Molecular identification of cultivable bacteria from infected root canals associated with acute apical abscess

The objective of this study was to investigate the bacterial composition present in root canals of teeth associated with acute apical abscess by molecular identification (16S rRNA) of cultivable bacteria. Two hundred and twenty strains isolated by culture from 20 root canals were subjected to DNA extraction and amplification of the 16S rRNA gene (PCR), followed by sequencing. The resulting nucleotide sequences were compared to the GenBank database from the National Center of Biotechnology Information through BLAST. Strains not identified by sequencing were submitted to clonal analysis. The association of microbiological findings with clinical features and the association between microbial species were also investigated. Fifty-nine different cultivable bacteria were identified by 16S rRNA gene sequencing, belonging to 6 phyla, with an average number of 6 species per root canal. Molecular approaches allowed identification of 99% of isolates. The most frequently identified bacteria were Prevotella spp., Pseudoramibacter alactolyticus, Parvimonas micra, Dialister invisus, Filifactor alocis, and Peptostreptococcus stomatis. Positive association was found between Prevotella buccae and Pseudoramibacter alactolyticus and between Parvimonas micra and Prevotella nigrescens (both p<0.05). It was concluded that the microbiota of infected root canals associated with acute apical abscess is diverse and heterogeneous, composed mainly of anaerobic Gram-negative bacteria, with the great majority belonging to the phyla Firmicutes and Bacteroidetes273318324CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302575/2009; 308162/2014-5Sem informação2009/07760-5; 2011/09047-4O objetivo deste estudo foi investigar a composição bacteriana de canais radiculares associados com abscesso apical agudo através de identificação molecular (16S rRNA) de bactérias cultiváveis. Duzentas e vinte cepas, de 20 casos, isoladas por cultura foram submetidas a extração de DNA e amplificação do gene 16S rRNA (PCR), seguido de sequenciamento. As sequências de nucleotídeos obtidas foram comparadas com o banco de dados (GenBank) do National Center of Biotechnology Information através do BLAST. Cepas não identificadas pelo sequenciamento foram submetidas à clonagem. Associação de achados microbiológicos e características clínicas e associação entre espécies bacterianas também foram investigadas. Cinquenta e nove bactérias cultiváveis diferentes foram identificadas pelo sequenciamento do gene 16S rRNA, pertencentes a 6 filos, numa média de 6 espécies por canal. Método molecular permitiu identificação de 99% das cepas isoladas. As bactérias mais frequentes foram Prevotella spp., Pseudoramibacter alactolyticus, Parvimonas micra, Dialister invisus, Filifactor alocis, Peptostreptococcus stomatis. Associação positiva foi encontrada entre Prevotella buccae e Pseudoramibacter alactolyticus, e entre Parvimonas micra e Prevotella nigrescens (p<0,05). Foi concluído que a microbiota de canais radiculares infectados associados com abscesso apical agudo é diversa e heterogênea, composta principalmente por anaeróbios Gram-negativos, pertencentes aos filos Firmicutes e Bacteroidete

Effects of Contemporary Irrigant Activation Schemes and Subsequent Placement of an Interim Dressing on Bacterial Presence and Activity in Root Canals Associated with Asymptomatic Apical Periodontitis

New tools for activating endodontic irrigants have evolved, yet their impact on root canal disinfection, in comparison to the passive placing of an inter-visit medication, have not yet been fully elucidated. The use of DNA- and rRNA-based methods may cast some new light on this issue, as they allow a comparison to be made between microbial presence and activity. Therefore, the aim of this single-arm intervention trial is to evaluate the antibacterial effect of endodontic procedures using both molecular methods. Root canal samples were obtained from 20 patients with asymptomatic apical periodontitis after each treatment step: access cavity, chemo-mechanical preparation, adjunctive procedures (XP-endo Finisher file and passive ultrasonic irrigation), calcium hydroxide medication, and 2nd-visit root canal preparation. DNA and cDNA from the samples were subjected to quantitative polymerase chain reaction with universal primers for the bacterial 16S rRNA gene. Chemo-mechanical preparation promoted a drastic reduction in bacterial levels and activity, whereas the adjunctive procedures did not make a significant contribution to further disinfection. At the 2nd visit, bacteria were active after the use of calcium hydroxide medication; however, they were significantly reduced after a 2nd-visit preparation. Consequently, the lowest bacterial levels were found at the end of the treatment. This clinical trial, which used an rRNA and rDNA combined approach, confirmed previous studies showing that root canal preparation represents the main strategy for root canal disinfection

Next-generation sequencing to assess potentially active bacteria in endodontic infections

