Analysis of Metabolically Active Bacteria After Adjunctive Steps for Disinfection of Teeth With Primary Endodontic Infections: RNA- and DNA-based Molecular Study
Phase: NA
Conditions: Periapical Periodontitis, Bacterial Infections
Sponsor: University of Sao Paulo

 
 
 
 
 
Analysis of Metabolically Active Bacteria After Adjunctive Steps for 
Disinfection of Teeth With Primary Endodontic Infections: RNA- and DNA-
based Molecular Study 
 
NCT03537664 
 
20th of July 2020 
 
 

STUDY PROTOCOL 
BACKGROUND 
Bacteria and their products in the root canals of necrotic teeth are the 
main cause of apical periodontitis. Therefore, the success of endodontic 
treatment in teeth with apical periodontitis (i.e. apical repair) depends on the 
reduction of viable microorganisms of root canals by endodontic disinfection 
procedures (1). Since the endodontic microbial community comprises many as-
yet-uncultivated/difficult-to-culture bacteria, molecular methods that target viable 
cells are the choice methods for endodontic microbiology studies (2). As active 
bacteria present higher abundance of ribosomal rRNA than DNA (rRNA gene), 
the rRNA/DNA ratio is considered a useful strategy for monitoring bacterial load 
during endodontic treatment (3-6).  
The endodontic treatment is usually performed at 2 visits. Root canal 
preparation at the 1rst visit represents the main strategy for bacterial reduction by 
the mechanical action of the endodontic instrument along with irrigation (7). On 
the other hand, the use of an inter-appointment medication to maximize root 
canal disinfection after root canal preparation is one of the most controversial 
issues in Endodontics (8). DNA-based studies to evaluate the antimicrobial 
effectiveness of calcium hydroxide [Ca(OH)2] as an intracanal medicament, have 
shown divergent results (9-13). Comparing DNA and rRNA may cast some new 
light on this issue, as these methods allow bacterial levels and activity to be 
compared.  
OBJECTIVES 
This study aimed to use the rRNA/DNA ratio to assess the total viable 
bacterial load in root canals during a treatment protocol, which uses a new 
irrigation tool during root canal preparation at the 1rst visit, and a subsequent two-
visit antibacterial approach with the use of an intracanal medicament for 14 days. 
Moreover, the success (apical repair) of the treatment protocol was assessed 

after 1 year. In addition to the total bacteria, the metabolic activity of the following 
species/taxa was investigated: Bacteroidaceae sp. HOT-272 (synonym, 
Bacteroidetes oral clone X083), Cutibacterium acnes (formerly known as 
Propionibacterium acnes), Selenomonas spp. and Enterococcus faecalis. 
Moreover, the composition of the active microbiome was investigated by Next 
Generation Sequencing (NGS) analysis. 
STUDY DESIGN 
This interventional study compared the antimicrobial effects of endodontic 
procedures (clinicaltrials.gov ID: NCT03537664). The interventions tested at the 
first and second visits are described in Figure 1. 
 

Figure 1. Flow chart of the interventions. 
METHODS 
Patient selection 
The study was conducted in accordance with the Declaration of Helsinki and 
the research protocol was approved by the Institutional Ethical Committee 
(#2.201.768). All the selected patients gave signed informed consent before the 
treatment. Inclusion criteria were patients with single- rooted teeth and 
asymptomatic apical periodontitis. Exclusion criteria were: patients who had 
received antibiotic therapy during the previous 3 months or had general disease, 
teeth that could not be properly isolated with a rubber dam, non-restored teeth, 
periodontal pockets depths greater than 4 mm, and radiographic evidence of 
previous root canal filling, open apex, crown/ root fracture, root resorption, or 
narrow canals. 
Interventions and microbiologic samples  
After the initial root canal samples (S1, baseline), chemo-mechanical 
preparation (CMP) was performed using Reciproc NiTi instruments (VDW GmbH, 
Munich, Germany) according to the manufacturer’s instructions. A total of 40 mL 
of 2.5% NaOCl was used by the end of root canal preparation. Then, the root 
canal was irrigated with 5 mL of 5% sodium thiosulfate and filled with sterile 
saline before the post-instrumentation sample was taken (S2).  
The root canal was filled with 2 mL of 2.5% NaOCl and then activated for 30 
s with the XPF instrument followed by irrigation/aspiration with 2 mL of 2.5% 
NaOCl. Next, 2 mL of 17% EDTA was inserted into the root canal and activated 
with XPF for 30 s, again followed by irrigation/aspiration with 2 mL of 2.5% 
NaOCl. The canal was dried using paper points and flushed with 5 mL of 5% 
sodium thiosulfate for 1 min. The root canal was filled with sterile saline and a 
root canal sample was taken (S3a).  
The Passive Ultrasonic Irrigation (PUI) protocol following XPF activation was 
similar to that described above. Both 2.5% NaOCl and 17% EDTA were activated 

