"Magnet" Based on Activated Silver Nanoparticles Adsorbed Bacteria to Predict Refractory Apical Periodontitis Via Surface-Enhanced Raman Scattering.

Refractory apical periodontitis (RAP) is an endodontic apical inflammatory disease caused by Enterococcus faecalis (E. faecalis). Bacterial detection using surface-enhanced Raman scattering (SERS) technology is a hot research topic, but the specific and direct detection of oral bacteria is a challenge, especially in real clinical samples. In this paper, we develop a novel SERS-based green platform for label-free detection of oral bacteria. The platform was built on silver nanoparticles with a two-step enhancement way using NaBH4 and sodium (Na+) to form "hot spots," which resulted in an enhanced SERS fingerprint of E. faecalis with fast, quantitative, lower-limit, reproducibility, and stability. In combination with machine learning, four different oral bacteria (E. faecalis, Porphyromonas gingivalis, Streptococcus mutans, and Escherichia coli) could be intelligently distinguished. The unlabeled detection method emphasized the specificity of E. faecalis in simulated saliva, serum, and even real samples from patients with clinical root periapical disease. In addition, the assay has been shown to be environmentally friendly and without secondary contamination through antimicrobial assays. The proposed label-free, rapid, safe, and green SERS detection strategy for oral bacteria provided an innovative solution for the early diagnosis and prevention of RAP and other perioral diseases.

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

OBJECTIVES: This 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 METHODS: The 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. RESULTS: Samples 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. CONCLUSIONS: Both 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 RELEVANCE: The most dominant taxa that persisted after instrumentation were Fusobacterium, Porphyromonas, Staphylococcus, and Bacteroidaceae [G-1].

Bacteriologic Conditions of the Apical Root Canal System of Teeth with and without Posttreatment Apical Periodontitis: A Correlative Multianalytical Approach.

INTRODUCTION: This study used a correlative multianalytical approach to investigate the bacteriologic conditions in the apical root canal system of treated teeth with or without apical periodontitis and their correlation with the technical quality of the previous root canal obturation and the presence and volume of apical periodontitis lesions. METHODS: Root apexes were obtained from recently extracted root canal-treated teeth with (n = 23) and without (n = 22) apical periodontitis lesions as demonstrated by cone-beam computed tomographic examination. The root apexes were sectioned and subjected to micro-computed tomographic (micro-CT) scanning. The specimens were cryopulverized, and DNA extracted from the powder was used as a template in real-time polymerase chain reaction assays to quantify total bacteria and members of the Streptococcus genus and Actinobacteria phylum. The bacteriologic findings were compared between the 2 groups and also evaluated for associations with cone-beam computed tomographic and micro-computed tomographic data. RESULTS: Bacteria were detected in all apical canal samples except 1. The mean counts of total bacteria, streptococci, and actinobacteria did not differ significantly between teeth with or without apical periodontitis (P > .05). Streptococcus levels were significantly lower by 80% in the apical canals of teeth with small lesions compared with those without lesions (P < .05). The limit of filling >2 mm short was significantly associated with more total bacterial counts compared with canals filled 0-2 mm short (P < .05). An adequate coronal restoration was significantly associated with lesser counts of Streptococcus (P < .05). CONCLUSIONS: Comparable bacterial loads were observed in the apical canal system of treated teeth with and without apical periodontitis, suggesting that factors other than only the total bacterial levels may also influence the development and progression of apical periodontitis. Bacteria were found in the apical canal in virtually all cases with a high prevalence of streptococci and actinobacteria. Streptococci counts were significantly higher in the apical canal of teeth with inadequate restorations and teeth with no lesions. Underfilled canals showed higher bacterial counts.

Clinical efficacy of endodontic protocols on reducing cultivable bacteria and endotoxin in infected root canal in patients submitted to head and neck radiotherapy: a randomised clinical trial.

