Identification of keystone taxa in root canals and periapical lesions of post-treatment endodontic infections: Next generation microbiome research.

AIM: The aim of this study was to analyse and compare the microbiome present in root canals and periapical lesions of teeth with post-treatment infections, and to identify the presence of keystone taxa in both habitats using next-generation sequencing analysis. METHODOLOGY: Apices and periapical lesions of patients with post-treatment apical periodontitis were surgically extracted. Specimens were cryo-pulverized, bacterial DNA was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using the Illumina Miseq platform. Bioinformatic analysis was carried out with Mothur software, whilst diversity indices were obtained using operational taxonomic units (OTUs). The diversity indices were compared with the Kruskal-Wallis test, and community composition differences were explored with Permutational Multivariate Analysis of Variance (PERMANOVA). A bacterial functional study was performed with the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Co-occurrence network analyses were performed using the Sparse Correlations for Compositional data (SparCC). Eigencentrality, clr-based abundance and ubiquitousness were applied to infer keystone taxa. P values <.05 were considered statistically significant. RESULTS: Thirty-two apices and thirty-nine periapical lesions were sequenced and analysed. A similar alpha-diversity (p < .05) and community composition (p = .91) was observed for apices and lesion samples. The most abundant OTUs identified amongst all samples included Fusobacterium nucleatum, Prevotella loescheii, Streptococcus intermedius, Porphyromonas gingivalis, Parvimonas micra, Synergistetes bacterium, Tannerella forsythia and Peptostreptococcus stomatis. The metabolic pathways with >0.81% abundances included membrane transport, genetic information processing and metabolic pathways. F. nucleatum was identified as a keystone taxon as it showed ubiquitousness, an eigenvector centrality value of 0.83 and a clr-based abundance >4. CONCLUSIONS: The microbiome in apices and periapical lesions of post-treatment endodontic infections showed a similar diversity and taxonomic composition. Co-occurrence network analyses at OTU level identified F. nucleatum as a keystone taxon candidate in these infections.

Prevalence of the colonization of Helicobacter pylori among students of the school of dentistry, University of Granada, Spain

BACKGROUND: The oral cavity is a reservoir for Helicobacter pylori, and occupations that involve close contact with it, like Dentistry, could entail a higher risk of colonization. The aim of this study was to evaluate if the length of stay at the School of Dentistry of the University of Granada could influence colonization by H. pylori, and to furthermore correlate colonization by H. pylori, with the state of oral health and oral hygiene habits. MATERIAL AND METHODS: The study groups were: Group 1, 35 students of Odontology in their first year of studies; Group 2, the same 35 students when they were in their fifth academic year; Group 3, 35 students from University study programs unrelated with Health and of the same age as the group 2 students. All underwent H. pylori, colonization determinations by means of the urea breath test (UBT), stool antigen test and a serological test. Also studied were the variables plaque index, gingival index and the number of times teeth were brushed per day. The Student t test was used for comparisons among the three studied groups. The Chi-squared test and Pearson correlation coefficient were used to determine any connection between colonization by H. pylori, and the variables studied. RESULTS: Comparisons between groups 1 and 2 and between groups 2 and 3 showed significant differences regarding colonization by H. pylori, plaque index, gingival bleeding index and tooth brushing. A positive correlation was found between being colonized by H. pylori, and having a gingival index higher than 10% and tooth brushing once a day or less, in all the studied groups. CONCLUSIONS: Colonization by H. pylori, among Dentistry students at the University of Granada decreased over a four-year time period at the University. Factors related with better oral health, such as a lower gingival index and more frequent tooth brushings, would explain these results

Serologic control against hepatitis B virus among dental students of the University of Granada, Spain

BACKGROUND: To evaluate the immunological situation against hepatitis B virus (HBV) of a cohort of dentistry students, to analyze the behavior of the levels of hepatitis B surface antigen (anti-HBs) after the administration of one or three vaccine doses, and to determine the influence of age and sex on the immune response. MATERIAL AND METHODS: This retrospective cohort study included students attending the School of Dentistry of the institution where the study was performed from 2005 to 2012 who had completed the public health vaccination calendar for HBV at the age of 12-13. Data on age, sex, basal anti-HBs levels, post-vaccination anti-HBs results and final anti-HBs levels were collected. Comparisons of the basal and final levels, as well as associations regarding age and sex, were performed by means of the Student t and Chi-square tests. RESULTS: Of the 359 students, 97 (27.02%) had basal antibody concentrations

Ex vivo microbial leakage after using different final irrigation regimens with chlorhexidine

Objective: To assess the influence of final irrigation protocols with chlorhexidine in the coronal leakage of Enterococcus faecalis in filled root canals. Material and Methods: Seventy single-root canals from extracted teeth were prepared using ProTaper instruments. The irrigation protocol accomplished an alternating irrigation with 5 mL of 2.5% sodium hypochlorite (NaOCI) and 17% EDTA between each file. The teeth were randomly divided into four experimental groups (n=15) according to the final irrigation regimen: group 1, without final irrigation; group 2, irrigation with 10 mL 2.0% chlorhexidine (CHX); group 3, with a final application of EC40™; and group 4, irrigation with the combination (1:1) of 0.2% CHX + 0.1% cetrimide (CTR). All the teeth were mounted in a two-chamber apparatus and the coronal access was exposed to E. faecalis. The presence of turbidity in the BHI broth over a period of 180 days was observed. The Friedman test was used for statistical analysis. Results: EC40™ varnish showed the least leakage at 180 days, and was statistically similar to 2% CHX. No significant differences were observed between the group without final irrigation and the 2% CHX group or 0.2% CHX + 0.1% CTR. Conclusions: In this ex vivo study, EC40™ showed the longest delayed coronal leakage of E. faecalis, although without significant differences from 2% CHX.

