Introduction

This study investigated the bacterial communities residing in the apical portion of human teeth with apical periodontitis in primary and secondary infections using a culture-independent molecular biology approach.

Methods

Root canal samples from the apical root segments of extracted teeth were collected from 18 teeth with necrotic pulp and 8 teeth with previous endodontic treatment. Samples were processed for amplification via polymerase chain reaction (PCR) and separated with denaturing gradient gel electrophoresis (DGGE). Selected bands were excised from the gel and sequenced for identification.

Results

Comparable to previous studies of entire root canals, the apical bacterial communities in primary infections were significantly more diverse than in secondary infections (p=0.0003). Inter- and intra-patient comparisons exhibited similar variations in profiles. Different roots of the same teeth with secondary infections displayed low similarity in bacterial composition, while an equivalent sample collected from primary infection contained almost identical populations. Sequencing revealed a high prevalence of fusobacteria, Actinomyces sp. and oral Anaeroglobus geminatus in both types of infection. Many secondary infections contained Burkholderiales or Pseudomonas sp. both of which represent opportunistic environmental pathogens.

Conclusion

Certain microorganisms exhibit similar prevalence in primary and secondary infection indicating that they are likely not eradicated during endodontic treatment. The presence of Burkholderiales and Pseudomonas sp. underscores the problem of environmental contamination. Treatment appears to affect the various root canals of multi-rooted teeth differently, resulting in local changes of the microbiota.

In situ microzone X-ray diffraction analysis of natural teeth is presented. From our experiment, layer orientation and continuous crystal variations in teeth could be conveniently studied using fast online measurements by high-resolution X-ray microdiffraction equipment.

The main component of natural teeth was determined many years ago as calcium phosphate, mostly in the form of hydroxyapatite with different crystallites. In the past, the method used in tooth crystal investigation has been mainly powder X-ray diffraction analysis, but this method has its drawbacks, i.e. the destruction of the natural tooth structure and the difficulty in examining the preferred orientation in different layers of the tooth. During the last century, microzone X-ray diffraction on the tooth surface was carried out, but, as the technology was less sophisticated, the results obtained were not very detailed. The newly developed microdiffraction equipment permits analysis of the microzone of teeth in situ. To test this new microdiffraction equipment, microdiffraction analysis of one natural healthy deciduous molar tooth and one carious deciduous molar tooth has been performed, using a Bruker D8 instrument. Phase analysis of the two teeth was performed; the crystal size at six test points in the natural healthy tooth was calculated by reflection (211), and the crystal preferred orientation of reflection (300) and reflection (002) at six test points in the natural healthy tooth were compared. The results showed that the tooth was a kind of biological mixed crystal composed of several crystal phases, the main crystal phase being hydroxyapatite. The crystal size grew larger going from the dentin to the enamel. The crystal preferred orientation mainly existed in the enamel, especially in the reflection (002). From our experiment, layer orientation and continuous crystal variations in teeth could be conveniently studied using fast online measurements by high-resolution X-ray microdiffraction equipment.