diagnosis and periapical diagnosis [Table 2] and [Table 3].
|Table 1: Bacterial species significantly associated with signs and symptoms|
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|Table 2: Summary of bacterial species associated significantly with pulpal diagnosis|
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|Table 3: Summary of bacterial species associated significantly with periapical diagnosis|
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Some strains were found to be significantly associated with the gender of the patients, such as Streptococcus mitis (P = 0.01), Actinomyces naeslundii (P = 0.02) and Bacteroides spp. (P = 0.00) which were associated with more male patients than female patients.
| Discussion|| |
The present study aimed to investigate the microbiological profiles of clinical cases of endodontic infection including irreversible, necrotic and pulpless teeth with different periapical diagnosis which consisted of: normal; acute apical periodontitis; chronic apical periodontitis; apical periodontitis with sinus; and apical periodontitis with abscess, by using the MicroSeq500 16S-rDNA technique. In addition, the study aimed to find if there is an association between the presence of some bacterial species in the infected root canal and the presence of clinical signs and symptoms of endodontic infections.
Primary infected root canal refers to the presence of bacteria in the untreated infected root canal. While secondary infected root canal refers to the failure of endodontic treatment. In this study, we investigated the types of bacterial species in the cases which had been classified clinically as irreversible pulpitis with normal or acute apical periodontitis, which (to the best of our knowledge) no single study has investigated all these variables together. Most of these studies included primary (necrotic) and secondary (failed root canal treatment) with or without periradicular lesion either asymptomatic or symptomatic. ,
The results of the microbiological analysis of this study revealed that bacteria were found in all infected root canals. Most of the bacterial species found belong to the phylum of Actinobacteria followed by Firmicutes, Proteobacteria and Bacteroidetes. Other studies found that most of the bacterial species belonged to the Firmicutes and Bacteroidetes phyla. , This difference in the phyla can be due to the number of the samples, type of the root canal infection involved in the study and the type of microbiological tests used.
This study revealed that, the bacterial species of the primary (irreversible and necrotic) endodontic infection is clearly different from the secondary (pulpless) endodontic infection. Irreversible pulpitis was associated with the presence of Actinomyces viscosus, while the necrotic pulp associated with Actinomyces odontolyticus, Corynebacterium spp. and Bacteroides spp. The difference in the types of bacterial species between irreversible and necrotic endodontic infections has not been studied previously.
Also, this study found that, Enterococcus faecalis was the most frequently isolated species from the pulpless root canal. This finding agrees to a large extent with the findings of several studies, ,,,,, which found that Enterococcus faecalis was associated with persistent endodontic infections. Other studies ,,,,, found that Enterococcus faecalis was a frequent species in root canal treated teeth with apical periodontitis lesions, which are similar to our findings, as we found about 78.6% of them associated with chronic periapical periodontitis. The frequent occurrence of Enterococcus faecalis in cases of failed endodontic therapy suggested that this species can play a relevant role in the etiology of persistent periradicular lesions. This is due to the fact that, Enterococci are able to survive for a long time with a minimum amount of nutrients.  In addition, Enterococcus faecalis has the ability to invade the dentinal tubules, which can allow them to resist the chemomechanical preparation and intracanal medication such as calcium hydroxide.  At the same time, our results showed that 71.4% of Enterococcus faecalis was found as a single species in the infected root canal which is inline with other studies. , This species does not depend on commensalism with other bacterial species and can survive without other species. These factors help to explain the presence of these species in the secondary infected root canal (failed root canal treatment). 
Other bacterial species were found in the pulpless root canal infection in this study. However, these species were not significantly associated with pulpless root canal. These species were Achromobacter spp., Actinomyces spp., Neisseria elongate, Propionibacterium acnes, Pseudomonas aerugionosa, Pseudomonas boreopolis, Streptococcus angionosus, Streptococcus sanguis, and Veillonella spp. Most of these species were found significantly associated with failed root canal treatment by other studies. ,,,
In this study, we also found that there was small percentage of Enterococcus faecalis (7.1%) in primary root canal infection (irreversible and necrotic pulp). This result is in line with findings of Baumgartner and Falker (1991) and Souza et al. (2005), who found this species in lower percentage in primary endodontic infections (necrotic). , On the other hand, other studies found high prevalence of this species in primary endodontic infection. 
