ORIGINAL RESEARCH


https://doi.org/10.5005/jp-journals-10005-2706
International Journal of Clinical Pediatric Dentistry
Volume 16 | Issue 06 | Year 2024

Evaluation of Presence of Enterococcus faecalis in Root Canals of Deciduous Molars with Necrotic Pulp by Agar Culture and Polymerase Chain Reaction


Triveni M Nalawade1, Kishore G Bhat2, Alka D Kale3, Suma Sogi4, Shivayogi M Hugar5https://orcid.org/0000-0003-3657-6709, Vijay M Kumbar6https://orcid.org/0000-0001-6261-1665, Rachappa M Mallikarjuna7https://orcid.org/0000-0001-8793-5842

1,7Department of Paediatric Dentistry, Oman Dental College, Muscat, Oman

2Department of Microbiology, Dr Prabhakar Kore Basic Science Research Center, Belagavi, Karnataka, India

3Department of Oral Pathology, KLE Academy of Higher Education and Research (Deemed to be University), V K Institute of Dental Sciences, Belagavi, Karnataka, India

4Department of Pedodontics & Preventive Dentistry, M M College of Dental Sciences and Research, Ambala, Haryana, India

5Department of Pedodontics and Preventive Dentistry, KLE Academy of Higher Education and Research (Deemed to be University), V K Institute of Dental Sciences, Belagavi, Karnataka, India

6Department of Basic Science (Molecular Biology), Dr Prabhakar Kore Basic Science Research Center, Belagavi, Karnataka, India

Corresponding Author: Triveni M Nalawade, Department of Paediatric Dentistry, Oman Dental College, Muscat, Oman, Phone: 91990834, e-mail: triveni_nalawade@rediffmail.com

ABSTRACT

Objective: To assess the presence of Enterococcus faecalis in root canals of deciduous molars with necrotic pulp by agar culture and polymerase chain reaction (PCR) assay.

Materials and methods: This is an experimental study, where a total of 120 endodontic samples were taken from deciduous molars with necrotic pulps. The presence of Enterococcus faecalis was assessed by culture, using Enterococcus confirmatory agar, and by PCR assay. Statistical analysis of the results was performed using McNemar’s test.

Results: The presence of Enterococcus faecalis was detected in 20 samples (16.67% of total) by microbial culture and in 45 samples (37.5% of total) by PCR assay, with a statistically significant difference between the two methods (p < 0.001). Microbial culture and PCR both detect Enterococcus faecalis, with the latter detecting an additional 25 positive samples.

Conclusion: In this study, PCR assay was significantly more sensitive than agar culture method in detecting the presence of Enterococcus faecalis in root canals of deciduous molars with necrotic pulp, that is, 37.5% of all samples.

Clinical significance: Importance of presence of Enterococcus faecalis in necrotic pulps of deciduous teeth, as it is primarily responsible for failure of endodontic treatment, thus helping clinicians to advocate the use of local drug delivery in primary teeth endodontics and also aids clinicians in choosing the most effective intracanal medication.

How to cite this article: Nalawade TM, Bhat KG, Kale AD, et al. Evaluation of Presence of Enterococcus faecalis in Root Canals of Deciduous Molars with Necrotic Pulp by Agar Culture and Polymerase Chain Reaction. Int J Clin Pediatr Dent 2023;16(6):816–819.

Source of support: Nil

Conflict of interest: None

Keywords: Deciduous tooth, Dental pulp necrosis, Enterococcus faecalis, polymerase chain reaction, Root canal medicaments

INTRODUCTION

Successful endodontic therapy of teeth with necrotic pulp depends upon reduction or elimination of infecting bacteria.1 Necrotic pulp tissue harbors an array of microbiota, with anaerobes like Porphyromonas gingivalis, Prevotella intermedia, Porphyromonas endodontalis, and Enterococcus faecalis, to name a few.25Enterococcus faecalis, most commonly recovered from teeth with failed endodontic therapy,68 was recently also found in primarily infected teeth.9,10

