Resumen de Bacteria Community Study of Combined Periodontal-Endodontic Lesions Using Denaturing Gradient Gel Electrophoresis and Sequencing Analysis
Hong Li, Rui Guan, Sun Jinghua, Benxiang Hou
Background: The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared.
Methods: Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction�based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs.
Results: Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P <0.001). The average Cs of the RC and PP samples was 93.81% ± 10.26%. Overall, 60 genera/species were identified by sequencing. Of these, the predominant genera in RCs were Porphyromonas sp. (13.9%), Filifactor sp. (12.5%), and Parvimonas sp. (11.1%), similar to the genera obtained from PP samples. In total, 43 genera/species were common to the RC and PP samples. The most prevalent bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia.
Conclusions: The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.
Combined periodontal-endodontic lesions are a clinical dilemma because making a differential diagnosis and deciding on a prognosis are difficult.1,2 Periodontal attachment loss caused by some untreated periodontal-endodontic lesions is responsible for tooth mortality and even tooth loss.3 The relationship between periodontal and pulpal diseases has been the subject of speculation, for example, whether periodontal disease has an effect on the pulp before it involves the apex.2 Although toxins and microbial agents in the pulp space can in some cases induce periodontal inflammation through lateral and accessory canals,4 the effect of these irritants from the periodontal space on the pulp is less clearly defined.2,3 The pathogenesis of combined periodontal-endodontic lesions is not completely understood. Previous studies5-8 analyzed the influence of the presence of periodontal disease on pulpal pathosis microbiologically and hypothesized that the periodontal pocket (PP) may be a possible source of root canal (RC) infection. However, these studies used conventional culture methods and investigated only cultivable microbes. Because of the limitations of conventional culture methods, these methods do not lead to comprehensive conclusions. Greater understanding of the relationship of the microorganisms in the PPs and RCs of patients with combined periodontal-endodontic lesions will be helpful in discovering the etiology of this disease.
Compared with conventional culture methods, molecular techniques have the advantage of detecting uncultivable bacteria or ones that are difficult to grow. However, only expected species have been investigated with any frequency because the number of target bacteria was restricted for investigations using polymerase chain reaction (PCR).9-11 For example, Rupf et al.10 used molecular techniques to detect several expected species in combined periodontal-endodontic lesions of the same tooth; they concluded that periodontal pathogens often accompany endodontic infections.
An alternative molecular technique, such as PCR-based denaturing gradient gel electrophoresis (PCR-DGGE), enables investigators to survey entire bacterial communities without cultivation.12 The technique has become an important tool for studying complex bacterial communities in a variety of infectious oral diseases, including caries,13,14 endodontic infections,15 and periodontitis.16 Given these previous studies,5-11 a hypothesis arose that the adjacent PP is a likely source for non-carious RC infection associated with combined periodontal-endodontic lesions. The aim of the present study is to identify the entire microbial population obtained from RCs and from subgingival plaque samples of teeth associated with combined periodontal-endodontic lesions, using PCR-based 16S ribosomal RNA (rRNA) gene DGGE and sequencing. The predominant microflora from the RCs are identified and compared with those from the adjacent PPs.