In addition, our study revealed that irreversible pulpitis and necrotic pulp are polymicrobial infections. This supports the findings of other studies. ,,,, Alternatively, we found that most pulpless teeth harbor one species and few teeth harbor two species, which confirm the monoinfection of secondary root canal infection. , Interestingly, there were some bacterial species found in canals which clinically had a normal periapical condition or had a radiograph that showed normal periapical tissues with no periapical lesions. This finding can suggest that the lack of visible periapical radiolucency in radiograph or the lack of signs and symptoms of infected periapical tissues clinically does not necessary imply the absence of periapical pathosis. Simply, this means that normal appearance of periapical tissues of infected root canals (in clinical and radiograph) does not necessarily mean that the root canal is free from bacteria. For that, clinicians should not depend on X-ray only to exclude the presence of periapical lesions in infected root canals. 
The apical periodontitis with abscess was found to be associated with Corynebacterium spp., Streptococcus mitis, Actinomyces naeslundii, Propionibacterium acnes, Bacteroides spp., and Veillonella spp. This result confirmed the findings of Khemaleelakul et al. (2002). 
In the present study, asymptomatic cases were associated with Neisseria spp., Bacteroides spp., and Actinomyces odontolyticus. While Sassone et al. (2008) found high level of Propionibacterium acnes in similar cases.  Interestingly, acute symptoms of pain were associated with the presence of Actinomyces viscosus and Streptococcus sanguis which was not detected by any previous reports. ,
Also, we found that, the pain to palpation was associated with Staphylococcus haemolyticus and Veillonella spp., which has not been observed in previous studies such as those of Jacinto et al. (2003) and Sassone et al. (2008). , On the other hand, teeth which are tender to percussion in our study were associated with Actinomyces spp., Actinomyces naeslundii and Actinomyces viscosus which are in line with the findings of Jacinto et al. (2003). 
In this study, when the endodontic infection presented with swelling, it was associated with Streptococcus mitis. This finding is consistent with some previous studies. , Also, this study revealed that other species (Propionibacterium acnes, Corynebacterium spp., Actinomyces spp., Actinomyces naeslundii, Bacteroides spp., Streptococcus mitis, Veillonella spp.) were found in canal infection with swelling, which were not reported before, therefore, further investigations are needed to confirm their role in these cases.
Wet canal refer to the presence of clear, hemorrhagic, or purulent exudates. In our study, the samples of root canal exudates are associated with Propionibacterium acnes.  In addition, our study found wet canals associated with Bacteroides spp. and Veillonella spp..  Interestingly, we found Corynebacterium spp., Actinomyces naeslundii and Streptococcus mitis were associated with wet canal, to the best of our knowledge this result was not found in any previous study. Also, in this study, the sinus tract was associated with Actinomyces naeslundii. This result is in line with the study of Pinheiro et al. (2003a).  We also found wet canal associated with Staphylococcus haemolyticus and Neisseria spp. which was not detected by similar studies like those of Haapasalo et al. (1987a), Gomes et al. (1994) and Pinheiro et al. (2003a). ,,
The present study, showed that there is an association between positive response to cold, heat and electrical pulp test with Actinomyces viscosus while the negative response to these vitality tests was associated with Corynebacterium spp. and Actinomyces odontolyticus. Only Chin-LoHahn et al. (1993)  associated the presence of Actinomyces viscosus associated with the positive and prolonged response to cold test, which is similar to our finding.
Interestingly, some important endodontic species, such as Fusobacterium nucleatum and the black-pigmented anerobes Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella nigrescens, and Peptostreptococcus micros, could not be detected in our study. This can be due to sampling technique, transport media or due to the molecular method which we used.
The microbiologic sampling procedure used in the present study was effective. , In our study, we found one species of Actinomyces C84 which has not been detected in any other study before. Routine culture technique helps us to get an overview of the cultivable bacteria of an individual infected root canal sample and the need to detect multiple organisms. PCR has been widely applied to study of bacterial species in the endodontic infections. Also, DNA sequencing of the 16S rRNA gene using MicroSeq 500 is a good method for identifying bacteria at the species level. 
| Conclusion|| |
In conclusion, the results of this study confirm the association of some species with clinical signs and symptoms of endodontic infections. The bacterial species of primary endodontic infections is clearly different from that of secondary endodontic infection. In addition, the polymicrobial nature was found in primary root canal infection, while mono-infection was found in secondary root canal infection. We also conclude that the absence of periapical changes in the radiograph does not mean that the bacteria and infection of root canal and its periapical tissue are absent, since bacteria were found in cases with normal periapical tissue on the radiograph and in cases that were classified clinically having normal periapical status.
| Acknowledgements|| |
We would like to thank Advance Biotechnology Center (ABC) in United Arab Emirates (Dubai) and we express our sincere appreciation to Dr. Sanjeet Mishra for his technical support. This research was supported by a grant from the Jordan University of Science and Technology (Grant Number 105/2008) Irbid, Jordan.
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[Table 1], [Table 2], [Table 3]