Root Canal Samples Research Articles

In-vitro and In-silico evaluation of antimicrobial and antibiofilm effect of Neem oil and Calcium hydroxide nanoparticles against Mutans Streptococci and Enterococcus faecalis isolated from endodontic infections

Different Streptococcal species including Streptococcus mutans, Streptococcus sobrinus and Enterococcus faecalis are commonly isolated in root canal infections including refractory, recurrent, and persistent cases. Calcium hydroxide (Ca (OH)2) has been widely used in endodontics as an intracanal medicament. However, using new antimicrobial herbal alternatives offers promising potentials which can be additionally enhanced by using nanoparticles (NPs). In this study, we evaluate the antimicrobial efficacy and antibiofilm effect of Neem oil including its NPs preparations and we compare the effect of conventional Ca (OH)2 to Ca (OH)2 NPs using standard disc diffusion method and quantitative microtitre dish biofilm formation assay against common pathogens isolated from root canal samples. Molecular docking was used to test the binding of 10 Streptococcal macromolecules to 5 candidate neem active constituents. Neem NPs 0.125 mg/ml showed better antibacterial effect than both Neem 15 mg/ml and Neem 0.15 mg/ml. Ca (OH)2 NPs 0.125 mg/ml also showed better antibacterial effect than each of Ca (OH)2 10 mg/ml and Ca (OH)2 0.1 mg/ml. Best biofilm mass inhibition was achieved by Neem oil 0.15 mg/ml at 74.55% ( IQ: 67.36–87.65) and Neem NPs 0.0125 mg/ml at 59.33% (IQ: 51-–75.27). For Ca (OH)2, the best biofilm mass inhibition was observed with Ca (OH)2 NPs 0.125 mg/ml at 54.7% (IQ: 42.37– 77.25). Both neem oil and neem NPs show promising antibacterial and antibiofilm potential against Mutans Streptococci group at low concentrations and hence are good candidates for use as endodontic medications. In silico analysis shows that both Sitosterol and Gedunin appear to be important active constituents of neem and possible drug candidates. Additionally, Ca (OH)2 NPs showed significantly higher antimicrobial effect against Mutans streptococci group than conventional Ca (OH)2 preparations.

Clinical Investigation of Bacteriome in Primary Endodontic Infections With Apical Periodontitis Using High-Throughput Sequencing Analysis

IntroductionThis study characterized the bacteriome in primary endodontic infection (PEI) with apical periodontitis (AP), identified core and rare bacteriome species and community diversity metrics, and analyzed the relationship between the bacteriome composition, diversity and features, and patient variables. MethodsTwenty-seven patients with PEI and AP were sampled. The DNA was extracted and quantified using quantitative polymerase chain reaction. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants, and taxonomic assignment of the amplicon sequence variants up to the species level was done against the Human Oral Microbiome Database. Core bacteriome and differential abundance analyses were performed using ANCOM. Alpha diversity was determined using Chao1, Shannon, and Simpson indexes. LeFse analysis was used to identify abundant taxa. Sparse Estimation of Correlations among Microbiomes analysis estimated linear and nonlinear relationships among bacteria. ResultsOf 27, 24 root canal samples were analyzed, and 3 root canal sampling were filtered out with a low read count. The bacterial phyla with top mean relative abundance were Bacteroidetes, Firmicutes, Synergistetes, Fusobacteria, and Actinobacteria. A total of 113 genera and 215 species were identified. The samples were gathered into 3 clusters. LefSe analysis identified differences in abundant taxa between distinct age, gender, symptomatology, and lesion size groups. Sparse Estimation of Correlations among Microbiomes distance analysis indicated Slackia exigua as the node with the highest degree. ConclusionsThe bacteriome in PEI with AP among the patients in this study was complex and displayed high microbial heterogeneity. Moreover, age, gender, symptomatology, and lesion size were associated with differences in bacteriome features in PEI with AP.

Comparative evaluation of selected concentrations of sodium hypochlorite on the outcome of endodontic therapy among Ghanaians.