OBJECTIVES: Assess the efficacy of biomechanical preparation using a reciprocating system followed by final irrigation protocols, then intracanal medication, on reducing endotoxins and cultivable bacteria of infected teeth in irradiated patients. MATERIALS AND METHODS: Twenty-two infected single-rooted canals in patients submitted to head and neck radiotherapy were prepared by reciprocating motion and 2.5% NaOCl. Patients were randomly divided into two groups of 11 patients before the final irrigation protocol: apical positive pressure (APP) or passive ultrasonic activation (PUA). Both groups were treated in two sessions, using Ca(OH)2 as intracanal medication for 14 days. Root canal content sampling was performed after canal access (S1), after biomechanical preparation plus the irrigation protocol (S2), and after intracanal medication (S3). Chromogenic limulus amoebocyte lysate assay measured endotoxin levels (EU/mL), and bacterial load was determined by culture techniques (CFU/mL). RESULTS: Treatment protocols reduced bacterial counts after S2 in both groups (p = 0.01). S3 differed from S1 (p = 0.01), but not from S2 (p = 0.4). Endotoxin levels were reduced in both groups after S2 (P = 0.03) and were lower in S3 than in S2, with significant differences in the APP group (p = 0.03). CONCLUSIONS: Biomechanical preparation using a reciprocating system and 2.5% NaOCl in irradiated teeth, followed by the irrigation protocol (APP or PUA), demonstrated efficacy in reducing endodontic contaminants. Ca(OH)2 as intracanal medication should be performed in irradiated patients with infected root canals. CLINICAL RELEVANCE: This clinical study demonstrated that endodontic treatment in irradiated patients is efficacious at reducing bacterial load and endotoxin levels.

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

PURPOSE: To characterize the bacterial community in the primarily infected root canals. METHODS: A 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. RESULTS: A 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. CONCLUSION: There 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.

TNF-α-TNFR1 Signaling Mediates Inflammation and Bone Resorption in Apical Periodontitis.

INTRODUCTION: The aim of this study was to investigate the role of the proinflammatory axis TNF-α-TNFR1 in experimentally induced periapical inflammation and bone resorption in mice. METHODS: After receiving Ethics Committee Approval (2019.1.139.58.0), experimental apical periodontitis was induced by means of inoculating oral microorganisms into the root canals of molars of mice. Genetically deficient tumor necrosis factor-α receptor-1 mice (TNFR1-/-; n = 50) response was compared with that of C57Bl6 wild-type mice (wild-type; n = 50) after 7, 14, 28, and 42 days. The analyses performed were micro-computed tomographic, histopathologic, histomicrobiological, and histometric evaluation, tartrate-resistant acid phosphatase staining, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction. Data were analyzed by using one-way analysis of variance, followed by Tukey or Bonferroni tests (α = 5%). RESULTS: TNFR1-/- mice exhibited lower recruitment of neutrophils at 14, 28, and 42 days (P < .05), which resulted in reduced area and volume of apical periodontitis at 42 days (P < .05). The number of osteoclasts was also lower in TNFR1-/- animals at 14 and 42 days (P < .01), along with reduced synthesis of CTSK, MMP-9, and COX-2. Expression of RANKL, but not OPG, was reduced at 14 and 42 days (P < .001). The highest RANKL expression over OPG (ratio > 1) was found in wild-type animals at 7 (P < .0001) and 42 days (P < .001). CONCLUSIONS: Periapical inflammation and bone resorption were exacerbated in wild-type animals compared with TNFR1-/- mice, demonstrating that the TNF-α-TNFR1 signaling pathway mediated catabolic events in bone after root canal contamination.

Bacterial quantity and interleukin-17 expression in necrotic teeth with apical periodontitis from type II diabetic patients.

Diabetes mellitus (DM) increases the susceptibility to infection. A plausible association between apical periodontitis (AP) and DM has been reported, but the underlying mechanism is not yet elucidated. AIM: To investigate the bacterial quantity and the expression of interleukin-17 (IL-17) in necrotic teeth with AP in type 2 DM (T2DM), pre-diabetic and non-diabetic control patients. METHODOLOGY: In all, 65 patients with necrotic pulp and AP [periapical index (PAI) scores ≥3] were included. The age, gender, medical history and medications list, including metformin and statin intake, were recorded. Glycated haemoglobin (HbA1c) was analysed, and the patients were divided into three groups: T2DM (n = 20), pre-diabetics (n = 23) and non-diabetic (n = 22). Bacterial samples (S1) were collected by file and paper points. Bacterial DNA was isolated and quantified using 16S ribosomal RNA gene-targeted quantitative real-time polymerase chain reaction (qPCR). For IL-17 expression, (S2) samples were collected from the periapical tissue fluid using paper points passing through the apical foramen. The IL-17 total RNA was extracted, and reverse transcription (RT-qPCR) analysis was performed. Comparisons between the three study groups were conducted using one-way anova and Kruskal-Wallis test to explore the relationship between bacterial cell counts and IL-17 expression in each group. RESULTS: The distributions of PAI scores were equivalent among the groups, p = .289. T2DM patients had higher bacterial counts and IL-17 expression than other groups, but these differences were not statistically significant, p = .613 and p = .281, respectively. T2DM patients taking statin appear to have lower bacterial cell count than those who do not take statin, approaching the significance level, p = .056. CONCLUSION: T2DM patients had a non-significant higher bacterial quantity and IL-17 expression compared to pre-diabetic and healthy controls. Although these findings indicate a weak association, it may impact the clinical outcome of endodontic diseases in diabetic patients.

