Bacterial Analysis Of Combined Periodontal-Endodontic Lesions Using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE)

Objective For periodontitis and pulpal-periapical diseases, the aetiological factors and pathological process are not completely the same. On the other hand, they are both proved to be a mixed anaerobic infection, and there are many similarities in the inflammatory and immune responses. In fact, the two diseased conditions can influence each other, leading to combined periodontal-endodontic lesions (peri-endo lesions). Periodontitis and pulpal-periapical diseases as independent lesions, the researches on microbiology are plentiful. However, when it comes to combined periodontal-endodontic lesions, the study is mostly based on the bacterial cultures, which restrains the detection of the culture-difficult or as-yet-unknown taxa..The denaturing gradient gel electrophoresis (DGGE) technique can been used to evaluate the microbial community and it allows the visualization of profiles of bacterial communities in multiple clinical specimens at a time, including the unpredictable bacteria. The present study was undertaken to compare the bacterial communities present in periodontium and root canals of the same tooth diagnosed as combined periodontal-endodontic lesions by using a16S rRNA gene-based broad-range PCR-DGGE approach. Materials and methods1. DGGE profiling and analysisSamples were collected from13extracted teeth with late-phase periodontitis, endodontic samples from root tip1/3root canal, and periodontal samples from the corresponding neighboring periodontium. Genomic DNA was collected for the following universal bacterial primers-PCR. The PCR products are then loaded on the DGGE gels to gain separate bands. The DGGE banding patterns are conveyed to the Quantity One software for the analysis like similarity matrix and cluster-analysis, and to the statistical software SPSS17.0for the paired t-test and spearman rank correlation analysis.2. Band excision, PCR-cloning and sequencingThe typical DGGE bands were excised and the DNA fragments eluted. They were amplified by another PCR and then cloned and sequenced. Taxa were identified through BLAST (Basic Local Alignment Search Tool).Results1.13paired samples yielded several bands depicting the community profiles of bacteria in DGGE images. The number of bands, which is indicative of the number of bacterial species, was compared intra-group (periodontal and pulpal specimen from the same tooth). The difference was statistically significant (P<0.01), but there was no positive correlation between them.The similarity (Dice Coefficient) between them was13.1%-62.5%.2. Taxa identified through BLAST (≧98%identity) was Campylobacter, Fusobacterium, Neisseria, Peptostreptococcus, Veillonella, Aggregatibacter, Enterobacter, Haemophilus in the periodontium, and Mogibacterium, Corynebacterium, Neisseria, Actinomyces in the root canals. Approximately half the BLASTs’results <95%(identity) Conclusions1. For combined periodontal-endodontic lesions (periodontal source), there was no positive correlation between the number of strains in periodontal tissue and neighboring pulpal tissue. Common bacteria existed between them, but not all of the periodontal bacteria would appear in neighboring root canal; and the bacteria in the root canal were not completely from neighboring periodontal tissue. The original bacteria in the root canals may resuscitate and enrich the bacterial community.2. In combined periodontal-endodontic lesions (periodontal source), it was probable that new strains existed to be confirmed either in the periodontium or in the root canal.