Decalcifying effects of antimicrobial irrigating solutions on root canal dentin

Objective: The aim of this study was to determine the decalcifying efficacy of 7% maleic acid (MA), 2% chlorhexidine (CHX), and combinations of 7% MA + 0.2% cetrimide (CTR) and 2% CHX + 0.2% CTR, in four time periods. Study Design: Four specimens per tooth were obtained from a 2-mm thick slice of the cervical third of the root of ten human incisors. At 1, 2, 3 and 5 minutes of immersion, the concentrations of Ca2+ were measured by atomic absorption spectrophotometry. The results were analyzed using the Mann-Whitney U-test. Results: Statistically significant differences were seen for the extracted calcium in all time periods. The amount of calcium extracted by 7% MA was the highest at all four immersion times, followed by 7% MA + 0.2% CTR. Two percent CHX and its combination with 0.2% CTR extracted virtually no calcium. Conclusions: The decalcifying capacity of 7% MA and 2% CHX diminished when combined with 0.2% CTR (AU)

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Ex vivo study of bacterial coronal leakage in indirect pulp treatment

Objective: The aim of this study was to evaluate, ex vivo, bacterial coronal leakage with different antimicrobial agents applied to the dentine for indirect pulp treatment (IPT).Study Design: Sixty extracted teeth were prepared and randomly distributed into 5 groups (n=10): Group 1: no antimicrobial dentine treatment; group 2: 1% chlorhexidine (CHX)+1% thymol varnish (Cervitec®); group 3: 2% CHX solution; group 4: 40% CHX varnish (EC40™) and group 5: Clearfil™ Protect Bond (CPB). Ten teeth served as controls. The teeth were restored using a resin-modified glass ionomer cement (GIC) and then mounted in a two-chamber device. The coronal access was exposed to Streptococcus mutants for 45 days. The appearance of turbidity in the BHI broth of the lower chamber was considered as specimen leakage. Results: Survival analysis, determined by non parametric Kaplan-Meier and log-rank tests, showed that the best results were for groups EC40™+GIC and GIC alone; yet there were not statistically significant differences between them. All specimens of CPB+GIC and 2% CHX+GIC, leaked at 45 days. Conclusions: In IPT the use of GIC without pretreatment of the dentine and pretreatment with 40% CHX varnish resulted in a significant delay of bacterial coronal leakage (AU)

Eradication of enterococci biofilms by lactic acid aloneand combined with chlorhexidine and cetrimide

Objective: The antimicrobial activity of lactic acid (LA) alone or in combination with chlorhexidine (CHX) and cetrimide (CTR) against three Enterococcus faecalis strains, E. faecalis ATCC 29212, E. faecalis EF�D1 and E. faecalis U�1765, one Enterococcus durans strain and one dual�species biofilm was investigated.Study Design: The irrigating solutions tested were 20%, 15%, 10%, 5% and 2.5% LA, alone and in combination with 2% CHX and with 0.2% CTR. The biofilms were grown in the MBECTM high�throughput device for 24 hours and exposed to the solutions for 30 seconds and 1 minute. "Eradication" was defined as 100% bacterial kill.Results: Twenty percent LA eradicated all enterococci biofilms after 30 seconds contact time. The association of LA + 0.2% CTR achieved better results than LA alone, in contrast with the results obtained using LA + 2% CHX. E. durans was eradicated by all the tested solutions at 1 minute. The dual�species biofilm, E. faecalis ATCC 29212 + E. durans, gave intermediate values of the pure cultures. Conclusions: LA is capable of eradicating enterococci biofilm at a concentration of 20%. The combination of lower concentrations with 0.2% CTR achieved eradication after 1 minute (AU)

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In vitro enterococcus faecalis biofilm formation on five adhesive systems

Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness.Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness.Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond (AU)

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Antimicrobial activity and enterococcus faecalis biofilm formation on chlorhexidine varnishes

Objective: To evaluate, in vitro, the antimicrobial activity and biofilm formation of three chlorhexidine varnishes in four Enterococcus faecalis strains: E. faecalis ATCC 29212, E. faecalis EF-D1 (from failed endodontic treatment), E. faecalis 072 (cheese) and E. faecalis U-1765 (nosocomial infection), and one Enterococcus durans strain (failed endodontic treatment). Study Design: The direct contact test was used to study the antimicrobial activity. Bacterial suspensions were exposed for one hour to EC40, Cervitec (CE) and Cervitec Plus (CEP) varnishes. "Eradication" was defined as 100% bacterial kill. The formation of enterococci biofilms was tested on the surface of the varnishes after 24 hours of incubation and expressed as percentage of biofilm reduction. Results: EC40 eradicated all strains except E. faecalis ATCC 29212, where 98.78% kill was achieved. CE and CEP showed antimicrobial activity against all the strains, but most clearly against E. durans and E. faecalis 072. EC40 completely inhibited the formation of biofilm of E. faecalis ATCC 29212, E. faecalis 072 and E. durans. CE and CEP led to over 92% of biofilm reduction, except in the case of E. faecalis U-1765 on CEP (76.42%). Conclusion: The three varnishes studied were seen to be effective in killing the tested strains of enterococci and in inhibiting the formation of biofilm, the best results being observed with EC40 (AU)

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