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

Aim: 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. Methodology: Sixty-seven patients were divided into five groups based on pulpal and 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, sterility control 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. Results: 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). Conclusion: 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.

Host-microbiome interactions in apical periodontitis: The endodontic microbiome in relation to circulatory immunologic markers.

AIM: To explore microbial differences in the endodontic infection of teeth with primary or secondary apical periodontitis (AP), with or without symptomatology. Additionally, to investigate if these differences are depicted in immunologic markers in blood. METHODOLOGY: Twenty-nine teeth with primary or secondary AP were extracted and cryo-pulverized. Blood was drawn from the subjects at three different time-points before and three time-points after the extraction in a time period of four months. The V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq. The microbial profiles were ordinated using principal component analysis and tested for differences between groups with permutational multivariate analysis of variance using the Bray-Curtis distance. If significantly different, the microbial profiles were further analysed using the LDA effect size (LEfSe) biomarker discovery tool. A broad panel of inflammatory mediators in blood was examined longitudinally in all subjects during the six visits with mixed models. The Spearman correlation between these mediators and the zOTUs was calculated, and significant correlations (p < .05) were used as input for significant analysis of microarrays (SAM) using MeV. RESULTS: After subsampling, the 467 zOTUs were classified into 9 phyla and 99 genera or higher level taxa. The predominant genus in the entire sample set was Fusobacterium with a relative abundance of 12.3%, followed by Prevotella (9.9%), Actinomyces (7.7%) and Streptococcus (6.7%). The microbiomes of the endodontic infections were significantly associated with endodontic status (primary/secondary infection; p = .015) as well as with the presence or absence of pain (p = .011). There was also a difference in the concentration of inflammatory mediators, namely, C-reactive protein, Interleukin (IL)-8, IL-10, IL-12p70, RANKL and TNF-α, depending on the existence of pain. In addition, the presence of specific bacteria (zOTUs) was correlated, positively or negatively, with the expression of several circulating inflammatory markers. CONCLUSIONS: The microbial profiles and the concentration-time relationship of systemic inflammatory mediators of primary endodontic infection differed from those of secondary, and of symptomatic from those of asymptomatic cases. The fingerprint of associations between the immunological and microbiological profiles differed between asymptomatic and symptomatic patients.

Microbiological profile of root canals indicated for endodontic retreatment due to secondary endodontic infections or for prosthetic reasons.

OBJECTIVE: The objective of this study was to evaluate the microbiological profile of root canals indicated for endodontic retreatment due to secondary endodontic infections evidenced by the presence of chronic apical periodontitis (G1) or for prosthetic reasons, without clinical and radiographic signs of endodontic reinfection (G2). METHODS: Microbiological samples were collected from thirty teeth (N=30) out of which 15 were indicated for retreatment due to the presence of chronic apical periodontitis (G1) and 15 were indicated exclusively for prosthetic reasons (G2). Samples were collected from root canals before (S1), after chemomechanical preparation (S2), and after 30 days of intracanal medication composed of calcium hydroxide and 2% chlorhexidine gel (S3). The molecular analysis was performed using Nested-PCR for the detection of 17 bacterial species. The efficacy of each stage of the retreatment in reducing the microbial load was verified by counting colony-forming units (CFU). The statistical analysis considered a significance level of 5%. RESULTS: The results showed that bacteria were detected in 100% of the cases in S1, in both groups, by Nested PCR. The most frequently found species in S1 in both groups were Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Parvimonas micra. The microbial load of G1 was higher than G2 in the initial samples (S1). Endodontic retreatment was effective in reducing the microbial load in G1 and G2. Statistically significant associations were found between specific bacteria and clinical signs and symptoms. CONCLUSION: It was concluded that the microbial community present in the root canal of teeth indicated for endodontic retreatment is mixed and heterogeneous. G1 and G2 differ in the number of species per case and microbial load. CLINICAL RELEVANCE: Endodontic retreatment was effective in reducing the microbial load. Statistically significant associations were found between specific bacteria and clinical signs and symptoms.