Introduction: Because active bacteria present a higher abundance of ribosomal RNA (rRNA) than DNA (rRNA gene), the rRNA/DNA ratio of next-generation sequencing (NGS) data was measured to search for active bacteria in endodontic infections. Methods: Paired complementary DNA and DNA samples from 5 root canals of teeth with apical periodontitis were subjected to polymerase chain reaction with bar-coded primers amplifying the 16S rRNA gene hypervariable regions V4-V5. High-throughput sequencing was performed using MiSeq (Illumina, San Deigo, CA), and data were analyzed using Quantitative Insights Into Microbial Ecology and Human Oral Microbiome Database. Statistical analysis was performed for relative abundance of bacteria in the DNA- and rRNA-based NGS data using the Mann-Whitney test, whereas differences in the diversity and richness indexes were assessed using a nonparametric 2-sample t test (P < .05). For bacterial taxa detected in both approaches, the rRNA/DNA ratios were calculated by dividing the average abundance of individual species in the respective analysis. Results: Although no significant difference was found in the indexes of bacterial richness and diversity, the relative abundance of bacterial members varied in both analyses. Comparing rRNA with DNA data, there was a significant decrease in the relative abundance of Firmicutes (P < .05). The bacterial taxa Bacteroidales [G-2] bacterium HMT 274, Porphyromonas endodontalis, Tannerella forsythia, Alloprevotella tannerae, Prevotella intermedia, Pseudoramibacter alactolyticus, Olsenella sp. HMT 809, Olsenella sp. HMT 939, Olsenella uli, and Fusobacterium nucleatum subsp. animalis were both dominant (DNA ≥ 1%) and active (rRNA/DNA ≥ 1). Conclusions: The integrated DNA- and rRNA-based NGS strategy was particularly important to disclose the activity of as-yet-uncultivated or difficult-to-culture bacteria in endodontic infections. Keywords: 16S ribosomal RNA sequencing; apical periodontitis; endodontic infection; next-generation sequencing

Effects of Contemporary Irrigant Activation Schemes and Subsequent Placement of an Interim Dressing on Bacterial Presence and Activity in Root Canals Associated with Asymptomatic Apical Periodontitis

New tools for activating endodontic irrigants have evolved, yet their impact on root canal disinfection, in comparison to the passive placing of an inter-visit medication, have not yet been fully elucidated. The use of DNA- and rRNA-based methods may cast some new light on this issue, as they allow a comparison to be made between microbial presence and activity. Therefore, the aim of this single-arm intervention trial is to evaluate the antibacterial effect of endodontic procedures using both molecular methods. Root canal samples were obtained from 20 patients with asymptomatic apical periodontitis after each treatment step: access cavity, chemo-mechanical preparation, adjunctive procedures (XP-endo Finisher file and passive ultrasonic irrigation), calcium hydroxide medication, and 2nd-visit root canal preparation. DNA and cDNA from the samples were subjected to quantitative polymerase chain reaction with universal primers for the bacterial 16S rRNA gene. Chemo-mechanical preparation promoted a drastic reduction in bacterial levels and activity, whereas the adjunctive procedures did not make a significant contribution to further disinfection. At the 2nd visit, bacteria were active after the use of calcium hydroxide medication; however, they were significantly reduced after a 2nd-visit preparation. Consequently, the lowest bacterial levels were found at the end of the treatment. This clinical trial, which used an rRNA and rDNA combined approach, confirmed previous studies showing that root canal preparation represents the main strategy for root canal disinfection

Pathogen levels and clinical response to periodontal treatment in patients with Interleukin 8 haplotypes

The aim of this study was to investigate the effect of non-surgical treatment of periodontitis on the levels of periodontopathogens and clinical parameters in patients with different genetic backgrounds produced by polymorphisms in the Interleukin (IL8) gene. Thirty patients grouped according to IL8 ATC/TTC or AGT/TTC haplotypes were submitted to non-surgical periodontal treatment. Levels of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were determined in 240 subgingival plaque samples by qPCR. The association between IL8 haplotypes and the levels of periodontopathogens and clinical parameters was investigated by multilevel analysis accounting for the clustering of diseased sites analyzed within patients. It was observed that neither levels of periodontopathogens nor non-surgical treatment was associated with the IL8 haplotype. The clinical parameters after periodontal treatment were similar in diseased and healthy sites, independently of the IL8 haplotype. Nonetheless, in the same period, diseased sites of AGT/TTC patients harbored higher levels of P. gingivalis, T. denticola, T. forsythia, and red complex than those of ATC/TTC patients. However, the non-surgical periodontal therapy decreased the levels of these periodontopathogens and of the tested clinical parameters of diseased sites in both groups. Non-surgical therapy is equally effective in improving clinical parameters and decreasing the levels of periodontopathogens, independent of the genotype groups produced by the IL8 haplotype.Foundation for Research Support of São Paulo State - FAPESP, 2003/10424-0Foundation for Research Support of São Paulo State - FAPESP, 2009/08773-3Foundation for Research Support of São Paulo State - FAPESP, 2009/11371-