using a smooth wire with 0.2 mm diameter and .01 taper (Irrisonic - Helse, 
Ribeirão Preto, SP, Brazil), driven by an piezoelectric ultrasonic device (Piezo 
Light D5 Led, Olsen, SC, Brazil) in accordance with the manufacturer’s 
recommendations. Finally, 2.5% NaOCl was inactivated using 5% sodium 
thiosulfate and a new sample was taken at the end of the first visit (S3b). 
UltraCal XS Calcium Hydroxide Paste (Ultradent Products Inc., South 
Jordan, UT, EUA) was used as an intracanal medication for 14 days. The access 
cavities were filled with 2 mm of temporary restorative material (Dentalvile, 
Joinville, SC, Brazil) and glass ionomer cement (Riva light cure, SDI limited, 
Bayswater, Victoria, Australia). 
At the second visit, the intracanal medicament was removed using 10 mL of 
17% EDTA and agitation with K-files, and a fourth sample was taken (S4). Next, 
the root canal was irrigated with 10 mL of 2.5% NaOCl and the second visit 
instrumentation was performed using the same Reciproc file selected for the first 
visit. A final irrigation was performed with 10 mL of 2.5% NaOCl followed by 10 
mL of 17% EDTA. A root canal sample was obtained at the end of the treatment, 
as described above (S5). 
Root canal filling was performed, and the access cavity restored. An intraoral 
radiograph and cone beam computed tomography (CBCT) scans were taken to 
allow future analysis of the treatment outcome (after 1 year). 
Nucleic Acids Extraction and cDNA synthesis  
DNA and RNA were extracted using the MasterPure Complete DNA and 
RNA Purification Kit (Epicentre Technologies, Madison, WI), as described 
previously. The complementary DNA (cDNA) was synthesized using the 
SuperScript® III First-Strand Synthesis System (Invitrogen) for reverse 
transcription (RT), in accordance with the manufacturer’s instructions. The DNA 
and cDNA were stored at −20 °C until use.  
Total bacteria analysis: DNA levels and metabolic activity 
Root canal samples from 20 patients were used to assess differences in the 

total bacteria between the first and second visit endpoints (S3b and S5). 
DNA and cDNA samples were used as templates for qPCR assays, which 
targeted conserved regions of the 16S rRNA gene of the Bacteria domain. The 
qPCR reactions (20 µL) contained: 10 µL of Power SYBR Green Master Mix 
(Applied Biosystems, Foster City, CA, USA), 2 µL of template, and 100 nM of 
each primer. Plasmid standard dilutions (from 107 to 10 DNA copies), DNA and 
cDNA samples were run in triplicate. The assay’s limit of quantification was 102 
DNA or cDNA copies; samples below the qPCR limit of quantification were 
considered negative.  
Specific bacterial species analysis: DNA levels and metabolic activity 
The analysis of total bacteria in the first 20 patients showed that the root 
canal preparation was the main step to reduce bacterial levels and activity. 
Therefore, the analysis of specific bacterial species was performed only in the 
initial (S1) and post-instrumentation samples (S2). Additional 25 patients were 
necessary for this analysis (n=45 patients). The metabolic activity of the following 
species/taxa was investigated: Bacteroidaceae sp. HOT-272 (synonym, 
Bacteroidetes oral clone X083), Cutibacterium acnes (formerly known as 
Propionibacterium acnes), Selenomonas spp. and Enterococcus faecalis. The 
oligonucleotide primers and cycling conditions for qPCR reactions are listed in 
Table 1. 
 
