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.

Microbiota of periodontal pockets and root canals in induced experimental periodontal disease in dogs

“…Even considering the limitations of sampling biofilms by using endodontic files/paper points, this approach is a well‐established protocol because all floating species present in the main RC/PP are or were members of the endodontic/periodontal biofilm . For assessment of microbiota in PP and/or RC, the literature has reported several studies using sterile paper points for sample collection . In addition, VMGA III is frequently used as transport medium, which avoids bacterial loss from PP and RC samples until laboratorial procedures …”

“…For assessment of microbiota in PP and/or RC, the literature has reported several studies using sterile paper points for sample collection . In addition, VMGA III is frequently used as transport medium, which avoids bacterial loss from PP and RC samples until laboratorial procedures …”

“…The methodology used 16S RNA gene sequencing performed according to Nóbrega et al After DNA extraction, the universal amplification by polymerase chain reaction (PCR) was performed in a total volume of 50 µL as follows: 5 µL of template DNA, 5 µL of 10× PCR buffer, 1.5 µL of 25 mmol/L MgCl 2 , 4.0 µL of DNTP solutions (25 mmol/L each), 1 µL of 25 pmol forward universal primer (D88, 5′‐GAGAGTTTGATYMTGGCTCAG‐3′) and reverse universal primer (E94, 5′‐GAAGGAGGTGWTCCARCCGCA‐3′), 0.5 µL of 5 U/mL platinum Taq polymerase and 32 µL of sterile distilled water.…”

The Use of Different Irrigation Techniques to Decrease Bacterial Loads in Healthy and Diabetic Patients with Asymptomatic Apical Periodontitis

“…Despite, the rapid evolution in irrigating materials, devices, and tools, the persistence of bacteria remains questionable. The diversity of root canal anatomy and the organisation of microorganisms hindered in the dentinal tubules, isthmuses and ramifications complicate the complete bacterial eradication from root canal which usually ends with apical periodontitis [2, 14]. It was confirmed that there is an actual relationship between the presence of specific bacterial taxa in filled root canal and treatment failure, which suggested that some taxa, such as streptococci, Olsenella uli, Propionibacterium acnes , and Fusobacterium nucleatum , might have the potentiality to be the initiator of risk factors and cause periapical diseases [15].…”

“…Diabetes mellitus may act as a precursor for inducing pulp necrosis and successive periapical lesions and failed endodontic treatment cases due to altered wound repair, immune and vascular functions [1, 2]. …”