Bacteria associated with apical periodontitis promotes in vitro the differentiation of macrophages to osteoclasts.

OBJECTIVE: To analyze the possible in vitro effect of the cytokine RANKL and bacteria involved in apical periodontitis on the differentiation of macrophages into osteoclasts. MATERIAL AND METHODS: Bacteria were isolated (mainly E. faecium and E. faecalis) from the root canal of fifty patients with apical periodontitis, the possible effect of these bacteria on the phagocytic activity of the monocyte cell line THP-1 was analyzed by flow cytometry. Furthermore, the effect of these bacteria (alone or in combination with the cytokine RANKL) on the differentiation of THP-1 macrophages into osteoclasts was analyzed through the expression of the receptor RANK and the tartrate-resistant acid phosphatase TRAP. Finally, the release of different cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) by THP-1 cells induced to differentiate into osteoclasts was also analyzed. RESULTS: We observed a significant proportion of THP-1 cells were able to internalize E. faecium and E. faecalis. Furthermore, these bacteria were able to induce (alone or in combination with RANKL) a significant expression of RANK by THP-1 macrophages; accordingly, E. faecium and E. faecalis induced very significant levels of TRAP in these cells. Finally, during the differentiation of THP-1 macrophages induced by RANKL or bacteria, a significant release of the pro-inflammatory cytokines IL-6 and TNF-α was observed. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggest that the causative agents of apical periodontitis can induce the differentiation of osteoclasts as well as the release of pro-inflammatory cytokines, phenomena that may have an important role in the bone damage observed in this condition.

Microbiome in paired root apices and periapical lesions and its association with clinical signs in persistent apical periodontitis using next-generation sequencing.

AIM: To assess and compare the microbiome of paired root apices and periapical lesions from cases with failed endodontic treatment and to associate the microbiome and bacterial metabolic pathways in both sites with asymptomatic apical periodontitis (AAP) and symptomatic apical periodontitis (SAP), using next-generation sequencing (NGS). METHODOLOGY: Matched root apices and periapical lesions of patients with failed root canal treatments were surgically extracted. Specimens were cryopulverized, bacterial DNA was extracted and the V3-V4 hypervariable regions of the 16 S rRNA gene were amplified and sequenced using the Illumina Miseq platform. Diversity and community composition were studied in the paired samples, as well as in AAP and SAP cases. Diversity indices were compared in each case by means of the Wilcoxon matched-pairs signed rank and Mann-Whitney U tests. Differences in the community composition were explored with multivariate statistical analysis and Linear discriminant analysis Effect Size (LEfSe). Bacterial functional study was performed through the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. RESULTS: Twenty-one paired apices and lesions were successfully sequenced and analysed, identifying a total of 21 phyla and 600 genera. A higher alpha-diversity was observed in the periapical lesions, although no global differences in the community composition between the two sites were found (p = .87), the most prevalent genera being Fusobacterium, Porphyromonas and Streptococcus. Prevotella, Clostridiales_vadinBB60_group, Bosea, Phreatobacter, Afipia and Xanthobacteriaceae_unclassified were enriched in SAP samples, while Pseudopropionibacterium, Campylobacter and Peptoniphilus were significantly more abundant in AAP cases (p < .05). Metabolic pathways involved in the amino acid metabolism or degradation and flagellum assembly were more abundant in SAP samples, whereas glucose metabolism-related pathways were associated with AAP. CONCLUSIONS: The bacterial community composition was similar in the apices and periapical lesions. The microbiome was different in AAP and SAP samples, gram-negative bacteria showing higher relative abundances in SAP cases. An association was observed between amino acid degradation and flagellum assembly pathways, and the development of tenderness to percussion or palpation.

Impact of N-acetylcysteine (NAC) and calcium hydroxide intracanal medications in primary endodontic infection: a randomized clinical trial.