Endodontic treatment is one of the main dental treatments to manage inflamed or infected root canal systems of teeth. The success of endodontic treatment principally depends on eradicating microorganisms in the root canal by chemo-mechanical debridement with irrigation solutions like sodium hypochlorite (NaOCl). NaOCl has been used in concentrations ranging from 0.5% to 5.25%. This study determined the antimicrobial effectiveness of selected concentrations (0.5%, 1.0%, 2.6%, and 5.2%) of NaOCl in endodontic treatment. The study sites were the University of Ghana Dental School (UGDS) and Noguchi Memorial Institute for Medical Research (NMIMR). Sixty infected single-rooted single-canal teeth were used. Before (S1) and after (S2), root canal samples during the endodontic treatment with the selected concentrations of NaOCl were examined via anaerobic and aerobic cultures. The isolates were identified using Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). All S1 samples were positive for cultivable bacteria. Fifty-three (53) different microbial species belonging to 20 different microbial genera were isolated. Streptococcus viridans was the most frequently isolated microbe. There were zero isolates in the root canals irrigated with 2.6% and 5.2% NaOCl. Two teeth had isolates in the groups irrigated with the lower concentrations (0.5% and 1.0%) of NaOCl. The persistent bacteria were one species each of Streptococcus mitis and Streptococcus oralis, respectively. Root canal treatments using chemo-mechanical preparation with the selected concentrations (0.5%, 1.0%, 2.6%, and 5.2%) of NaOCl were effective in significantly reducing the microbial load, and for the 5.2% and 2.6% concentrations, in eliminating all the microorganisms.

Prevalence and virulence factors of haemolytic Enterococcus faecalis isolated from root filled teeth associated with periradicular lesions: A laboratory investigation in Thailand.

Previous endodontic research has provided limited understanding of the prevalence and roles of haemolytic and non-haemolytic Enterococcus faecalis strains in root filled teeth. This study aimed to determine the prevalence of these strains in root filled teeth with periradicular lesions and investigate their associated virulence factors. A total of 36 root canal samples were collected from 36 subjects. The prevalence of E. faecalis was determined using culture and PCR methods. Antibiotic susceptibility of haemolytic and non-haemolytic E. faecalis strains was assessed using the broth dilution assay. The cytokine stimulation in periodontal ligament (PDL) cells and neutrophil migration were evaluated using real-time PCR and migration assay, respectively. Cell invasion ability of the strains was assessed using a cell culture model. Additionally, the virulence gene expression of the haemolytic and non-haemolytic strains was investigated using real-time PCR. The Mann-Whitney U and Spearman's ρ tests were used to examine the significant difference between the two strains and to analyse the correlation between phenotype and gene expression, respectively. Enterococcus faecalis was detected in 33.3% and 88.9% of samples by culture and real-time PCR, respectively. Haemolytic strains were found in 36.4% of subjects. Non-haemolytic strains exhibited susceptibility to erythromycin and varying susceptibility to tetracycline, while all haemolytic strains were resistant to both antibiotics. Haemolytic strains significantly upregulated the expression of IL-8, OPG and RANKL in PDL cells (p < .05). Notably, the fold increases in these genes were higher: IL-8 (556.1 ± 82.9 vs. 249.6 ± 81.8), OPG (2.2 ± 0.5 vs. 1.3 ± 0.2) and RANKL (1.8 ± 0.3 vs. 1.2 ± 0.1). Furthermore, haemolytic strains had a greater effect on neutrophil migration (68.7 ± 15.2% vs. 46.9 ± 11.4%) and demonstrated a higher level of internalization into oral keratinocyte cells (68.6 ± 0.4% vs. 33.8 ± 0.5%) (p < .05). They also showed enhanced expression of virulence genes associated with haemolysin, surface proteins, collagen-binding and aggregation substances. Gelatinase activity was only detectable in non-haemolytic strains. This study revealed that haemolytic strains E. faecalis possessed enhanced abilities in host invasion and a higher abundance of virulence factors, suggesting their potential contribution to more severe disease manifestations.

The Effect of Different Compositions and Concentrations of Etidronate-Containing Irrigants on the Antibacterial Activity of Sodium Hypochlorite against Enterococcus faecalis and Candida albicans.

