Molecular characterization of intact cell-derived and cell-free bacterial DNA from carious dentine samples

• Published in: Journal of microbiological methods Vol. 158; pp. 33 - 43
• Main Authors: Schmidt, Jana, Krohn, Sandra, Buenger, Laura, Zeller, Katharina, Schneider, Hartmut, Treuheit, Maud, Kaiser, Thorsten, Ziebolz, Dirk, Berg, Thomas, Haak, Rainer
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: 16S rRNA gene based PCR; Bacteria - classification; Bacterial DNA; Cell-Free Nucleic Acids - isolation & purification; Clinical Studies as Topic; Dental Caries - microbiology; dental materials; Dentine caries; DNA; DNA, Bacterial - isolation & purification; genes; Humans; microbial communities; Microbiota; patients; quantitative polymerase chain reaction; restriction fragment length polymorphism; ribosomal RNA; Specimen Handling - methods; T-RFLP; teeth; therapeutics; Dentine caries; Bacterial DNA; 16S rRNA gene based PCR; T-RFLP
• ISSN: 0167-7012; 1872-8359; 1872-8359
• DOI: 10.1016/j.mimet.2019.01.012

Microbial analyzes of carious dentine samples, especially in terms of interventions, represent a challenge due to difficulties in carious dentine sampling particularly with small bacterial DNA contents. Therefore, information about the quantitative reduction of bacterial DNA as well as microbial shifts and differences in diversity correlating with treatment interventions are scarce. In this study, carious dentine samples were collected in a first step in the course of a selective caries excavation at two different deep dentine caries lesions in three patients. Second, after selective caries excavation and sampling of carious dentine, an intervention was performed by applying dental materials onto the remaining carious dentine followed by a restoration of the study teeth with composite fillings. After 8 weeks, remaining carious dentine was sampled and analyzed as described above. The microbial community before and after therapy was analyzed by conventional culture compared to bacterial DNA analyses using 16S rRNA gene based real-time PCR and terminal restriction fragment length polymorphism (T-RFLP) for fingerprinting community changes within carious dentine samples. An ultra-pure workflow allowed the valid comparison of even small carious dentine samples with low DNA contents and the differentiation between intact cell-derived and cell-free bacterial DNA. Intra- and inter-subject related differences in the bacterial DNA content and its composition in deep dentine caries were determined considering the first visits. The ratio of cell-free bacterial DNA and DNA from intact cells decreased in two of three subjects included in the current study from visit 1 to visit 2 with the test substance (1:200 to 1:17) and the control substance (1:82 to 1:7). T-RFLP revealed changes in the bacterial diversity and composition shifts after treatment as well as between cell-free bacterial DNA and DNA derived from intact cells. The approach of differentiation and quantification of cell-free and intact cell-derived bacterial DNA is reasonable within the investigation of carious dentine samples, especially when considering the effect of an intervention. T-RFLP is principally suitable for the analysis of microbial shifts within carious dentine samples.