Recovery of Enterococcus faecalis after single- or multiple-visit root canal treatments carried out in infected teeth ex vivo.

Abstract

To assess the presence of Enterococcus faecalis after root canal treatment in single or multiple visits in an ex vivo model.
Forty-five premolar teeth were infected ex vivo with E. faecalis for 60 days. The canals were then prepared using a crowndown technique with System GT and Gates-Glidden burs and irrigated with 2% chlorhexidine gel. The specimens were divided into five groups (G1, G2, G3, G4 and G5) according to the time elapsed between chemical-mechanical preparation and root canal filling, the irrigant solution used and the use or nonuse of a calcium hydroxide intra-canal medicament. The teeth were then root-filled and incubated for 60 days at 37 degrees C. Dentine chips were removed from the canal walls with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing Brain-Heart Infusion broth. These samples were placed onto agar plates and colony forming units were counted after 24 h at 37 degrees C. Data were ranked and analysed using the Kruskal-Wallis statistical test.
Enterococcus faecalis was recovered from 20% (three of 15 specimens) of G1 (chlorhexidine irrigation and immediate root filling in a single visit), 25% (four of 15 specimens) of G2 (chlorhexidine irrigation and filling after 14 days use of a calcium hydroxide dressing in multiple visits), 40% (two of five specimens) of G3 (chlorhexidine irrigation and filling after 7 days), 60% (three of five specimens) of G4 (saline irrigation and filling after 7 days) and from 100% (five of five specimens) of G5 (saline irrigation and immediate filling without sealer).
Neither single- nor multiple-visit root canal treatment ex vivo, eliminated E. faecalis completely from dentinal tubules. Up to 60 days after root filling, E. faecalis remained viable inside dentinal tubules. When no sealer was used, E. faecalis presented a higher growth rate.

Full text

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Recovery of Enterococcus faecalis after single- or
multiple-visit root canal treatments carried out in
infected teeth ex vivo
N. Vivacqua-Gomes1,2, E. D. Gurgel-Filho3, B. P. F. A. Gomes1, C. C. R. Ferraz1, A. A. Zaia1
& F. J. Souza-Filho1
1Dental School of Piracicaba – State University of Campinas, UNICAMP, Piracicaba-SP;2Department of Endodontics – Dentistry
Brazilian Association-Ceara `, Fortaleza-CE; and3Department of Endodontics, University of Fortaleza, UNIFOR, Fortaleza-CE, Brazil
Abstract
Vivacqua-Gomes N, Gurgel-Filho ED, Gomes BPFA,
Ferraz CCR, Zaia AA, Souza-Filho FJ. Recovery of Entero-
coccus faecalis after single- or multiple-visit root canal treat-
ments carried out in infected teeth ex vivo. International
Endodontic Journal, 38, 697–704, 2005.
Aim To assess the presence of Enterococcus faecalis
after root canal treatment in single or multiple visits in
an ex vivo model.
Methodology Forty-five premolar teeth were infec-
ted ex vivo with E. faecalis for 60 days. The canals were
then prepared using a crowndown technique with
System GT and Gates–Glidden burs and irrigated with
2% chlorhexidine gel. The specimens were divided into
five groups (G1, G2, G3, G4 and G5) according to the
time elapsed between chemical–mechanical prepar-
ation and root canal filling, the irrigant solution used
and the use or nonuse of a calcium hydroxide intra-
canal medicament. The teeth were then root-filled and
incubated for 60 days at 37 ?C. Dentine chips were
removed from the canal walls with sequential sterile
round burs at low speed. The samples obtained with
each bur were immediately collected in separate test
tubes containing Brain–Heart Infusion broth. These
samples were placed onto agar plates and colony
forming units were counted after 24 h at 37 ?C. Data
were ranked and analysed using the Kruskal–Wallis
statistical test.
Results Enterococcus faecalis was recovered from 20%
(three of 15 specimens) of G1 (chlorhexidine irrigation
and immediate root filling in a single visit), 25% (four of
15 specimens) of G2 (chlorhexidine irrigation and
filling after 14 days use of a calcium hydroxide dressing
in multiple visits), 40% (two of five specimens) of G3
(chlorhexidine irrigation and filling after 7 days), 60%
(three of five specimens) of G4 (saline irrigation and
filling after 7 days) and from 100% (five of five
specimens) of G5 (saline irrigation and immediate
filling without sealer).