We assessed the effect of different compositions and concentrations of two etidronate-containing irrigants on the antibacterial activity of sodium hypochlorite (SH) against Enterococcus faecalis and Candida albicans in vitro. Pure cultures of C. albicans and E. faecalis were isolated from root canal samples. The disc diffusion method was used to compare the antibacterial effect of pure SH and SH mixed with 9%, 15%, and 18% etidronate of two manufactures (dual rinse (DR); IsraDent (ID)) and EDTA. The pH and temperature of the solutions were measured immediately after mixing and within 40 min. The ANOVA revealed a significant influence of the type of irrigating solution on the C. albicans and E. faecalis inhibition zone diameters that ranged from 6.6 to 51.6 mm and from 6.4 to 12.4 mm, respectively. SH with DR 9% exhibited the highest effect against C. albicans. The antifungal activity of the other irrigants was SH = SH + DR15% = SH + DR18% = SH + ID9% > SH + EDTA > SH + ID15% > SH + ID18%. No significant differences in the anti-E. faecalis effect were revealed between the tested solutions except for the mixtures of SH and 15% and 18% ID, which exhibited no antiseptic effect. There was a strong positive correlation between antiseptic activity against both microorganisms and the pH values of the tested solutions. In conclusion, most etidronate formulations did not significantly hamper sodium hypochlorite activity against C. albicans and E. faecalis. The effect was concentration- and manufacturer-dependent.

Impact of root canal preparation using two single-file systems on the intra-radicular microbiome of teeth with primary apical periodontitis

ObjectivesThis study aimed to describe the effects of two single-file systems on the diversity of the endodontic microbiome of teeth with primary asymptomatic apical periodontitis.Materials and methodsThe root canals from single-rooted teeth with apical periodontitis were prepared using either the Reciproc Blue (RB) or the XP-endo Shaper (XPS) instrument system. The latter was followed by a supplementary step with the XP-endo Finisher (XPF) instrument. For irrigation, 5.25% sodium hypochlorite was used. Root canal samples were taken at the baseline (S1), after preparation (S2), and after the supplementary step (S3). DNA was extracted and subjected to high-throughput sequencing using the MiSeq Illumina platform.ResultsSamples from 10 teeth from the RB and 7 from the XPS group were subjected to DNA sequencing. Initial samples differed significantly from post-preparation samples in bacterial diversity, with no significant difference when comparing the two instrument systems. The most dominant phyla in S2 were Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, and Actinobacteria. The same phyla were found to dominate baseline samples and samples taken after using XPF, but with differences in the ranking of the most dominant ones. At the genus level, the most dominant genera identified after RB instrumentation were Bacteroidaceae [G-1], Fusobacterium, and Staphylococcus, while the most dominant genera after XPS instrumentation were Fusobacterium and Porphyromonas. These genera were also dominant in the initial samples.ConclusionsBoth treatment protocols had measurable effects on the root canal microbial diversity, with no significant differences between them. Most of the dominant taxa involved in the primary infection and probably in the aetiology of apical periodontitis were eliminated or substantially reduced.Clinical relevanceThe most dominant taxa that persisted after instrumentation were Fusobacterium, Porphyromonas, Staphylococcus, and Bacteroidaceae [G-1].

Prevalence of Enterococcus faecalis and Candida albicans in endodontic retreatment Cases: A comprehensive study

IntroductionEndodontic treatment failures often stem from the presence of microbial pathogens, particularly Enterococcus faecalis and Candida albicans. This study systematically assesses the prevalence of E. faecalis and C. albicans in endodontic retreatment cases, aiming to explore their impact on treatment outcomes. MethodsEmploying a systematic sampling approach, 30 patients with a history of previous endodontic treatment were selected. Rigorous clinical and radiographic assessments were conducted, following standardized protocols for root canal sample collection. Microbiological analysis, utilizing selective culture media, was employed to identify and quantify E. faecalis and C. albicans. Statistical analyses, including chi-square and logistic regression tests, were performed. ResultsThe study involved 30 patients undergoing endodontic retreatment, with comprehensive clinical and radiographic evaluations for cases with and without periradicular lesions. Microbiological analysis unveiled a significant prevalence of E. faecalis and C. albicans, establishing a robust association between these pathogens and retreatment failure. These findings underscore the critical need for targeted antimicrobial interventions to enhance the overall success rates of endodontic retreatment procedures. ConclusionThis study highlights the substantial prevalence of E. faecalis and C. albicans in endodontic retreatment cases, emphasizing the importance of identifying and effectively managing these pathogens for successful treatment outcomes. The notable association between these microbial agents and retreatment failure underscores the imperative for tailored antimicrobial strategies to enhance the efficacy of endodontic retreatment procedures.

