Molecular Identification of Cultivable Bacteria From Infected Root Canals Associated With Acute Apical Abscess

Abstract 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 Bacteroidetes.

Resumo O 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 Bacteroidetes.

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 Bacteroidetes.

Histomorphometric and microbiological assessment of photodynamic therapy as an adjuvant treatment for periodontitis: a short-term evaluation of inflammatory periodontal conditions and bacterial reduction in a rat model.

OBJECTIVE: The aim of this study was to investigate the short-term effects of photodynamic therapy (PDT) in periodontal tissue when it is used as an adjuvant treatment for periodontitis. BACKGROUND DATA: PDT has been used as an adjuvant in the combat of local infections, such as periodontitis, and combines a photosensitizer (PS) with a light source to induce reactive oxygen species (ROS) and kill microbial cells. METHODS: Fifty healthy male rats were used in this study. Periodontitis was induced by placing a cotton ligature around the upper left second molar in a subgingival position. Posterior maxillas were removed and histologically prepared with hematoxylin & eosin (H&E) staining techniques. PDT was performed with a diode laser (λ=660 nm) with an output power of 100 mW. Methylene blue aqueous solution (100 µM) was used as the PS while control group used phosphate buffered saline (PBS). Collagen organization, inflammatory infiltrate, and bone loss were evaluated. Bacterial samples were collected before and immediately after treatment to determine bacterial reduction. RESULTS: The experimental group that was treated with PDT presented better periodontal healing, as measured by collagen organization, inflammatory infiltrate, and bone loss. Significant bacterial reduction was achieved following treatment with or without PDT compared to control, with a higher microbial reduction observed in the PDT group. CONCLUSIONS: PDT used as an adjuvant treatment showed effective short-term control of periodontitis infection.

Exploring bacterial diversity of endodontic microbiota by cloning and sequencing 16S rRNA.

INTRODUCTION: The characterization of microbial communities infecting the endodontic system in each clinical condition may help on the establishment of a correct prognosis and distinct strategies of treatment. The purpose of this study was to determine the bacterial diversity in primary endodontic infections by 16S ribosomal-RNA (rRNA) sequence analysis. METHODS: Samples from root canals of untreated asymptomatic teeth (n = 12) exhibiting periapical lesions were obtained, 16S rRNA bacterial genomic libraries were constructed and sequenced, and bacterial diversity was estimated. RESULTS: A total of 489 clones were analyzed (mean, 40.7 ± 8.0 clones per sample). Seventy phylotypes were identified of which six were novel phylotypes belonging to the family Ruminococcaceae. The mean number of taxa per canal was 10.0, ranging from 3 to 21 per sample; 65.7% of the cloned sequences represented phylotypes for which no cultivated isolates have been reported. The most prevalent taxa were Atopobium rimae (50.0%), Dialister invisus, Prevotella oris, Pseudoramibacter alactolyticus, and Tannerella forsythia (33.3%). CONCLUSIONS: Although several key species predominate in endodontic samples of asymptomatic cases with periapical lesions, the primary endodontic infection is characterized by a wide bacterial diversity, which is mostly represented by members of the phylum Firmicutes belonging to the class Clostridia followed by the phylum Bacteroidetes.