The goals of endodontic therapy are to establish and maintain
an aseptic condition inside the root canals and to treat periradicular
diseases. The success rate for this therapy ranges
between 53% and 95%, which is probably due to differences
in treatment modalities such as post-endodontic treatment
follow up and diagnostic criteria [1,2]. Endodontic retreatment
is still commonly performed by dentists in Brazil.
Endodontic instruments and chemical agents are necessary
for cleaning and shaping the root canal . Sodium hypochlorite
(NaOCl) is commonly used as an irrigating solution
in endodontic procedures due to its antimicrobial action
[4,5], and a combination of NaOCl with calcium hydroxide
and/or chlorhexidine shows excellent disinfection when applied
to the root canal [6,7].
The microbial contamination plays a major role in the periapical
etiopathogenesis. Many microorganisms that are resistant
to multiple antimicrobials can infect the dentinal tubules
and persist in the apical portion of the root canal; thus endodontic
treatment is used to reduce the microbial load in these
tissues. Studies using culture have demonstrated the presence
of anaerobic bacteria in endodontic infections including the
pulp cavities and periradicular abscesses [8,9].
Enterococcus faecalis is often found in a canal with a necrotic
pulp and during persistent or secondary endodontic infections,
and this persistence in treated root-canals reflects its
substantial resistance to hostile environments and nutritional
The complex anatomy of the root canal, with the isthmus and
the deltas, facilitates the persistence of this pathogen in the
deeper areas that are beyond the reach of endodontic instruments
and/or irrigating solutions; this explains the limited
effectiveness of antimicrobial solutions in reducing or eliminating
pathogens such as E. faecalis into root canal system
Thus, the purpose of this study was to evaluate the effectiveness
of specific antimicrobials in association against Enterococcus
faecalis in an in vitro model of root-canal-treated
Materials and Methods
This study was approved by the Ethics Committee of the Federal
University of Espirito Santo (Process No. 290/10).
Thirty-four molar teeth with three canals and completely
formed apices were selected. Teeth having between 14 and
16 mm of root length, as measured from the cervical margin
(cement-enamel junction) to the apex, were used; and teeth
displaying calcifications, root resorption or a fourth canal were
excluded of this study. All teeth were first submerged in 0.5%
NaOCl for 12 hours to achieve surface disinfection and then
kept in 0.85% phosphate buffered saline (PBS) until further
use. Caries were removed using a digger's dentin with spherical
steel bits (Duflex, SS-White, Rio de Janeiro, RJ, Brazil). Conventional
cavities were performed using a spherical diamond
bur (KG Sorensen Ind Com Ltd, Rio de Janeiro, RJ, Brazil) and
a Endo Z drills (Dentsply; Maillefer-Ballaigues, Switzerland).
The crowns were removed close to the cement-enamel junction
using a diamond disk (No. 7020; KG Sorensen Ind Com
Ltd, Rio de Janeiro, RJ, Brazil).
A dental K-file #10 (Dentsply) was inserted into each canal
using a magnifying glass, and its progress from the tip and
through the apical foramen was carefully monitored. The
working length was restricted to 1 mm less than the length of
the file at the point where it exited the root. Canals were subsequently
cleaned and shaped with K-files #10 and #15 to the
last 1 mm of their working length. Canals were then irrigated
with a solution containing 0.85% PBS and 17% EDTA (Biodynamic
Products, Sao Paulo, SP, Brazil) to remove the smear
layer. The teeth were placed into tubes containing 10 mL of
trypticase soy broth (TSB) (Himedia Laboratories, Mumbai,
India), subjected to ultrasonic agitation for 1 min to facilitate
TSB penetration, removed and sterilized at 121 °C for 20 min.
Adequate dental sterility was confirmed by incubation in TSB
at 37 °C overnight.
1 mL of a 24 hour-culture of E. faecalis ATCC 29212 was used
to inoculate 200 mL of TSB. The teeth were immersed in the
inoculated TSB and kept for a maximum of 14 days with gentle
shaking (60 rpm, 37 °C) . After 7 days of incubation,
the broth medium was renewed as above. After 14 days of experimental
contamination, 34 teeth were used for experiments
and two additional teeth were analyzed by scanning electron
microscopy (SEM, JEOL, Akishima, Japan).
Twenty-six teeth were subjected to chemo-mechanical processes
on a rotary nickel-titanium system (ProTaper Universal,
Dentsply) according to the operative sequence recommended
by the manufacturer. The canals were irrigated five times,
during 30 s each time, with 1 mL of 5.25% NaOCl (Pharmic
pharmaceutical, Vitoria, ES, Brazil) using a 5 mL syringe with
a 30-gauge needle, while the eight teeth of the control group
were irrigated five-times with 0.85% PBS.