OBJECTIVES: This RCT investigated the impact of N-acetylcysteine (NAC) and calcium hydroxide [Ca(OH)2] intracanal medications (ICMs) in primary endodontic infection with apical periodontitis (PEIAP). MATERIALS AND METHODS: Thirty-six teeth with PEIAP were randomly divided into groups according to the ICM: NAC, Ca(OH)2 + saline solution (SSL), and Ca(OH)2 + 2% chlorhexidine-gel (2% CHX-gel) (all, n = 12). Root canal samples (RCSs) were collected before (s1) and after instrumentation (s2) and after 14 days of ICM (s3). Chemomechanical preparation (CMP) was performed with a Reciproc file and 2.5% NaOCl. Checkerboard DNA-DNA hybridization was used to assess 40 target bacteria species. RESULTS: At s1, bacterial DNA was detected in 100% of RCSs (36/36). All 40 bacterial species were found in PEIAP. The mean number of species per RCS was 17.92 ± 13.18. The most frequent bacteria were S. mitis (65%), E. nodatum (63%), E. faecalis (63%), F. nucl sp vicentii (58%), T. forsythia (58%), and F. periodonticum (56%). CMP reduced the mean number of species per RCS to 6.8 ± 2.36 (p < 0.05). At s3, the intragroup analysis revealed a broader antimicrobial activity for Ca (OH)2 + 2% CHX-gel and NAC than Ca(OH)2 + SSL (p < 0.05). NAC eliminated 8/12 bacteria species resistant to both Ca (OH)2 ICMs, including P. micra, P. nigrescens, T. denticola, A. israelii, P. endodontalis, P. acnes, C. ochracea, and E. corrodens. CONCLUSIONS: Ca (OH)2 + 2% chlorhexidine gel (2% CHX gel) showed a greater bacterial elimination over the number of bacterial species; however, NAC eliminated 8/12 bacteria species resistant to both Ca (OH)2 ICMs (RBR-3xbnnn). CLINICAL RELEVANCE: The use of intracanal medication with a broad antimicrobial activity can optimize root canal disinfection. Ca(OH)2 + 2% CHX gel and NAC showed a broader antimicrobial activity than Ca(OH)2 + SSL against endodontic pathogens in primary root canal infection. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials (REBEC), No. RBR-3xbnnn.

Effects of Calcium Hydroxide Paste in Different Vehicles on Bacterial Reduction during Treatment of Teeth with Apical Periodontitis.

INTRODUCTION: This clinical study evaluated the antibacterial effects of calcium hydroxide associated with different vehicles during the treatment of infected teeth with apical periodontitis. METHODS: Bacteriologic samples were taken from 90 necrotic root canals of teeth with apical periodontitis before (S1) and after preparation with a rotary nickel-titanium instrument system and 2.5% sodium hypochlorite irrigation (S2). The teeth were distributed in 3 groups according to the intracanal medication used, which consisted of a calcium hydroxide paste in glycerin, camphorated paramonochlorophenol/glycerin (CHPG), or 2% chlorhexidine for 1 week, and then another sample was taken (S3). The frequency of bacteria-positive cases and the reduction of bacterial counts were evaluated by quantitative real-time polymerase chain reaction. RESULTS: Substantial intracanal bacterial reduction was observed after preparation in the 3 groups (P < .001). After calcium hydroxide paste in glycerin medication, the number of bacteria-positive cases decreased from 20/29 (69%) to 17/29 (59%); however, the mean bacterial counts increased 8.4% from S2 to S3. Medication with CHPG reduced the number of bacteria-positive cases from 17/29 (59%) to 15/29 (52%), with a significant mean S2-S3 reduction of 71% (P < .05). In the chlorhexidine group, the number of bacteria-positive cases decreased from 21/30 (70%) to 17/30 (57%) after medication, with a mean S2-S3 reduction of 55%, which, however, was not statistically significant (P > .05). Intergroup comparisons showed no significant differences (P > .05). CONCLUSION: Comparison between the 3 calcium hydroxide pastes showed no significant differences in antibacterial effectiveness in the main root canal. However, only the CHPG paste showed a significant reduction in bacterial counts when postpreparation and postmedication samples were compared.

Antimicrobial resistance in patients with endodontic infections: A systematic scoping review of observational studies.