An Ex-vivo Comparative Study to Evaluate the Efficacy of Three Root Canal Irrigation Systems in Reduction of Enterococcus Faecalis.

The purpose of this ex-vivo study is to evaluate the efficacy of three irrigation techniques in the reduction of E. faecalis in root canal. Eighty healthy maxillary anteriors were employed in this investigation. The bacteria E. faecalis was left on teeth for 7 days while they were kept. Once teeth were contaminated, they were randomly divided into three groups of 20 for experimentation and a control group of 20 for comparison. NaviTip FX was used to irrigate Group I, the Max-I-probe was used to water Group II, and the Endovac system was used to water Group III. The number of colony forming units (CFUs) was determined by growing bacteria from root canal samples for 2 days in Brain heart infusion agar after chemo-mechanical operations. There was a substantial decrease in bacterial numbers across all experimental groups compared to group IV (control). Group III (Endovac) had the fewest bacterial colonies among the test groups. There were no discernible changes between Group I (the Navitip FX) and Group II (the Max I probe). Endovac was the most effective of the three irrigation methods for reducing CFUs. This study's findings lend credence to the idea that the apical negative pressure approach has more potential than the conventional irrigation delivery system for achieving an enhanced antibacterial impact.

Bacterial diversity in primary infected root canals of a Chinese cohort: analysis of 16 S rDNA sequencing

PurposeTo characterize the bacterial community in the primarily infected root canals.MethodsA total of 13 samples were collected from the primarily infected root canals. 16 S rDNA sequencing was performed to define bacterial community. Taxonomic annotation, bacterial hierarchical structures, community richness and diversity, and inter-subject variability of the bacterial community in the root canal samples were analyzed. Gender, age, and duration of the toothache-specific bacterial community associated with the patient groups were analyzed.ResultsA total of 359 Species were annotated and identified in the whole study cohort. The Alpha diversity analysis showed that the species diversity and detection rate of the 13 samples were high, which reflected the authenticity of sequencing results. The Beta diversity analysis was used to compare the degree of difference between different root canal samples. The 13 samples were divided into two groups according to the results, group A was samples I1-I12, and group B was samples I13. The bacterial species of group A samples were analyzed with the clinical characteristics of patients, and it was found that gender, and duration specific differences in bacterial species, and there was no significant difference in species types among different ages of patients.ConclusionThere were a wide diversity and inter-subject variability in the bacterial community in the primary infected root canals. While Porphyromonas gingivalis was the most abundant species, Fusobacterium nucleatum was the most variable species in the bacterial community of the root canal. The bacterial community at different taxonomic levels varied from sample to sample, despite consistent disease diagnoses. There was gender, duration-specific differences in the bacterial species in the primary infected root canals.

Evaluating the use of self-conditioning adhesive combined with dual curing resin cement as an endodontic sealer: An in vitro study.

Objectives To compare infiltration, sealing and microleakage in root dentin with a self-conditioning adhesive system combined with dual curing resin (resin-based cement) to a conventional epoxy-resin-based sealer using confocal microscopy imaging. Methods 26 roots were enlarged and disinfected. Dentin tubules of 24 teeth were labelled with a red fluorophore (Rhodamine B) (two samples served as controls). Root canal samples were sealed in group AH (n = 11) with a conventional sealer (AH Plus Root Canal Sealer, Dentsply DeTrey) and in group RC (n = 11) with a resin-based cement (Parabond combined with Paracore, Coltène). Roots were then sectioned horizontally and immersed in H2O2 to remove the Rhodamine B not fixed by the sealers. The empty dentin spaces were labeled with a green fluorophore (Fluorescein) enabling the evaluation of infiltration as well as microleakage by confocal microscopy. Two additional samples were fractured in vertical direction for observation under SEM. Results Group RC presented significantly more infiltration in the middle third than in the middle and apical thirds of group AH. Microleakage was significantly higher in group AH than in group RC. SEM images revealed more dentin plugs and a homogenous resin layer in group RC in contrast to group AH. Conclusion The resin-based cement revealed promising outcomes compared to a traditional epoxy resin based sealer.

