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.

Comparison of Fusobacterium nucleatum and Porphyromonas gingivalis Lipopolysaccharides Clinically Isolated from Root Canal Infection in the Induction of Pro-Inflammatory Cytokines Secretion

Abstract The aim of this study was to compare the biological activity of lipopolysaccharides (LPS) purified from Fusobacterium nucleatum and Porphyromonas gingivalis strains, both isolated from primary endodontic infection (PEI) in the levels of IL-1β and TNF-α released by macrophage cells. Moreover, LPS was purified from F. nucleatum and P. gingivalis American Type Collection (ATCC) and its biological activity was compared to respectively clinical isolates strains. F. nucleatum and P. gingivalis strains clinically isolated from PEI had their identification confirmed by sequencing the 16S rRNA gene. LPS from F. nucleatum and P. gingivalis and their respective ATCC strains were extracted by using Tri-reagent method. Macrophages (Raw 264.7) were stimulated with LPS at 100 ng/mL for 4, 8 and 12 h. Secretion of IL-1 β and TNF-α was also determined. Paired t-test, repeated measures ANOVA and one-way ANOVA were employed. All LPS induced significant production of IL-1β and TNF-α, with the former being secreted at higher levels than the latter in all time-points. F. nucleatum induced a higher expression of both cytokines compared to P. gingivalis (p<0.05). No differences were observed between clinical and ATCC strains, as both presented the same potential to induce pro-inflammatory response. It was concluded that F. nucleatum and P. gingivalis LPS presented different patterns of activation against macrophages as seen by the IL-1β and TNF-α production, which may contribute to the immunopathogenesis of apical periodontitis. Moreover, clinical and ATCC strains grown under the same in vitro environment conditions presented similar biological activity.

Resumo O objetivo deste estudo foi comparar a atividade biológica de lipopolissacarídeos (LPS) purificados a partir de linhagens de Fusobacterium nucleatum e Porphyromonas gingivalis, ambas isoladas de infecções endodônticas primárias (IEP) nos níveis de IL-1β e TNF-α produzidos por macrófagos. Adicionalmente, LPS foi purificado de F. nucleatum e P. gingivalis "American Type Collection" (ATCC) e sua atividade comparada às respectivas linhagens clinicamente isoladas. Linhagens de F. nucleatum e P. gingivalis isoladas clinicamente de IEP tiveram sua identificação confirmada por sequenciamento do gene 16S rRNA. LPS de F. nucleatum e P. gingivalis e das respectivas linhagens foram extraídos com o uso do método "Tri-reagent". Macrófagos (Raw 264.7) foram estimulados com LPS a 100 ng/mL por 4, 8 e 12 h. A secreção de IL-1β e de TNF-α foi determinada. Foram usados os testes t-pareado, ANOVA de medidas repetidas e ANOVA de um fator. Todos os LPS induziram a produção significante de IL-1β e TNF-α, sendo o primeiro secretado em mais altas concentrações que o último em todos os tempos avaliados. F. nucleatum induziu uma maior expressão de ambas as citocinas comparativamente ao P. gingivalis (p<0,05). Não foram observadas diferenças entre as linhagens clínica e ATCC, uma vez que ambas apresentaram o mesmo potencial de indução da resposta pró-inflamatória. Conclui-se que F. nucleatum e P. gingivalis possuem diferentes padrões de ativação dos macrófagos, como visto pela produção de IL-1β e TNF-α, o que pode contribuir para a imunopatogênese da periodontite apical. Ainda, linhagens clínica e ATCC mantidas no mesmo ambiente in vitro apresentaram ativação biológica semelhante.