In this study, we investigated the microbiota that colonizes the apical portion of root canals in primary and secondary infections. Ricucci et al.
) demonstrated the presence of biofilm bacteria in this section of the root in the vast majority of cases with apical periodontitis. While successfully healed teeth often still contain bacteria in the coronal part of root canal, the apical sections are typically free of microorganisms (30
). Even though the apical portion of infected root canals is considered most difficult to treat and thus most prone to harbor the residual infection (6
), few studies have performed separate analyses of the microbiota located in this section of the tooth. Alves et al.
) demonstrated that the bacterial profiles of the middle portion of teeth with primary and secondary endodontic infections differed significantly from the apical portion of the same teeth. A follow-up checker-board analysis examined these samples for the presence of a panel of 28 suspected endodontic pathogens (31
). Two additional studies investigated the apical third of primary infected teeth for a number of species via species-specific PCR (14
) and checker-board analysis (21
We used a PCR-DGGE approach to obtain bacterial profiles for the apical portions of teeth derived from different individuals, different teeth of the same individuals as well as different roots of the same tooth. Both primary and secondary infections with chronic apical periodontitis were examined. While Alves et al.
) found similar species diversity for both types of infections in similar samples, the primary infections analyzed here harbored significantly more diverse (p=0.0003) microbial profiles than the secondary infections () with an average of 33±8 vs
16±6 bands, respectively. This observation is consistent with previous studies examining species diversity of the entire root canal (20
) and the difference to above study could be due to a number of reasons including geographical location (32
) and the smaller sample size examined by their group. Consistent with findings by other groups (18
), intra-individual and inter-individual variations were similar (). Collection of several pairs of apical portions derived from the same multi-rooted tooth allowed for the first time separate analysis of the species diversity present in the specific microenvironments of individual roots. Despite our small sample size it was apparent that the disturbance of the microbial biofilm during endodontic treatment produced conditions which resulted in a disparate recovery of the individual apical root-associated microbiota ( – samples S6-1a/S6-1b and S7-1a/S7-1b). Among the identified microorganisms (), disparities were most obvious for Fusobacterium nucleatum
sp. and Burkholderiales
. We believe that these differences in the respective microbiotas are most likely the result of incomplete eradication of the original infection. We propose that re-infection via coronal leakage should equally introduce a similar bacterial population to both roots and thus result in more comparable profiles as we observed for the two roots taken from the primary infected mandibular molar ( – samples P2-1a/ P2-1b).
Phylotypic identification of over 50 excised bands revealed the high prevalence of fusobacteria, Actinomyces
sp. and Anaeroglobus geminatus
in both, primary and secondary infections. The suspected endodontic pathogen Pseudoramibacter alactolyticus
) was also present in both types of infection (). Other species found in the majority of primary infections which were less abundant or not identified in previously treated teeth included Porphyromonas endodontalis
, Parascordavia denticolens
sp. Dialister invisus
, and Synergistetes
. Most of these species/taxa have been previously associated with endodontic disease (13
). A striking observation in the samples from secondary infections was the high prevalence of Pseudomonas
sp. as well as Burkholderiales
which includes the familiesBurkholderiaceae
( and ). These microorganisms are typically not abundant in the oral cavity and thus could indicate a problem with environmental contamination introduced during treatment. E. faecalis
was present in less than half of the secondary infections as a relatively minor band consistent with previous observations that this species is unlikely a prime pathogen for apical periodontitis (9
In conclusion, the samples examined in our study exhibited a similar difference in bacterial profile diversity between primary and secondary infections in the apical root section as previously described for samples obtained from the entire root canal. In addition to environmental opportunistic pathogens, Fusobacterium, Actinomyces and Anaeroglobus are candidate genera for involvement in re-emergence/persistence of endodontic infections. Examination of different roots from the same teeth indicated that disturbance of the microenvironment during treatment can lead to the development of divergent communities within the respective apical portions.