Microbiota present in combined endodontic-periodontal diseases and its risks for endocarditis.

Infective endocarditis (IE) is an inflammatory disease usually caused by bacteria that enter the bloodstream and establish infections in the inner linings or valves of the heart, including blood vessels. Despite the availability of modern antimicrobial and surgical treatments, IE continues to cause substantial morbidity and mortality. Oral microbiota is considered one of the most significant risk factors for IE. The objective of this study was to evaluate the microbiota present in root canal (RC) and periodontal pocket (PP) clinical samples in cases with combined endo-periodontal lesions (EPL) to detect species related to IE using NGS. Microbial samples were collected from 15 RCs and their associated PPs, also from 05 RCs with vital pulp tissues (negative control, NC). Genomic studies associated with bioinformatics, combined with structuring of a database (genetic sequences of bacteria reported for infective endocarditis), allowed for the assessment of the microbial community at both sites. Functional prediction was conducted using PICRUSt2. Parvimonas, Streptococcus, and Enterococcus were the major genera detected in the RCs and PPs. A total of 79, 96, and 11 species were identified in the RCs, PPs, and NCs, respectively. From them, a total of 34 species from RCs, 53 from PPs, and 2 from NCs were related to IE. Functional inference demonstrated that CR and PP microbiological profiles may not be the only risk factors for IE but may also be associated with systemic diseases, including myocarditis, human cytomegalovirus infection, bacterial invasion of epithelial cells, Huntington's disease, amyotrophic lateral sclerosis, and hypertrophic cardiomyopathy. Additionally, it was possible to predict antimicrobial resistance variants for broad-spectrum drugs, including ampicillin, tetracycline, and macrolides. Microorganisms present in the combined EPL may not be the only risk factor for IE but also for systemic diseases. Antimicrobial resistance variants for broad-spectrum drugs were inferred based on PICRUSt-2. State-of-the-art sequencing combined with bioinformatics has proven to be a powerful tool for conducting studies on microbial communities and could considerably assist in the diagnosis of serious infections. Few studies have investigated the microbiota in teeth compromised by combined endo-periodontal lesions (EPL), but none have correlated the microbiological findings to any systemic condition, particularly IE, using NGS techniques. In such cases, the presence of apical periodontitis and periodontal disease can increase IE risk in susceptible patients.

Clinical Study of Antimicrobial Efficacy of Laser Ablation Therapy with Indocyanine Green in Root Canal Treatment

IntroductionLaser ablation (LA) therapy is used as an adjunct to endodontic treatment to improve microbial reduction. However, studies evaluating the impact of LA with indocyanine green (ICG) are scarce. This study aimed to evaluate the antimicrobial efficacy of LA therapy with ICG in root canal treatment. MethodsSixty patients with periapical lesions in teeth with a single canal and absence of pain, edema, and previous treatment were selected. Patients were randomly allocated into 3 groups according to the apical sizes used (n = 20); 25/04, 30/04, and 35/04 were the final sizes used. In half the patients of each group, 2.5% sodium hypochlorite was used as an irrigating solution, and in the other half, saline solution was used. After instrumentation, all patients received LA therapy with ICG. Root canal sampling was performed before (S1) and after (S2) root canal instrumentation and immediately after LA therapy with ICG (S3). Colony-forming units were counted, and statistical tests were applied (P < .05). ResultsThere was a significant reduction in colony-forming units from S1 to S2 in all treatment protocols (P < .05); 2.5% sodium hypochlorite as an irrigating solution showed a greater microbial reduction compared with saline solution (P < .05). LA therapy with ICG further reduced the microbial counts significantly (S2 to S3 and S1 to S3) whether sodium hypochlorite or saline was used (P < .05). ConclusionsLA therapy with ICG significantly increased microbial reduction in root canals regardless of instrumentation sizes or the irrigation solution used.

The presence of Enterococcus faecalis in saliva as a risk factor for endodontic infection.

