Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries

Abstract

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65?2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10?9 %), Spirochaetes (4?3 %), Bacteroidetes (6?5 %), Actinobacteria (2?2 %) and Deferribacteres (2?2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa. INTRODUCTIONAlthough more than 150 species of bacteria have been identified in infected root canals, only a restricted number can be found simultaneously in the same tooth and a considerable variation of species is expected when analysing distinct clinical conditions, individuals or populations (Sundqvist, 1976;Molander et al., 1998;Baumgartner et al., 2004). Cultivation studies have shown a predominance of facultative and strict anaerobes in the endodontium, including representatives of Eubacterium, Fusobacterium, Peptococcus, Peptostreptococcus, Porphyromonas, Prevotella and (Sundqvist, 1992b;Le Goff et al., 1997). Bacteria inside the canal are the major cause of periapical pathologies (Kakehashi et al., 1965) and, if not adequately treated, can give rise to dentoalveolar abscess, a condition that has ability to initiate morbidity, life-threatening illness (Walsh, 1997), and to predispose to transient bacteraemia during therapy (Savarrio et al., 2005). Previous reports suggested that endodontic bacteria might be involved in extra-oral complications, such as chronic maxillary sinusitis (Melen et al., 1986), orbital cellulitis (Ngeow, 1999), infective endocarditis (Bate et al., 2000), rheumatoid arthritis (Breebaart et al., 2002) and brain abscess (Henig et al., 1978). In this regard, substantial understanding of the endodontic microbiota is an important requirement for both oral and medical microbiologists. StreptococcusWhile it is common knowledge that the development of efficient treatment strategies ...

Root Canal Microbiota of Teeth with Chronic Apical Periodontitis

“…D. invisus was originally found in infected root canals of teeth with chronic apical periodontitis (20). Findings for this species are in line with findings from several other recent studies that demonstrated the presence of D. invisus in association with both asymptomatic and symptomatic endodontic infections (24,27,29,34). P. stomatis (or Peptostreptococcus sp.…”

“…P. stomatis (or Peptostreptococcus sp. clone CK035) is a newly named species that had been previously detected in infected canals only by studies using clone library analysis (29)(30)(31). This is the first study evaluating the occurrence of this species in a large number of root canal samples.…”

“…Endodontic bacteria fall into eight bacterial phyla, namely, Firmicutes, Bacteroidetes, Spirochaetes, Fusobacteria, Actinobacteria, Proteobacteria, Synergistes, and TM7 (20,29,30,34). The panel of taxa targeted in this study included representatives of all these phyla except TM7.…”

Diversity of Spirochetes in Endodontic Infections

“…Most of the previous molecular studies focused on the detection of selected Treponema species or phylotypes (4, 11, 13, 15, 25-27, 38, 40, 43, 46). Of the several studies that have performed broad-range analyses for overall bacteria in primary endodon- (41) 2 (6) 8 (11 (14, 22, 29, 31-33, 52, 53), only two reported the occurrence of spirochetes, and even those were a few species in a few cases (31,32). These studies using universal bacterial primers may underestimate spirochetal populations, especially if they are in low numbers in the environment (5,16).…”

Detection of Mogibacterium timidum in subgingival biofilm of aggressive and non-diabetic and diabetic chronic periodontitis patients

“…Prior to sampling, supragingival plaque was removed with sterile cotton pellets and the sites were isolated with cotton rolls to avoid saliva contamination. DNA was extracted as previously described by Saito et al (18). After DNA extraction, the presence of M. timidum was determined by a Nested Polymerase Chain Reaction (Nested PCR).…”