The aim of this study was to assess the prevalence and proportions of antimicrobial-resistant species in patients with endodontic infections. A systematic scoping review of scientific evidence was accomplished involving different databases. Nine investigations were selected including 651 patients. Enterococcus faecalis was resistant to tetracycline (30%-70%), clindamycin (100%), erythromycin (10%-20%), ampicillin (9%) and azithromycin (60%). On the contrary, Prevotella spp., Fusobacterium spp., Peptostreptococcus spp. and Streptococcus spp. were resistant to penicillin, tetracycline, doxycycline, ciprofloxacin, amoxicillin, erythromycin, metronidazole and clindamycin in different proportions. Fusobacterium nucleatum showed high resistance to amoxicillin, amoxicillin plus clavulanate and erythromycin. Prevotella oralis presented a predisposition to augment its resistance to clindamycin over time. Tanerella forsythia exhibited resistance to ciprofloxacin and rifampicin. Lactococcus lactis presented robust resistance to cephalosporins, metronidazole, penicillin, amoxicillin and amoxicillin-clavulanic acid. It was observed high levels of resistance to antimicrobials that have been utilised in the local and systemic treatment of oral cavity infections.

Endodontic Microbial Communities in Apical Periodontitis.

INTRODUCTION: Apical periodontitis (AP) represents an inflammatory condition of peri-radicular tissues due to invasion and colonization of bacteria in the root canals. Primary apical periodontitis (PAP) is associated with untreated necrotic root canal and can be efficiently treated with endodontic treatment to remove bacteria. Persistent/secondary apical periodontitis (SAP) is a perpetual periapical lesion due to unsuccessfully treated root canals after an initial apparent healing of the tooth. The aim of the study was evaluating the microbial communities associated with root canals using Nanopore sequencing. METHODS: Seventeen samples from the root canals of 15 patients with AP were Polymerase Chain Reaction-amplified for 16s ribosomal DNA gene and sequenced. Information regarding the presence or absence of AP symptoms, PAP and SAP, and periapical index of patients were recorded. RESULTS: Firmicutes, Bacteroidetes, and Actinobacteria were the most abundant phyla detected and Phocaeicola, Pseudomonas, Rothia, and Prevotella were the most prominent genera. In samples of patients with AP symptoms, the most frequent detected genera were Cutibacterium, Lactobacillus, Pseudomonas, Dialister, Prevotella, and Staphylococcus. In PAP samples, the most represented genera were Cutibacterium, Lactobacillus, Pseudomonas, and Prevotella, whilst in SAP cases were Cutibacterium, Prevotella, Atopobium, Capnocytophaga, Fusobacterium, Pseudomonas, Solobacterium, and Streptococcus. CONCLUSIONS: The results provide additional information on the microbiota of root-canals. These data evidence the complexity of the microbiota and the relationship with many clinical and endodontic conditions. Future studies must evaluate these conditions and identify their role in inducing bone damage and local and systemic disease, aiming to better elucidate the relationship between microbes and endodontic pathologies.

Taxonomic abundance in primary and secondary root canal infections.

AIM: To evaluate the root canal microbiome composition in cases of primary and secondary apical periodontitis. METHODOLOGY: Thirty-nine samples from patients with primary root canal infections obtained before root canal treatment, and 40 samples obtained during root-end resection procedures from previously filled cases with apical periodontitis were evaluated using 16S rRNA next-generation sequencing analysis (NGS). Demographic and clinical factors included age, sex, infection type, percussion sensitivity, and presence of pain. Differences in abundances of genera were evaluated using Kruskal-Wallis test. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bonferroni correction for multiple comparisons. RESULTS: Significantly fewer operational taxonomic units values were observed from samples from secondary infections (p < .0001). While no significant differences were observed in the Chao 1 index between primary and secondary infections, the Shannon alpha diversity was significantly lower in secondary relative to primary infections (p = .008). Among samples, sex, age (adult vs. older adult), percussion sensitivity, and presence of pain all showed no significant effects on community composition via an analysis of similarity (ANOSIM). However, community composition was significantly different depending on whether the sample was from a primary or secondary infection (R = .051, p = .03). Nine microbial genera comprised the predominant taxa observed among samples (>3.3%) and included Parvimonas, Fusobacterium, Campylobacter, Arachnia, Eubacterium, Prevotella, Peptostreptococcus, Fretibacterirum, and Pseudoramibacter. Significantly greater relative abundances of Prevotella, Peptostreptococcus, Veillonella, Lactucaseibacillus, and Dialister were observed in primary infections. CONCLUSIONS: Primary endodontic infections are more diverse than secondary infections. The microbial composition is not associated with the clinical manifestations of apical periodontitis.