Enterococcus faecalis is a gram-positive facultative anaerobe occurring solitary, in pairs or short chains, and can thrive in extreme conditions like alkaline pH of 9.6 and temperature of 60°C for as long as 30 minutes. It is a virulent bacteria due to the presence of lytic enzymes like cytolysin, pheromones, and lipoteichoic acid, suppressing the action of lymphocytes, hence, potentially contributing to endodontic failure.11,12Enterococcus faecalis, being small in size, invades dentinal tubules and can endure prolonged periods of starvation.13 It can also utilize serum originating from alveolar bone and periodontal ligament as a source of nutrition and synthesize an array of stress proteins when exposed to adverse environmental conditions.14 Such a microorganism situated in inaccessible parts of root canals of deciduous tooth with necrotic pulp may be difficult to eradicate due to the complex root canal morphology and anatomical irregularities.15

A recent study by Fabris et al. using polymerase chain reaction (PCR) found that Enterococcus species and Porphyromonas gingivalis were prevalent in the endodontic samples from deciduous teeth with necrotic pulp.16 Studies to date suggest that Enterococcus faecalis constitutes a small percentage of the microbial species isolated from endodontic samples of necrotic teeth.7 Until recently, agar culture has been a predominant method of detecting microorganisms in microbiological samples. With the advance of molecular biology, more sensitive, accurate, and rapid methods like PCR are becoming a standard,7,17 although the difference in ability of detecting Enterococcus faecalis by means of culture and PCR in endodontic samples was reported not to be significant in isolated studies.7

As there are few published studies assessing presence of highly pathogenic Enterococcus faecalis by both culture and PCR in necrotic pups of deciduous dentition,7 the present study was undertaken to determine its presence in the latter and to compare both methods’ capability in detecting Enterococcus faecalis.

MATERIALS AND METHODS

A total of 120 endodontic samples were taken from 40 teeth (three root canals per tooth) from 38 (17 females and 21 males) 5–8-year-old children attending the Outpatient Department of Pedodontics and Preventive Dentistry. Ethical committee approval No. Ethic/14-15/D-73 was taken prior to start of the study. Informed consent was taken from each parent, and assent was taken from children, too. A proforma was recorded for each patient comprising of case history recording, along with investigations and diagnosis. Deciduous molars with necrotic pulps, including ones with abscesses or draining sinuses, were included in the study. The procedures followed were in accordance with the ethical standards of the Human Ethical Committee and with the Helsinki Declaration of 1975, as revised in 2000.

Patients willing to participate in the study and fulfilling the inclusion and exclusion criteria were included for the study.

Selected teeth had to be fully isolated with rubber dam. Patients with systemic disease or treated with antibiotics within last 3 weeks were excluded. Teeth with open carious lesions and teeth with resorption of more than one-third of the root, internal resorption, or external resorption were excluded from the study.

Sample Collection

All samples were collected by an experienced pediatric dentist. A pilot study consisting of six samples was carried out to standardize all procedures, including disinfection, isolation, sample collection, culture, and deoxyribonucleic acid (DNA) isolation, but were not included in the final study.

After disinfection of the mucosal surface with 0.12% chlorhexidine swabs, sample teeth were cleansed with pumice and isolated with rubber dam under local anesthesia. Sample teeth and the surrounding field were disinfected with 1% chlorhexidine gluconate.1

Access cavities were prepared using new sterile burs cooled with sterile saline drops. Each of three root canals in every tooth was sampled as follows—K-file (#15) used to agitate the canal content to by radiograph determined working length was collected by cutting off the handle and placing working part in 2 mL Eppendorf tube containing reduced transport fluid (RTF). Subsequently, two paper points were placed in root canal one after another for 30 seconds to the same length and transferred to the same Eppendorf tube. In dry root canals, one drop of sterile RTF was placed, avoiding flooding prior to endodontic sample collection.17

All the samples were immediately taken to the Basic Science Research Laboratory for further processing. After thoroughly vortexing the endodontic sample for 60 seconds—necessary quantity was used for culture, and the remaining were stored immediately at –20°C until the PCR assay.

Microbial Culture and Identification

After centrifuging each sample for 60 seconds, samples were streaked on Enterococcus confirmatory agar. The plates were incubated in a candle jar for 48–72 hours, and the colony-forming units (CFU) were counted. The purity of cultures was confirmed by Gram’s staining.