All the 26 teeth were randomly grouped. Group 1, 13 teeth
filled with 2% chlorhexidine gel (Pharmic pharmaceutical, Vitoria,
ES, Brazil); group 2, 13 teeth filled with glycerinate calcium
hydroxide paste (Ca (OH)2) (Pharmic pharmaceutical,
Vitoria, ES, Brazil); and group 3, eight teeth filled with 0.85%
PBS (control group). All canals were dried with paper points
prior to being filled with the appropriate medication. A drill
lentulo (spiral drill #35, Dentsply) at low rotation speed was
used to place the antimicrobial inside each canal. Subsequently,
a needle tip was used to complete canal filling with 2% chlorhexidine
gel, while Ca (OH)2 was applied with an endodontic
syringe ML (Duflex-SSWhite). The canals were then sealed
using a sterile cotton ball. Treated teeth were maintained in a
tube with 50 mL of TSB at 37 oC until the end of the experiment.
Samples from each root canal were taken, as follows: after bacterial
contamination (S1); immediately after chemo-mechanical
preparation (S2); after 14 days of intracanal antimicrobial
(S3); and after seven days without any intracanal antimicrobial
(S4). S1 samples were taken after 14 days of contamination,
and each apex was sealed with cyanoacrylate cement (Superbonder,
Henkel Loctite, Belo Horizonte, MG, Brazil) after
sampling. For sampling, canals were filled with sterile 0.85%
saline solution and samples were obtained with three paper
points (Meta Biomed Co, Ltd) kept in place for 1 min. The
paper points were then transferred to a tube containing 1 mL
of 0.85% PBS, vortexed twice, and for 30 s each time. Finally,
the points were discarded and serial dilutions were performed.
S2 samples were obtained immediately after instrumentation,
and canals were filled with a 10% sodium thiosulfate solution
before sample collection to inactive the NaOCl used previously.
Canals were rinsed three times with PBS and a Hedstrom
#35 was used to soak the dentin walls for 1 min.Teeth filled
with chlorhexidine or Ca (OH)2 were sealed for 14 days, after
which the seals and antimicrobials were removed, and the S3
samples were collected. Teeth without any medication were
placed in a sterile dry tube for seven days, after which S4 samples
were collected. These samples were evaluated for the presence
of any residual bacteria.
Experimental device setup
After samples collection, each tooth was inserted individually
into tapering Eppendorf plastic tubes (Eppendorf-Elkay,
Shrewsbury, MA, USA). Each tooth was attached to the inside
of the tube with the crown enclosed and the root protruding
through the end. The teeth were sealed at the cement-enamel
junction with cyanoacrylate to prevent external contamination.
This set up, was then filled with 10 mL of TSB solution to
ensure complete immersion of the dental root .
After 14 days of dental exposure with E. faecalis, gram stain
and biochemical assays were performed to prove the purity
of strain. Bacterial quantification was performed on all teeth
using serial dilutions (10–1 to 10–6), which were then plated
(0.1 mL) onto blood agar and incubated at 37 °C for 48 hours.
Colonies were manually counted after the incubation period and CFU/mL determined.
Scanning electron microscopy
Two teeth were analyzed by SEM to confirm the bacterial colonization.
The roots were grooved at the long axis using a diamond
blade No. 7020 (KG Sorensen Ind Com Ltd) and broken
off with orthodontic pliers. The four hemi-parts were washed
with PBS to remove excess TSB, fixed in 25% glutaraldehyde
and 0.1 M cacodylate buffer, kept at room temperature for 2
hours, washed twice with 0.1 M cacodylate buffer (pH 7.2) for
10 min, and finally fixed with 1% osmium tetroxide (OsO4) in
0.1 M cacodylate buffer containing 1.25% potassium ferrocyanide.
The teeth were washed twice with ultrapure water (Milli-
Q) for 10 min and dehydrated using an ascending series of
ethanol concentration (30%, 50%, 70%, 90% and 100%) with
a 10 min incubation period at each concentration. Teeth were
desiccated using a critical point method appliance (Balzers
CPD-030, Electron Microscopy Sciences, USA) on aluminum
holders and covered with gold sputter (Balzers SCD-050, Electron
Microscopy Sciences, USA). The samples were examined
and photographed on a SEM JEOL 6100.
The Mann-Whitney test was used to compare differences between
the experimental and control groups in the S2 samples.
The Kruskal-Wallis test was used to compare values between
the PBS, chlorhexidine and Ca (OH)2 groups in the S3 samples.
The Tukey's test was used to detect differences between
the chlorhexidine and Ca (OH)2 groups in the S3 samples.
All teeth (S1 samples) showed similar levels of bacterial contamination
after 14 days of exposure to E. faecalis with no
significant difference of colonization. All teeth, irrespective of
being treated with chlorhexidine, Ca (OH)2, or TSB (control),
harbored E. faecalis and SEM revealed biofilm formation in
the dentinal tubules after 14 days of infection with E. faecalis
(Figure 1). After chemo-mechanical preparation of teeth from
control group, a reduction of the microbial load was observed.
Further, only 6 out of the 26 teeth showed bacterial growth in
TSB after this preparation.