Distinctive microbiota distribution from healthy oral to post-treatment apical periodontitis.

Post-treatment apical periodontitis (PoAP) occurs when root canal treatment has not adequately eliminated bacterial invasion and infection. Yet little is known about the bacterial composition and changes related to the etiology and pathogenesis of PoAP. In this study, clinical samples classified as root apex (HARD) and periapical granulation tissues (SOFT) were separately collected from 10 patients with PoAP. The microbiota of each sample was characterized by 16S rRNA gene sequencing, and the obtained dataset was coanalyzed with 20 NCBI sequence read archive (SRA) datasets of healthy oral (HO) and primary apical periodontitis (PAP). We observed 2522 operational taxonomic units (OTUs) belonging to 29 phyla, and all samples shared 86.5% of the sequence reads. The OTUs affiliated with Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria, were identified as core microbiota, which accounted for nearly 90% of 16S rRNA sequences in all samples. However, the principal coordinates analysis (PCoA) of the beta diversity demonstrated that the three periapical statuses have distinct microbial compositions. Compared with HO and PoAP, Actinomyces has a significantly increased abundance in PAP. The microbial diversities in PoAP were significantly lower than those in the HO and PAP (p<0.05). The relative abundance of most bacterial taxa was decreasing, except that Clostridia and Synergistia were increased. Furthermore, we explored the potential metabolic differences of the microbial communities by KEGG pathway prediction. We revealed that the microbiota of PoAP might have a more active metabolic capacity, including carbohydrate metabolism, energy metabolism, and enzyme cofactor/carrier biosynthesis (p<0.05). Our study revealed that invasion of opportunistic pathogens such as Clostridia and Synergistia might play a significant role in PoAP, thus guiding the further study of complex microbial-host interactions and the development of more effective diagnostic and therapeutic methods.

Evaluation of PCR primers to identify Porphyromonas endodontalis in apical periodontitis clinical samples.

We aimed to evaluate whether two commonly used PCR primers are effective to identify P. endodontalis and discriminate from other prevalent black-pigmented bacteria in apical periodontitis (AP). Endodontic canal samples from patients with asymptomatic AP (n = 20) were collected and cultured in anaerobiosis. Two primer sets to detect P. endodontalis were selected from the literature and first analyzed for their specificity in silico; and then tested on clinical isolates in vitro and finally, in apical exudates ex vivo. The identity of P. endodontalis was verified by PCR and Sanger sequencing with universal primers for bacterial V3-V6 regions 16S rDNA. Only one primer set showed specificity only for P. endodontalis clones in silico and also was specific for P. endodontalis in vitro and ex vivo.

Association between Endodontic Infection, Its Treatment and Systemic Health: A Narrative Review.

The 'Focal Infection Era in Dentistry' in the late 19th and early 20th century resulted in widespread implementation of tooth extraction and limited the progress of endodontics. The theory proposed that bacteria and toxins entrapped in dentinal tubules could disseminate systemically to remote body parts, resulting in many types of degenerative systemic diseases. This theory was eventually refuted due to anecdotal evidence. However, lately there has been increased interest in investigating whether endodontic disease could have an impact on general health. There are reviews that have previously been carried out on this subject, but as new data have emerged since then, this review aims to appraise the available literature investigating the dynamic associations between apical periodontitis, endodontic treatment, and systemic health. The available evidence regarding focal infection theory, bacteraemia and inflammatory markers was appraised. The review also collated the available research arguing the associations of apical periodontitis with cardiovascular diseases, diabetes mellitus, adverse pregnancy outcome and autoimmune disorders, along with the effect of statins and immunomodulators on apical periodontitis prevalence and endodontic treatment prognosis. There is emerging evidence that bacteraemia and low-grade systemic inflammation associated with apical periodontitis may negatively impact systemic health, e.g., development of cardiovascular diseases, adverse pregnancy outcomes, and diabetic metabolic dyscontrol. However, there is limited information supporting the effect of diabetes mellitus or autoimmune disorders on the prevalence and prognosis post endodontic treatment. Furthermore, convincing evidence supports that successful root canal treatment has a beneficial impact on systemic health by reducing the inflammatory burden, thereby dismissing the misconceptions of focal infection theory. Although compelling evidence regarding the association between apical periodontitis and systemic health is present, further high-quality research is required to support and establish the benefits of endodontic treatment on systemic health.