Conclusions Neither single- nor multiple-visit root
canal treatment ex vivo, eliminated E. faecalis com-
pletely from dentinal tubules. Up to 60 days after root
filling, E. faecalis remained viable inside dentinal
tubules. When no sealer was used, E. faecalis presented
a higher growth rate.
Keywords: calcium
gel, Enterococcus faecalis, root canal treatment, single
visit.
hydroxide,chlorhexidine
Received 1 November 2004; accepted 18 April 2005
Introduction
The role of bacteria and their by-products in the
initiation and perpetuation of pulpal and periapical
disease has been well established (Gomes et al. 2002b).
Most infecting bacteria, together with their principal
substrate of necrotic pulp debris, may be removed by
routine intra-canal procedures such as instrumentation
and irrigation of the pulp space and the use of an intra-
canal medicamenthaving
Nevertheless, this is not always fully achieved in
clinical practice. The anatomical complexities of
many root canals and consequent access limitations
antimicrobialactivity.
Correspondence: Dr Eduardo Diogo Gurgel-Filho, Rua Silva
Jatahy,1140apto2002,
60.165.070 Brazil (Tel.: +55 85 34866281; fax: +55 85
34866280; e-mail: gurgeleduardo@unifor.br).
Meireles,Fortaleza-CE CEP:
ª 2005 International Endodontic JournalInternational Endodontic Journal, 38, 697–704, 2005
697

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of instruments, irrigants and intra-canal medicaments
are well-recognized factors (Biffi & Rodrigues 1989,
Gomes et al. 2002b). Moreover, despite the antimicro-
bial effect of chemomechanical preparation and intra-
canal medicaments, the elimination of microorganisms
may not be uniform because of the vulnerability of the
species involved (Gomes et al. 1996). Therefore, con-
cern exists as to the fate and consequences of the
remaining microorganisms in the canal. They may
multiply rapidly, in some cases, to almost the initial
number in 2–4 days, if the canal is left empty (Bystro ¨m
& Sundqvist 1981).
Many clinicians believe that the bacteria remaining
after chemomechanical preparation can be eliminated
or be prevented from repopulating the root canal space
by placing an interappointment medicament. Another
approach is to eliminate the influence of the remaining
microorganisms or to render them harmless by
entombing them in a filling, immediately after prepar-
ing and irrigating the canal space in the same visit.
This way, the remaining microorganisms may be killed
by the antimicrobial activity of the sealer or the Zn2+
ions of gutta-percha (Moorer & Genet 1982, Kaplan
et al. 1999, Fuss et al. 2000, Siqueira et al. 2000,
Peters & Wesselink 2002).
Root canal treatment in single or multiple visits
should be viewed as part of a total endodontic
treatment spectrum, with the choice of one over the
other being determined by the circumstances peculiar
to each particular case. The technique chosen would
then best fit those circumstances (Ashkenaz 1984). The
healing potential for teeth treated in one or two visits
with an intra-canal disinfectant being applied appears
to be similar (Katebzadeh et al. 1999, Weiger et al.
2000). Some studies report the presence of remaining
microorganisms after cleaning and shaping, and then
dressing with calcium hydroxide. But they do not relate
this to healing (Bystro ¨m et al. 1985, Yared & Bou
Dagher 1994, Shuping et al. 2000).
Peters & Wesselink (2002) showed that positive
cultures obtained immediately before root filling did not
influence the outcome of treatment in either single or
multiple visits. However, Sjo ¨gren et al. (1997) did
report a strong relationship between bacterial contam-
ination and treatment outcome. If teeth are symptom-
free and the canals are dry, it should be possible to
complete root canal treatment in a single visit. Weiger
et al. (2000) observed that the success rate, after
5 years, in single-visit and two-visit root canal treat-
ments with calcium hydroxide was 92 and 93%
respectively. Complete radiographic healing was found,
up to 4.5 years after treatment, in 81% of the cases
treated in one-visit and 71% of the cases treated in two
visits with calcium hydroxide dressing (Peters &
Wesselink 2002).
