Quantification of Bacterial Colonization in Dental Hard Tissues Using Optimized Molecular Biological Methods

Abstract

Bacterial infections of root canals and the surrounding dental hard tissue are still a challenge due to biofilm formation as well as the complex root canal anatomy. However, current methods for analyzing biofilm formation, bacterial colonization of root canals and dental hard tissue [e.g., scanning electron microscopy, confocal laser scanning microscopy (CLSM) or determination of colony forming units (CFU)] are time-consuming and only offer a selective qualitative or semi-quantitative analysis. The aim of the present study is the establishment of optimized molecular biological methods for DNA-isolation and quantification of bacterial colonization via quantitative PCR (qPCR) from dental hard tissue. Root canals of human premolars were colonized with Enterococcus faecalis. For isolation of DNA, teeth were then grinded with a cryo mill. Since the hard tissues dentin and especially enamel belong to the hardest materials in the human organism, the isolation of bacterial DNA from root dentin is very challenging. Therefore, treatment steps for the isolation of DNA from grinded teeth were systematically analyzed to allow improved recovery of bacterial DNA from dental hard tissues. Starting with the disintegration of the peptidoglycan-layer of bacterial cells, different lysozyme solutions were tested for efficacy. Furthermore, incubation times and concentrations of chelating agents such as EDTA were optimized. These solutions are crucial for the disintegration of teeth and hence improve the accessibility of bacterial DNA. The final step was the determination of prior bacterial colonization of each root canal as determined by qPCR and comparing the results to alternative methods such as CFU. As a result of this study, optimized procedures for bacterial DNA-isolation from teeth were established, which result in an increased recovery rate of bacterial DNA. This method allows a non-selective and straightforward procedure to quantify bacterial colonization from dental hard tissue. It can be easily adapted for other study types such as microbiome studies and for comparable tissues like bones.

Comparative Evaluation of Smear Layer Removal by Apple Vinegar Using Laser Activated Irrigation and EndoVac System: Randomized Control Trial

“…Smear layer formed during root canals instrumentation consists of dentin chips, bacteria and their byproducts, microorganisms, and tissue remnants (30) . This layer coats root canal dentinal walls which preserves bacteria in the dentinal tubules, interferes with deep penetration of irrigating solution and root canal sealer, prevents adaptation between obturation material and root canal wall that can disturb the apical seal which subsequently affect success rate of endodontic treatment (31,32) . Sodium hypochloride (NaOCl) which has capability to remove organic components used alternatively with EDTA chelating agent concerned with inorganic part were used to remove smear layer completely, however this comments irrigation protocol has detrimental effect on dentinal structure (4,33) .…”

Quantification of Bacterial DNA from Infected Human Root Canals Using qPCR and DAPI after Disinfection with Established and Novel Irrigation Protocols

“…Molecular biological methods offer the opportunity to omit these problems; they allow the quantification of the complete material without bias due to sample collection. We recently optimized a method for the purification of DNA from infected dental hard tissue [ 34 ]. In the present study, we used the established and standardized method in a controlled study analyzing the effect of different irrigation protocols on infected root canals for the first time.…”

“…Preparation of the specimens and inoculation of the teeth have been described previously [ 10 , 34 ]. The crown of 78 human single-canal premolars was separated from the roots, and the roots were prepared with rotary nickel-titanium files Pro Taper Gold F1 and F2 (Dentsply, York, PA, USA) under irrigation with NaCl 0.9%.…”

“…The other half of each root was used for purification of DNA from the colonized root canals as previously described [ 34 ]. In short, the other root halves were grinded in a 6775 Freezer/Mill cryogenic grinder (SPEX) using the following parameters: precool: 10 min; run Time: 1 min; cool time: 1 min; impactor rate: 12; and cycles: 4.…”

“…Quantitative real-time PCR was done as previously described [ 34 ]. In short, two µL of isolated DNA from both the pellet and supernatant fraction were analyzed by qPCR in a CFX96 TM Real-Time System (Bio-Rad, Hercules, CA, USA) using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in triplicate.…”