The aim of the present study was to investigate and correlate the prevalence of Enterococcus faecalis in saliva and in root canals with different pulpal and periapical conditions. Sixty-seven patients were divided into five groups based on pulpaland periapical tissue status: healthy vital teeth (HVT, n=7), healthy treated teeth without lesion (HTT, n=9), irreversible pulpitis (IP, n=13), necrosis (N, n=18), and post-treatment apical periodontitis (PTAP, n=20). Saliva, rubber dam, sterilitycontrol and pre-treatment root canal samples were collected and microbiologically processed by culture method. The phylogenetic relationship of E. faecalis isolates collected from root canals and saliva were investigated by whole genome sequencing. Fisher's exact test was used to correlate the presence of E. faecalis in root canals or saliva with clinical and/or radiographic findings. Linear/logistic regression analyses were performed to establish the relationship between the presence of E. faecalis in root canals, saliva, and the status of periapical tissues. E. faecalis was found in 18 root canal and saliva samples. E. faecalis root canal isolates were recovered with the highest frequency from post-treatment apical periodontitis. The occurrence of E. faecalis in saliva was strongly associated with its detection in the root canals (P < 0.001). The pretreatment presence of E. faecalis in root canals was associated with significantly higher odds of having periapical lesions (OR=11.03; 95% CI, 1.27-95.70; p < 0.05). Saliva and root canal isolates from the same patient were highly correlated at the phylogenetic level (Jaccard index >0.95). This pilot study confirms the role of E. faecalis in developing peri-radicular lesions in secondary endodontic infections and suggests that saliva could be the main source of infection. Further studies are needed to investigate the exact origin of this bacteria and its true role in the pathogenesis of secondary/persistent endodontic infections.

Amplicon-based next-generation sequencing for comparative analysis of root canal microbiome of teeth with primary and persistent/secondary endodontic infections.

To compare the root canal microbiome profiles of primary and persistent/secondary infections using high-throughput sequencing with the help of a reliable bioinformatics algorithm. Root canal samples of 10 teeth in the primary endodontic infection (PEI) group and 10 teeth in the persistent/secondary endodontic infection (SEI) group were included resulting in a total of 20 samples. After DNA extraction from the samples, sequencing was performed on the Illumina MiSeq platform. Pair-end Illumina reads were imported to QIIME 2; amplicon sequence variants (ASVs) generated by DADA2 were mapped to GreenGenes database. Weighted UniFrac distances were calculated and principal coordinates analysis (PCoA) was used to compare beta diversity patterns. The multiple response permutation procedure (MRPP), the analysis of similarities (ANOSIM), and permutational multivariate analysis of variance (adonis) were conducted for testing group differences. Linear discriminant analysis effect size (LEfSe) analysis was utilized to identify differentially abundant taxa between the groups. The linear discriminant analysis (LDA) score threshold was set to 4.0. Within the Gram-negative facultative anaerobic Gammaproteobacteria class outgroup, two orders (Pasteurellales, Vibrionales) and two families (Pasteurellaceae, Vibrionaceae) were significantly more abundant in the PEI group, whereas Gram-positive bacteria, Actinomycetales order, and Gram-positive anaerobic taxa, one genus (Olsenella) and one species (Olsenella uli), were identified as significantly more abundant in the SEI group. A few taxa were differentially abundant within either the PEI or SEI group. Reliable bioinformatic tools are needed to define microbial profiles of endodontic infections. Based on a limited number of samples, no distinct variation was determined between the bacterial diversity of initial and recurrent endodontic infections.