Enterococcus faecalis, a facultative gram-positive
bacterium, has been considered one of the most
resistant species in the oral cavity and one possible
cause of post-treatment disease after root canal
treatment. Several studies have reported their low
susceptibility to irrigant solutions (Gomes et al. 2001,
Vianna et al. 2004) and to intra-canal medicaments
such as calcium hydroxide (Siqueira & Uzeda 1997,
Valera et al. 2001, Gomes et al. 2002a,b, 2003,
Menezes et al. 2004). Therefore, they may persist after
instrumentation, medication and even after root filling,
increasing the risk of post-treatment disease (Bystro ¨m
et al. 1987, Sjo ¨gren et al. 1991).
Root canal treatment has been described as the
disinfection of the root canal system, using endodontic
instruments aided by an antimicrobial agent, before
filling. Gomes et al. (2001) studied the disinfection time
of several concentrations of chlorhexidine (either in gel
or liquid vehicles) and sodium hypochlorite against
E. faecalis. The 2.0% chlorhexidine in both presentation
forms gave the shortest disinfection times, the same as
with 5.25% sodium hypochlorite. Chlorhexidine has a
broad antimicrobial activity against Gram-negative and
Gram-positive bacteria, especially E. faecalis (Gomes
et al. 2001, Vianna et al. 2004). Therefore, it has been
used as irrigant or intra-canal medicament to improve
disinfection.
Thus, the purpose of this study was to assess E. faecalis
survival after root canal filling of infected extracted
teeth in single and multiple visits, using chlorhexidine
as irrigating solution in an ex vivo laboratory model.
Material and methods
The method followed was a modification of one
described previously by Gomes et al. (2003). Forty-five
mandibular premolars freshly extracted for orthodontic
reasons with complete apex formation and foramen
diameter measuring up to a size 15 file were used. The
teeth were cleaned with curettes to remove periodontal
tissue and bone. The specimens were kept in 0.5%
sodium hypochlorite solution for no longer than
7 days.
The crowns were removed, to enhance access, with
carborundum disks (KG Sorensen, Barueri, Brazil) to
the level of the amelo-dentinal junction resulting in
roots 15 mm in length. The teeth were instrumented to
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International Endodontic Journal, 38, 697–704, 2005
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the apex using a size 25 file (Dentsply Maillefer,
Ballaigues, Switzerland) to create a patency and
facilitate the intra-canal contamination procedures.
The external root cementum was completely removed
using diamond burs (Haapasalo & Ørstavik 1987).
All teeth were submitted to an ultrasonic bath for
10 min in 17% EDTA, followed by 10 min in a 5.25%
NaOCl bath, according to Perez et al. (1993) and a
tampon phosphate bath (to eliminate EDTA and
hypochlorite residues) followed by a distilled water
bath (10 min each), in order to eliminate the smear
layer produced during the initial preparation.
The teeth were individually placed in bijoux bottles
containing 3.0 mL of Brain–Heart Infusion Medium
(BHI; Oxoid, Basingstoke, UK) and autoclaved at
121 ?C, 1 atm, for 15 min. They were then kept in
an incubator at 37 ?C for 24 h to check the efficacy of
the sterilization treatment.
Isolated colonies (24 h) of pure cultures of E. faecalis
(ATCC 29212) grown on 5% defibrinated sheep
blood + BHI agar plates were suspended in 5.0 mL of
BHI. The cell suspension was adjusted spectrophoto-
metrically to match the turbidity of 1.5 · 108colony
forming unit (CFU) mL)1(equivalent to ± 0.5 McFar-
land standard).
The bijoux bottles containing each specimen were
opened under laminar flow. Sterile pipettes were used
to remove 2.0 mL of sterile BHI and to replace it with
2.0 mL of the bacterial inoculum. The bottles were
closed and kept at 37 ?C for 60 days, with the
replacement of 1.0 mL of contaminated BHI for
1.0 mL of freshly prepared BHI every 2 days, to avoid
medium saturation. The turbidity of the medium
during the incubationperiod
growth. The purity of the cultures was confirmed by
Gram staining, catalase production, colony morphol-
ogy on BHI agar + blood and by the use of a
biochemical identification kit (API 20 Strep; Bio-
Me ´rieux SA, Marcy-l’Etoile, France). Bacterial penetra-
tion into the dentinal tubules using this technique was
confirmed using SEM in a pilot study (Fig. 1).