Polymerase Chain Reaction Assay

The DNA was isolated by proteinase K method. Microcentrifuge was centrifuged at 10,000 rpm for 5 minutes, and supernatant was discarded. After this, fresh 300 µL of Tris-ethylenediaminetetraacetic acid (TE) buffer was added and again centrifuged for 3 minutes. The above procedure was repeated three times with fresh TE buffer. Supernatant was discarded, and 100 µL of lysis buffer I was added and kept for 3 minutes. Afterward, 100 µL of lysis buffer II was added, kept for 3 minutes followed by 10 µL of proteinase K.

This was kept in water bath at 65°C for 2 hours and in boiling water for 10 minutes, and the DNA was stored at –20°C. Quantification and purity were determined using a ultraviolet light spectrophotometer (BioPhotometer) at 260 and 280 nm. The absorbance ratio at 260/280 nm was analyzed, and the DNA was considered of good purity if the ratio ranged between 1.8 and 2.0.

The primer sequences for Enterococcus faecalis were designed based on literature from Sedgley et al.18 The primers specificity was confirmed by National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis. Table 1 lists the PCR primers used in the current study.

Table 1: Sequence of specific primers for Enterococcus faecalis
Microorganism Primer
Enterococcus faecalis
(Forward primer)
5’ CCGAGTGCTTGCACTCAATTGG 3’
Enterococcus faecalis
(Reverse primer)
5’CTCTTATGCCATGCGGCATAAAC3’

To determine the temperature for optimal primer annealing for PCR specificity experiment, temperature gradient PCR assays were performed, using positive control DNA from Enterococcus faecalis ATCC 35550 and molecular grade water as negative control in the Veriti 96-Well Fast Thermal cycler (Applied Biosystems New York). A total of 100 ng total DNA template was prepared for 25 µL PCR amplification as follows 7.5 pmol of each primer, respectively, Taq DNA polymerase 2X Master Mix Red (Amplicon) as per manufacturer’s instructions.

The PCR steps were as follows—5-minute DNA denaturing step at 95°C was followed by 35 consecutive cycles at 94°C for 30 seconds, 60°C for 45 seconds, and 72°C for 15 seconds. The PCR amplicons were analyzed by electrophoresis in a 2% agarose gel and stained with 0.5 µg/mL ethidium bromide.

Polymerase chain reaction (PCR) products were visualized under ultraviolet light using Syngene gel documentation. A 100-bp DNA ladder served as the molecular weight marker. The identity of each band was concluded by visual comparison with a molecular weight ladder. Gel documentation of PCR-generated DNA bands of Enterococcus faecalis positive samples following standardization by electrophoresis is confirmed (Fig. 1).

Fig. 1: Gel documentation of PCR generated DNA bands of Enterococcus faecalis-positive samples following standardization by electrophoresis; 100-bpγ DNA ladder. γBasepairs

Statistical Analysis

Data collected for each sample were recorded into an Excel sheet and analyzed with Statistical Package for the Social Sciences (SPSS) software version 20. Presence of Enterococcus faecalis was determined as the percentage of the cases analyzed. The ability of microbial culture and PCR assay to detect Enterococcus faecalis from the same endodontic sample was compared using McNemar’s test. Level of significance was set at p < 0.05.

RESULTS

The children comprised of 17 girls and 21 boys. Among them, children having more than one deciduous molar with necrotic pulp were also included. The deciduous mandibular molar teeth showed a higher distribution of necrotic pulp (75%) as compared to deciduous maxillary molars (25%).

The presence of Enterococcus faecalis was detected in 20 samples (16.67%) by microbial culture and in 45 samples (37.5% of total) by PCR assay, with statistically significant difference between the two methods (p < 0.001; McNemar’s test). Microbial culture and PCR both detect Enterococcus faecalis; with the latter detecting additional 25 positive samples. Thus, PCR method was found to be more sensitive than culture method in detection of Enterococcus faecalis from root canal of deciduous molar with necrotic pulp.