Microbial identification from traumatized immature permanent teeth with periapical lesions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BackgroundThis study aims at identifying the microbiota in traumatized immature permanent teeth with periapical lesions using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).MethodsThe study included 16 immature maxillary central incisors with periapical lesions in 13 patients. Field decontamination and negative control samples were performed before and after access cavity preparation. Root canal samples were taken using sterile stainless-steel hand files following field decontamination. In-office inoculation and pure sub-cultures were performed. Bacterial isolates were prepared for MALDI-TOF MS (Bruker, Billerica, MA USA) analysis using the formic acid extraction method. A comparison of the prevalence of isolated microorganisms was done using a one-sample chi-square test. Comparisons between identified microbial species with the, cone beam computed tomography periapical index (CBCT PAI) scores and lesion volume were also conducted. The Chi-square test was applied to investigate the association between the categorical variables .ResultsOut of the forty isolates recovered from the 16 traumatized teeth included in the present study with the mean patients’ age of 10.93 ± 1.77, 37 isolates were reliably identified by MALDI-TOF MS. Twelve teeth (62.5%) were polymicrobial. The recovered bacteria belonged to five phyla, 15 genera and 25 species. Firmicutes were the predominant phylum (P < 0.001) over Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria. Gram positive bacteria were significantly more prevalent than Gram negative (p = 0.03). Facultative anaerobes were the most prevalent (P < 0.001) compared to the obligate anaerobes and the obligate aerobes. The latter were the least prevalent. Statistically, significant differences existed in the comparison between CBCT PAI scores according to bacterial gram staining.ConclusionTraumatized immature permanent teeth with periapical lesions showed a significant predominance of Gram-positive facultative anaerobes. MALDI-TOF MS provided accurate identification of numerous viable endodontic microbes.

Ex vivo evaluation of antibiotic sensitivity in samples from endodontic infections

ABSTRACT Objective To develop an in vitro model for real-time monitoring of endodontic biofilm growth and evaluate the ex vivo effect of antibiotics on biofilm growth. Material and Methods Root canal samples were taken from 40 patients and inoculated into 96-well plates in a system that measures biofilm growth through electrical impedance. Biofilm bacterial composition at the genus and species level was analyzed by Illumina sequencing. ANCOM-BC corrected data were used to compare bacterial composition after antibiotic treatment through compositional analysis, and to compare microbiological with clinical data. Results The stationary phase was reached at 8 hours. The biofilm formed had a similar bacterial composition to the inoculum, and Enterococcus faecalis was virtually absent from the samples. The bacterial composition and the effect of antibiotics were sample-dependent. Metronidazole was the antibiotic that most inhibited biofilm formation and azithromycin the one that inhibited it in the highest percentage of cases. The antibiotic effect could not be related to the biofilm original bacterial composition. Conclusions The impedance system allowed real-time monitoring of endodontic biofilm formation, and we propose it as a model for ex vivo evaluation of the whole biofilm susceptibility to antimicrobials, as opposed to evaluating antibiotic sensitivity of specific bacterial isolates.

An effective artificial testing model to reliability experiments for electronic apex locator devices used in endodontic practice.

This experimental study aimed to investigate the reliability of using electronic apex locator devices to determine the working length of artificial root canals. The experiments were performed using resin endoblocks and mandibular canine teeth (n = 20/group). After the same working length of root canal samples was provided, the teeth and artificial root canals were embedded in an alginate mold. The measurements with Root ZX® and Propex Pixi® apex locators were performed and recorded. The data were analyzed using SPSS software (SPSS V23; IBM Corp., Armonk, New York, USA) and the variance was set at P < 0.05. There was no significant difference between the groups; mean distance from the actual working length using different apex locators (P = 0.633, P = 0.474), and endpoint positioning distributions (P = 0.591). The results indicate that the artificial model could be a laboratory method of determining the accuracy of apex locators and efficient calibration of devices before their clinical use.

Streptococcus mutans Associated with Endo-Periodontal Lesions in Intact Teeth

A massive periodontal destruction can affect the root canal (RC) system and potentially expose the pulpo-dentinal complex to opportunistic bacteria. Streptococcus mutans is a major pathogen of human caries and periodontal diseases, and its virulence mostly resides in the ability to adhere to collagen and form biofilms, due to collagen-binding proteins (CBPs) Cnm and Cbm. Seventeen patients affected by severe endo-periodontal lesions without caries and/or exposure of pulpal tissue were subjected to tooth extraction and samples for microbiological investigation were collected from the root surface (RS) and RC. The collected swabs were cultivated and subjected to the quantitative real-time PCR (qPCR) for the detection of S. mutans and to the PCR for the cnm/cbm genes investigation, followed by next-generation sequencing (NGS). S. mutans DNA was detected in 12/17 (70.5%) RS samples and in 8/17 (47.0%) RC samples. In the CBPs screening of positive samples, the cnm gene was detected in 4/12 (33.3%) RS and in 1/8 (12.5%) RC samples, whilst all the samples tested negative for the cbm gene. The presence of the cnm gene could enhance the local virulence of the pathogens. Therefore, S. mutans have to be included as potential periodontopathogen bacterium in severe or refractory forms of periodontal diseases.