Following the contamination period, each specimen
was removed from its bijoux bottle under aseptic
conditions and mounted in a sterile aluminium appar-
atus (Fig. 2). The canal was irrigated with 3.0 mL of
0.85% sterile saline and microbial samples (Sample 1)
were collected with a sterile paper point. The paper
point samples were placed individually in a 1.5 mL
eppendorff tubes containing 1 mL BHI. The eppendorff
tubes were then shaken for 30 s, followed by dilutions
until 100 times. Fifty-microlitre aliquots of the dilutions
indicated bacterial
were inoculated in BHIA + 5% of sheep defibrinated
blood plates, which after 24 h at 37 ?C incubation
were inspected for CFU. The numbers counted were
multiplied by 2000 to obtain CFU mL)1. The purity of
the cultures was confirmed by Gram staining, catalase
production, colony morphology and by the use of a
biochemical identification kit (API 20 Strep; Bio-
Me ´rieux SA). These samples were used to confirm
homogeneous infection in specimens.
The canals were instrumented using Greater Taper
instruments (Tulsa Dentsply, Tulsa, OK, USA) from
tapers 12 through 6, tip size 20 (Buchanan 1994).
Gates–Glidden burs were then used from sizes 6 to 2 in
the crowndown technique, followed by manual apical
preparation up to a size 35 file (master apical file;
Figure 1 Dentin infection after 60 days.
Figure 2 Aluminium apparatus.
Vivacqua-Gomes et al. Dentin disinfection after single- or multiple-visit treatment
ª 2005 International Endodontic Journal International Endodontic Journal, 38, 697–704, 2005
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Maillefer Dentsply). Two test and three control groups
were distributed as follows:
G1. Single visit – 0.5 mL of 2% Chlorhexidine gel
(EndogelTM; Essencial Pharma, Itapetininga, SP, Brazil)
irrigation between each file and immediate filling (15
specimens).
G2. Multiple visit – 0.5 mL of 2% Chlorhexidine gel
(EndogelTM) irrigation between each file and filling after
14 days of calcium hydroxide intra-canal dressing (15
specimens).
G3. 0.5 mL of 2% Chlorhexidine gel (EndogelTM)
irrigation between each file and filling after 7 days of
BHI culture medium intra-canal dressing (five speci-
mens; Chlorhexidine substantivity control).
G4. 1 mL saline solution irrigation between each file
and filling after 7 days of BHI culture medium intra-
canal dressing (five specimens; positive control).
G5. 1 mL saline solution irrigation each file and
immediate filling only with gutta-percha cones and
without sealer (five specimens; positive control).
All groups were irrigated 5 mm short apical foramen
with 5 mL saline solution before filling (20 · 5.524 3/
4 needle), including the calcium hydroxide group. After
that, the specimens were dried with sterile medium
paper points (KonneTM, Belo Horizonte, Brazil) and
filled with KonneTMmedium gutta-percha points (taper
.06) and a few accessory points (when necessary).
Grossman sealer was also applied using warm vertical
condensation (De Deus 1992), except for G5 where
only gutta-percha points were used.
The teeth where intra-canal dressings were used (G2,
G3 and G5) were sealed coronally with CavitTM(3M-
Espe, Seefeld, Germany) and incubated at 37 ?C in
eppendorff tubes with BHI (without entirely covering
the root) for the established periods.
After being filled, all groups were sealed coronally
with CavitTMagain and incubated in eppendorff tubes
with 50 lL BHI for 60 days at 37 ?C. Every 72 h, the
BHI was exchanged.
Coronal seals were removed and the specimens were
transversally cut with a diamond disk (KG Sorensen) in
the middle to the apical third, to obtain a 9–10 mm
cervical fragment and a 5 mm apical fragment. The
coronal and apical fragments were mounted and fixed
in a sterile aluminium apparatus. The mean dentine
width was 2 mm in the coronal fragment and 1.6 mm
in apical one (Fig. 3).