In all groups with antimicrobials or PBS, no statistically significant
values were observed (P < 0.05) (Table 1). However, Ca
(OH)2 was significantly more effective than PBS in reducing
E. faecalis colony counts (P < 0.001). Teeth treated with chlorhexidine
(samples S3) for 14 days showed no bacterial growth
while 5 out of the 13 treated teeth with Ca (OH)2 showed bacterial
growth. At seven days, in the teeth without any intracanal
antimicrobials, a low residual activity was observed, since
E. faecalis grown in 63% of the teeth treated with Ca (OH)2 and
in 78% of the chlorhexidine treated teeth; but, this difference
was not statistically significant (P = 0.2723).
Enterococcus faecalis is a common persistent pathogen in endodontic
infections; however, its role in primary and secondary
infections is not well defined [13,14]. Studies have shown
that E. faecalis is present in teeth showing root canal treatment
failure and that it is highly resistant to endodontic treatment
[10,15]. In this study, the efficacy of a combination of NaOCl
and chlorhexidine or Ca (OH)2 in reducing E. faecalis from
root canal-treated molar teeth was evaluated in vitro.
The culture method is the gold standard for bacterial isolation and identification of viable cells, but this method is both
labor intensive and time consuming. Nonetheless, we used the
culture method to quantify E. faecalis in dental samples due to
this method has shown good reproducibility when used during
the various stages of treatment .
A 5.25% NaOCl solution is routinely used for irrigation due to
its efficacy in eliminating E. faecalis from the dentinal tubules
. Gomes et al.  have evaluated the antimicrobial action
of Ca (OH)2 when delivered with different vehicles and against
various microorganisms, including E. faecalis, and have concluded
that calcium hydroxide is the best antimicrobial agent
that can be used in endodontic treatments. Almyroudi et al.
 have also confirmed the effectiveness of Ca (OH)2 in reducing
E. faecalis counts from dentinal tubules after eight days
of antimicrobial; however, Ca (OH)2 was not efficient in reducing
E. faecalis counts after 14 days. Contrarily, we show that
Ca (OH)2 treatment for 14 days resulted in either a decrease
in bacterial number or their total absence. This might be explained
by the fact that different vehicles were used for delivering
Ca (OH)2; while we used glycerin as the delivery vehicle
based on its ability to slowly dissociate Ca (OH)2, Almyroudi
et al.  used aqueous vehicles.
Studies have shown that E. faecalis is able to penetrate the deep
dentinal tubules, and that it is highly resistant to antimicrobial
agents used in dental procedures . It is important to
note that the presence of Enterococcus spp. and its clinical relevance
in various endodontic infectious processes as well as
the high levels of resistance to antimicrobials particularly to
methicillin and vancomycin are not clear.
This microorganism is capable of growing even if under conditions
of nutritional scarcity and is generally considered viable
but not cultivable; however, it may become cultivable under
favorable conditions .
Chlorhexidine is the drug of choice in endodontic treatments
because it has a broad antimicrobial spectrum that also includes
the anaerobic bacteria associated with endodontic
treatment failure. Its substantial effectiveness and low tissue
toxicity further justify its use as an intracanal antimicrobial
[21,22]. Studies have shown that even though chlorhexidine is
an effective disinfectant, it is not a curative drug .
We evaluated the effect of chlorhexidine treatment on root
canals after 7 days without any intracanal antimicrobial and
found a logarithmic increase in CFU of E. faecalis; this result
is in accordance with those reported previously by Delgado
et al. . Importantly, no recontamination was observed in
the root-canal treated teeth that were not filled with any intracanal
antimicrobial for seven days. This is probably because
both chlorhexidine and Ca (OH)2 are capable of disintegrating
cell membranes on physical contact, and the presence of these
drugs in the root canal acts as an antimicrobial barrier.
In clinical practice, it is essential to place intracanal antimicrobial
as a complementary treatment in the root canals after mechanical
instrumentation. Possible failures in root canal filling
help bacteria remain within the canal, thus causing failure of
therapy. We therefore evaluated the residual effect of antimicrobials
in removing E. faecalis and found that after seven days without drugs there as a low reduction of this microorganism.
Electron microscopy revealed adequate bacterial colonization
after 14 days of contamination; however, it is possible that the
intracanal antimicrobials used did not sufficiently reach all
areas of the canal. Similar observations have also been previously
A limitation of this study is that it is an in vitro study, and thus,
the intraoral environment, comprising an extensive bacterial
diversity, could not be reproduced. It is also possible that, in
this in vitro model, sufficient nutrition owing to only a monoinfection
also favored bacterial survival.
In conclusion, we report that 5.25% sodium hypochlorite, either
with a 2% chlorhexidine gel or Ca (OH)2 is effective in
reducing the number of E. faecalis bacteria in root canals,
and that the use of chlorhexidine was more effective than Ca
(OH)2. It may be explained because chlorhexidine is adsorbed
on dental surface for long time displaying a close contact with
oral microorganisms. The absence of recontamination of the
main canal even in the absence of intracanal antimicrobial for
seven days provides further proof of their effectiveness against
The authors thank Mrs. Marcia Harumi for her technical
support. This study was supported by grants from CAPESPNPD
(No. 2009), FAPESP (No. 2013/13652-6) and FAPES