Antimicrobial potential of new diclofenac hydrogels for disinfection in regenerative endodontics: An in vitro and ex vivo study.

There is a need to explore new alternatives for root canal disinfection in regenerative endodontics, since the current strategies are far from ideal. Currently, the potential use of diclofenac (DC) is being investigated for controlling root canal infections. The objective was to evaluate the antimicrobial efficacy of novel DC-based hydrogels (DCHs) against polymicrobial biofilms grown in radicular dentine and root canals and to compare results with triantibiotic (TAH) and diantibiotic (DAH) hydrogels, and calcium hydroxide (Ca[OH]2 ). The in vitro antimicrobial activity of intracanal medicaments was evaluated against 3-week-old polymicrobial root canal biofilms grown on human radicular dentine. Dentine samples were obtained and randomly divided into the study groups (n=4/group): (1) 1mg/ml TAH; (2) 1mg/ml DAH; (3) 5% diclofenac (DCH); (4) 2.5% DCH; (5) 1.25% DCH; (6) 1mg/ml DAH + 5% DCH; (7) Ca(OH)2 paste; (8) positive control. The microbial viability, in terms of percentage of intact cell membranes, was assessed after 7 days by confocal scanning laser microscopy (CSLM). The ex vivo efficacy of intracanal medications was evaluated in root canals infected with a polymicrobial suspension. Intracanal microbiological samples at baseline (S1) and 7 days post-treatment (S2) were taken; microbial quantification and cell viability were assessed by quantitative polymerase chain reaction (qPCR) and flow cytometry (FC). The mean Log10 of bacterial DNA copies in root canal samples before (S1) and the Log10 reduction of DNA copies S1-S2 in qPCR were recorded. The absolute value of total cells stained, and the percentage reduction of intact membrane cells after treatment (S1-S2), were analysed by FC. Global comparison was done using the Kruskal-Wallis test, whilst the Mann-Whitney U test was used for pair-by-pair comparison. Confocal scanning laser microscopy analysis indicated that the greatest effectiveness was obtained with 5% DCH, showing significant differences with respect to the other groups (p < .001). In root canals, the highest Log10 DNA reduction S1-S2 was obtained with 5% DCH and TAH, with no differences between them. The results of FC showed that only 5% DCH proved significantly superior to the other treatments. Sodium DC hydrogels demonstrate antimicrobial efficacy against endodontic biofilms.

Proteomic analysis of infected root canals with apical periodontitis in patients with type 2 diabetes mellitus: A cross-sectional study.

This study aimed to quantitatively and qualitatively determine the proteomic profile of apical periodontitis (AP) in type 2 diabetes mellitus (T2DM) patients in comparison with systemically noncompromised patients and to correlate the protein expression of both groups with their biological functions. The sample consisted of 18 patients with asymptomatic AP divided into two groups according to the presence of T2DM: diabetic group-patients with T2DM (n=9) and control group-systemically healthy patients (n=9). After sample collection, the root canal samples were prepared for proteomic analysis using reverse-phase liquid chromatography-mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression between groups were calculated using t-test (p < .05). Biological functions were analysed using the Homo sapiens UniProt database. A total of 727 human proteins were identified in all samples. Among them, 124 proteins common to both groups were quantified, out of which 65 proteins from the diabetic group showed significant differences compared with the control: 43 upregulated (p < .05) and 22 downregulated (p < .05) proteins. No significant differences in protein expression were seen for the remaining 59 proteins (p > .05). Most proteins with differences in expression were related to immune/inflammatory response. Neutrophil gelatinase-associated lipocalin, Plastin-2, Lactotransferrin and 13 isoforms of immunoglobulins were upregulated. In contrast, Protein S100-A8, Protein S100-A9, Histone H2B, Neutrophil defensin 1, Neutrophil defensin 3 and Prolactin-inducible protein were downregulated. Quantitative differences were demonstrated in the expression of proteins common to diabetic and control groups, mainly related to immune response, oxidative stress, apoptosis and proteolysis. These findings revealed biological pathways that provide the basis to support clinical findings on the relationship between AP and T2DM.