Two sizes of round burs were used to sample the
internal dentine and gutta-percha on both cervical
and apical fragments. The first bur was adapted to
the root canal diameter. The shallow collection (SC)
was obtained by removing all filling material and
approximately200–250 lm
dentine over the complete extension of the fragment
(round bur ISO 10 for the apical fragment and 16 for
the cervical one). The second bur produced the deep
collection(DC) removing
650 lm of dentine from the apical and cervical
fragments (round bur ISO 18 in the apical fragment
and 25 in the cervical one) (Fig. 3). Debris was
collected in a 5 mL glass flask containing 1 mL of
BHI. These samples were shaken for 1 min and
100 lL was placed onto BHI agar plates that were
incubated for 24 h at 37 ?C to allow a count of CFU.
All counts were multiplied by 10 to obtain CFU mL)1
(Almyroudi et al. 2002).
All samples were identified by Gram staining,
catalase production, colony morphology on BHI
agar + blood and by the use of a biochemical identi-
fication kit (API 20; Strep – bioMe ´rieux SA). Miniapi
software (BioMe ´rieux SA) was used to visually read the
API range tests from BioMe ´rieux.
ofthecircumpulpal
uptoapproximately
Results
The Kruskal–Wallis statistical test was applied with 5%
significance. All initial samples presented high CFU
counts (mean of 106each specimen); there were no
statistically significant differences between the five
experimental groups (P > 0.05).
Up to 60 days following filling, E. faecalis was
viable inside dentinal tubules. E. faecalis cells were
recovered from 20% (three of 15 specimens) of G1
root blocks (chlorhexidine irrigation and immediate
root filling in a single visit), 25% (four of 15
specimens) of G2 root blocks (chlorhexidine irrigation
and filling after 14 days of calcium hydroxide dress-
ing in multiple visits), 40% (two of five specimens) of
G3 root blocks (chlorhexidine irrigation and filling
after 7 days), 60% (three of five specimens) G4 root
blocks (saline irrigation and filling after 7 days) and
100% (five of five specimens) of G5 root blocks
(saline irrigationand immediate
sealer).
No statistical differences were found between G1, G2
and G3 (P > 0.05). G4 had significantly higher CFU
counts than G1 and G2 (P < 0.05), but without a
difference to G3 group (P > 0.05). G5 was statistically
different to all other groups (P < 0.05), except for the
apical G3 shallow collection (P > 0.05), cervical G4
deep collection and all apical G4 collections (P > 0.05)
(Figs 4 and 5).
fillingwithout
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Discussion
The dentine block model, suggested by Gomes et al.
(2003), was used with some modifications. E. faecalis
has been detected within dentinal tubules up to
500–700 lm after 60 days of incubation. Saleh et al.
(2004) used 3 weeks of incubation and found micro-
organisms up to 300–400 lm within the tubules. The
results of the present study are not compatible with
those of Haapasalo & Ørstavik (1987) who reported
that the average depth of penetration of the tubules by
bacteria increased only slowly with time.
In the current study, all the experimental groups had
viable residual E. faecalis, either in gutta-percha and
sealer or in the inner dentine layers. However, the
number of cells recovered in G1 and G2 was smaller
than in other groups. In the single-visit group (G1),
E. faecalis was equally recovered from both root
ISO 10
ISO 18
ISO 16
ISO 25
5 mm
4 mm
5.5 mm
10 mm
2 mm
1.6 mm
A
B
Figure 3 Debris collection using round
burs. A: cervical and medium thirds;
B: apical third.
75.3
76
89.2
105.6
165.8
0
20
40
60
80
100
120
140
160
180
CFU mean ranks
G1G2G3G4G5
ababca
Figure 4 Ranked colony forming unit
(CFU) averages, per group. Values
followed by different letters presented
significant statistical differences using
the Kruskal–Wallis statistical test
(P < 0.05).
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fragments, whilst in the multiple-visit group (G2) they
were recovered more frequently in the apical fragment.