DISCUSSION

In this study, children exhibited high distribution of dental caries with necrotic pulp in deciduous mandibular molars. This is in accordance with a study by Fabris et al. due to retention of food and poor oral hygiene in children. Also, this study shows that Enterococcus faecalis has higher percentage of detection in deciduous teeth with necrotic pulp in accordance with recent studies.16Enterococcus faecalis is reported to be an organism responsible for developing resistance and the main cause for the failure of endodontic therapy, but its prevalence in primary endodontic infections of deciduous teeth is not yet established.

Microbial culture has its limitations as it can identify the predominant species and only those which are viable and not sensitive to favorable conditions for growth. On the contrary, PCR can detect unculturable and difficult-to-grow bacteria. Molecular methods like PCR assays can detect any organism, even if nonviable, that is, dead or in fewer numbers, as it amplifies virtually any given piece of DNA or gene. Thus, it is more sensitive, accurate, and rapid technique for detection of microorganisms than culture.7,17

Although many studies have been used to detect Enterococcus faecalis in permanent dentition by means of culture or PCR. Only two studies by Cogulu et al. are carried out in deciduous dentition using PCR, one of which compares detection of the highly pathogenic Enterococcus faecalis by both culture and PCR in necrotic pulps of deciduous dentition. Previous studies found a lower prevalence of Enterococcus faecalis using molecular techniques, whereas our study showed a relatively higher prevalence of 37.5%.11,19 Also, the prevalence of Enterococcus faecalis by means of microbiological culture was higher, that is, 16.67%.7

Contrary to the study by Cogulu et al. using culture and PCR to detect Enterococcus faecalis in deciduous molars with necrotic pulp, the difference between the culture and PCR is statistically significant (p < 0.001, McNemar’s test). This can be explained by the use of Enterococcus confirmatory agar, which is a more specific media, thus reducing the number of false positive detection by culture method, thus making the difference between detection by means of culture and PCR statistically significant.

The presence of Enterococcus faecalis in a primary endodontic infection of necrotic deciduous molar has many clinical implications during pulpectomy of these teeth. Not only should efficient debridement be performed, but also, use of intracanal medicaments might be used for successful pulp therapy of deciduous teeth.21 Also, judicious use of antibiotics in children for pulp therapy in deciduous teeth should be recommended and used only if systemic involvement exists in order to tackle the problems of developing antibiotic resistance.20,21 The systemic administration of antibiotics depends on patient compliance and is unreliable in children. It also undergoes absorption through the gastrointestinal tract and distribution via the circulatory system to deliver the drug to the infected site. Hence, the infected area requires a normal blood supply, which is no longer the case for teeth with necrotic pulps and for teeth without pulp tissue. Therefore, local application of antibiotics within the root canal system may be a more effective mode for delivering the drug.22,23 Also, as dosage required for intracanal medicament is less, the child suffers lesser side effects of systemic antibiotic ingestion.24

Enterococcus faecalis is a virulent and therapy-resistant bacterium;25 more studies need to be carried out in the deciduous dentition using culture and PCR as results are contrasting to studies in deciduous dentition7 but in accordance with those in permanent dentition.7,9

CONCLUSION

Within the limitations of the present study, we can conclude that PCR method is a sensitive method as compared to culture for detection of Enterococcus faecalis from root canals of deciduous molars with necrotic pulp, the difference being statistically significant (p < 0.001).

Clinical Significance

Importance of presence of Enterococcus faecalis in necrotic pulps of deciduous teeth, as it is primarily responsible for failure of endodontic treatment, thus helping clinicians to advocate the use of local drug delivery in primary teeth endodontics and also aids clinicians in choosing the most effective intracanal medication.

ACKNOWLEDGMENT

We would like to thank Dr Michael Peszkowski (DDS, PhD) for his intellectual help during the proofreading of the manuscript. We would also like to thank Dr Vinayak Kamath for his help in the statistical analysis.

ORCID

Shivayogi M Hugar https://orcid.org/0000-0003-3657-6709

Vijay M Kumbar https://orcid.org/0000-0001-6261-1665

Rachappa M Mallikarjuna https://orcid.org/0000-0001-8793-5842

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