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

ABSTRACT

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

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.