This may be due to the inherent difficulties of inserting
calcium hydroxide into the apical third of the root
canal and also because of the anatomical character-
istics of this region, where the dentine tubules are
smaller and less frequent making the diffusion of
hydroxyl ions more difficult. For these reasons bacterial
growth in that region between treatment visits may
have occurred. Haapasalo et al. (2000) stated that the
poor diffusion of hydroxyl ions into infected-dentine
and the buffering capacity of dentine can limit the raise
of pH. These are some of the reasons why hydroxide
pastes are ineffective against E. faecalis even after an
extended incubation time.
Chlorhexidine gel was chosen to irrigate the canals of
the test groups because of its broad-antimicrobial spec-
trum (Jeansonne & White 1994, White et al. 1997,
Gomes et al. 2001,2003, Vianna et al. 2004), good
lubrication (Ferraz et al. 2001) and especially its sub-
stantivity (i.e. residual antimicrobial activity) (Kuruvilla
& Kamath 1998, Tanomaru Filho et al. 2002). Such
residual antimicrobial activity may explain the reduced
numberofbacteria recoveredfromgroup G3(40%)after
irrigation with chlorhexidine and 1 week with BHI
medium intra-canaldressing. Ontheotherhand,forG4,
where the canals were irrigated with saline solution
followed by 7 days BHI medium intra-canal dressing,
bacteria were recovered from 60% of the canals.
The root canal sealer is also of great importance in
root canal filling, removing all space and nutrients
necessary to maintain bacterial viability. In G5, where
the canals were irrigated with saline and filled with
gutta-percha only, E. faecalis was recovered from 100%
of the canals. This result confirms the findings of Saleh
et al. (2004) that the use of Grossman sealer in vitro
killed all bacteria in the dentinal tubules within a
300 lm zone around the root canal.
Several studies (Bystro ¨m et al. 1985, Haapasalo &
Ørstavik 1987, Safavi et al. 1990, Saleh et al. 2004)
have shown that calcium hydroxide has limited capa-
bility to completely kill bacterial cells inside dentinal
tubules. The present study showed that after single and
multiple visits, the number of E. faecalis cells inside the
root canals can be reduced, but cannot be eliminated
completely. However, the E. faecalis cells were main-
tained viable because the cementum was intentionally
removed and the dentinal tubules were in contact in
the middle third of the canals containing the BHI
medium. The root canal cementum is of greater
importance in isolating patent dentinal tubules on the
periodontal side, preventing residual bacteria from
reaching exit portals to the periodontal space, except
in larger lateral canals.
Thus, when the cementum is present, residual
bacteria inside dentinal tubules will not survive with-
out nutrients and space for their growth (Haapasalo &
Ørstavik 1987, Safavi et al. 1990). The cementum was
confirmed by Berutti et al. (1997) to be a barrier
against the penetration of bacteria.
Clinically, the nutrition required for microbial cell
recovery may come from coronal leakage, from fluid
within dentinal tubules or from periapical tissues. This
demonstrates the importance of complete filling of the
root canal system after chemomechanical disinfection,
followed by a high quality coronal seal, in order to
prevent post-treatment failure.
Conclusion
Within the limits of this study:
1 No statistical differences in E. faecalis counts were
found ex vivo between single-visit and multiple-visit
root canal treatments.
2 Even after 60 days of root filling E. faecalis remained
viable inside dentinal tubules ex vivo.
0
20
40
60
Cervical SC
Cervical DC
Apical SC
Apical DC
Fragments
80
100
120
140
160
180
CFU mean ranks
G1
G2
G3
G4
G5
bc
bc
bc
c
ab
a
abc
abcabc
a
aa
a
a
a
a
a
a a a
Figure 5 Colony forming units (CFU) per
group, per fragment. SC, shallow collec-
tion; DP, deep collection. Values followed
by different letters presented significant
statistical differences using the Kruskal–
Wallis statistical test (P < 0.05).
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3 When no sealer was used E. faecalis had a higher
growth rate ex vivo.
Acknowledgements
The authors would like to thank Mr Adailton dos
Santos Lima for technical support, NAP MEPA-ESALQ-
USP, Piracicaba, Brazil for permitting the use of the
scanning electron microscope and Biolab Me ´rieux for
lending the MiniApi equipment. This work was sup-
ported by the Brazilian agencies FAPESP (2000/
13689-7, 04/05743-2) and CNPq (520277/99-6,
304282/2003-0, 141299/2001-0).
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