 

Table 1. Primers sequences and qPCR reactions. 
Taxa 
Primers Sequences 
Cycling 
conditions 
for 
qPCR reactions 
Reference 
16S 
rRNA 
universal 
CCA TGA AGT CGG AAT CGC TAG 
G CT TGA CGG GCG GTG T  
  
95°C 
for 
10 
minutes and 40 
cycles of 95°C 
for 15 seconds 
and 60°C for 1 
minute 
Shelburne 
et 
al., 
2000 (14) 
Bacteroidaceae 
sp. HOT-272 
AGAGTTTGATCCTGGCTCAG 
ACT TGA GTG GAG GGT AGG 
 
95°C 
for 
10 
minutes and 40 
cycles of 95°C 
for 1 minute and 
55°C 
for 
1 
minute 
Rôças 
et 
al., 
2014 
(15) 
Cutibacterium 
acnes 
GCGTGAGTGACGGTAATGGGTA 
TTCCGACGCGATCAACCA 
 
95°C 
for 
10 
minutes and 40 
cycles of 95°C 
for 15 seconds 
and 60°C for 1 
minute 
Eishi 
et 
al., 
2002 
(16) 
Selenomonas 
spp. 
TGGCATCATCCCCGGATA 
GCCCATCGACAGGCGATA 
 
95°C 
for 
10 
minutes and 40 
cycles of 95°C 
for 15 seconds 
and 55°C for 1 
minute 
This study 
Enterococcus 
faecalis 
CGCTTCTTTCCTCCCGAGT  
GCCATGCGGCATAAACTG 
95°C 
for 
10 
minutes and 40 
cycles of 95°C 
for 15 seconds 
and 60°C for 1 
minute  
Williams et 
al., 
2006 
(17) 
 
Microbiome analysis: Next Generation Sequencing (NGS) analysis 
A subset of 20 cDNA samples canals were subjected to PCR with 
barcoded primers (Bakt_341F CC TAC GGG NGG CWG CAG and Bakt_805R 
GAC TAC HVG GGT ATC TAA TCC) that amplify the V4–V5 region of the 16S 
rRNA gene (18). High-throughput sequencing was performed using Illumina 
MiSeq in a commercial facility (Macrogen, Seoul, Republic of Korea) according to 
the manufacturer’s protocols. 
 

Success rate of the treatment 
After 1 year of the endodontic treatment, sinus tract, pain, swelling, tenderness to 
percussion/ palpation were recorded. The absence of these signs/ symptoms 
indicated clinical success. 
Periapical radiograph images and CBCT scans were analyzed by 2 observers, 
and the apical lesion area and volume at baseline were compared with those at 
recall period. The success of the treatment was determined as absence or 
reduction (≥ 20%) of the radiolucency.  
 
Statistical Analysis Plan 
The DNA data was used for bacterial quantification, whereas rRNA data 
were used to estimate bacterial activity at samples positive for rDNA. Ratios 
between rRNA and rDNA levels were calculated to search for active bacteria 
(rRNA/DNA ≥ 1) in root canal samples.  
The nonparametric Wilcoxon signed rank test was used to determine 
differences in the bacterial levels before and after treatment procedures. 
Cochran's Q test and the Wilcoxon signed rank test were used for qualitative 
analysis (incidence of qPCR positive samples before and after treatment 
procedures).  
For each qPCR assay, the Wilcoxon signed rank test was used to 
compare the number of rRNA and DNA copies in root canal samples. McNemar’s 
test was used to compare the rRNA and DNA-based qPCR assays’ detection 
rates. Differences were considered statistically significant if P < 0.05. 
For the Next Generation Sequencing analysis, sequences were filtered 
using USEARCH tools (http://www.drive5.com/usearch/). Low quality sequences 
(score < 30) and reads shorter than 440 bp were excluded. Nonbacterial 
sequences, chimeras, and singleton reads were also removed. Sequences were 

clustered into operational taxonomic units (OTUs) at 97% similarity using the 
Quantitative 
Insights 
Into 
Microbial 
Ecology 
(QIIME) 
1.8.0 
pipeline 
(http://qiime.org/1.8.0/). All OTUs occurring <10 times were removed from the 
dataset. Taxonomic analysis was performed using the BLAST method against 
the 
Human 
Oral 
Microbiome 
Database 
(HOMD) 
version 
15.1 
(http://www.homd.org), and the relative abundance was measured using the 
QIIME pipeline.  
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